ABSTRACT entitled ROLE AND DISTRIBUTION OF SELECTIVE CELLULAR 3OMPONENTS OF OSMOREGULATORY TARGET ORGANS IN
SUBMITTED TOALIGARH MUSLIM UNIVERSITY FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
w SUBUHI ABIDI
DEPERTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA) 2013 `Y~ ~~, na Azad Lrh
r ^ rc. No....__._......
l ~N - &ii ~]il .' ABSTRACT
of the thesis entitled "Role and distribution of selective cellular components of osmoregulatory target organs in some freshwater teleost fishes'
Background
Fishes constitute a major vertebrate group and a predominant component of aquatic fauna. The body suface of all mutticellular organisms including fishes, is protected by epithelial lining wh!ch serves as a physical barder bebaeen the 'mlemal milieu and the external environment. Integument, gia and GI tract constitute a large surface area to serve as -contact surface for the passage of solute, water and also possible invasion of pathogens. The occurrence of a thick and prominent mucus layer over the epithelial surface of these organs, in addition to others, contribute a great deal to, lubrication for smooth passage, interlace for the passage of gases and ions, defense against predators, protecton against any frictional and mechanical injury and invasion of the environmental pathogens. Hence, mucus, an innocuous but complex srimy secretion contained predominantly in the cells called goblet cells and spread over various epithelial surfaces has assumed considerable biological significance due to its wide distribution in varlely of vital organs. In view of its diverse functional nature, the study on mucus has of late, gained considerable momentum amongst clinicians, biochemists, histologists, physical chemists and biologists.
Broadly mucus consists of 95% water and remaining 5'/o is made up of organic and inorganic constituents. The organic constituents are typically made-up of carbohydrates, proteins and lipids either in free or conjugated forms called glycococjugates. The mucus glycoprotein is of extremely high molecular weight and more than 50% of this molecule weight consists of carbohydrate. The structural organization is such that a polypeptide chain fors a central core to which oligoseccharide units are embedded along its length attached to almost every third amino acid. The tddert mucus functions include lubrication, water proofing and indeed protection. Even though mucus overlays the epitheliel surfaces of gill, skin and GIT but it plays a more critical role in case of integurnent and gills which is the first line of defense against the microbial exposure. In case of GIT, muss functions as the skin of the guP where it acts more effectively than the skin of the body to provide protection to gastric mucosa againstrowerful acid and proteolyflcenzymes.
The involvement of mucus in osmoregulatlon has been suggested in some earlier experiments in eel Anguilla anguifla where wiping of fish skin mucus compromised the osmoregulatory ability Hypophysectomy of these fishes reduced the lumber of goblet cells and replacement with prolactin, an important osrnoregulatory hormone in fresh water fish, reversed this effect Abundance of goblet cells on fish integument and gills may correlate with environmental salinity. That sulpha- and sialomucin of fish mucus have teen very intimately linked with the maintenance of homeostatic balance in divergent salinities. Hence, there is strongly suggestive evidence that fish muds in integument, gill and SIT plays an important role in osmoregulatory adjustment of lelecsis. The fresh water stenohalioe catfish H. fossils and C. hatrachusare important components of capture and culture fishery of the Indian subcontinent and are preferred consumers choice. The basic csmoregulatory mechanism of H. fossilis in high salinities as well as in deionized water involves the coordinated participation of the major target organs i.e. gill, skin, gut and kidney. However, despite the fact that mucus covers at least three major osm oregulatory target organs ''.. e. skin, gills and gut and is known to be a mulfduretional entity, its biochemical nature and pcsaible role in these osmoregulatory target organs have not been studied. Moreover, no detailed account with reference to cellular and other histological features of the above three important osmoregulatory target organs i.e. skin, gill and gut is documented in literature. Clearly, such an account is extremely important to understand the role of mucus 'is-i-vis these three target organs in the overall perspective of osmoregulatory adjustment. Hence, the present study is designed to ill this long standing gap in our knowledge to study the distribution of mucus in three important osmoregulatory target organs i.e. skin, gills and GIT, its glycoptotein components and temporal variations and functional role in csmoregulatory processes of these two important Indian catfishes H. fossils and C. batrachus.
The information thus generated will help us to build up a comprehensive overview of better and more precise understanding of the morphology, histology and celliiar composition of skin, gill and GI tact, the important osmoregulatory target organs in fshes. The consortium of such information in conjunction with the pre-existing documented information on these aspects will greatly enhance our current understanding of the role of mucus in csrnoregulatory adjustment in teleost in general and in tropical freshwaterstenohaline fishes in particular.
The organization of the thesis components
This thesis is divided into four district chapters which are preceded by an introductory preface on the significance and other important aspects of the mucus. The first chapter deals with the effects of handling stress on the skin and gill mucous cells and mapping of the relative abundance of mucous cells in different locational regions of skin and the gills. The next three chapters focuses on the general histological organization and role of mucus in integument, gills and GIT of these catfishes. The detailed protocol of the common methodology used in each chapter has been elaborately described under separate section captioned 'Materials and Methods' and the specific 'Experimental Protocor is included in each chapter. The chapter-wise salient Findings are described below,
(Chapter l)
Handling stress and mucous cells region-based distribution:
This chapter deals with Iwo important aspects to study the effects of experimental handling on the mucous cells population and also to ascertain the distributional pattern of mucous cells in different regions of the integument and different gills of the catfishes H fossils and C. batrachus, The fish and other aquatic vertebrates are subjected to greater variety of stressors compared to their terrestrial counterparts which will directly affect their horneostatic mechanism. The focal theme of the present Investigation is to explore the role of overlying mucus layer in three important osmomgulatory organs i.e. skin, gills and the GIT. This mucus is being secreted chiefly by the goblet cells I mucous cells in these organs. The sampling protocol, normally followed, is to net out fishes from aquaria, sacrifice them and excise the target tissues for histological and other investigations. However, considering the basic tenets of stress physiology, one wonders if this simple innocuous protocol of sampling is not exerting any stress on to the fishes and particularly on the mucous cells which are known to play a significant role in defense and protection of fishes against unfavourable conditions and invading pathogens. If the sampling protocol followed by us is stressful, then, any data generated based on this protocol of sampling will be 'grossly suspect' until the issue of perceived stressful conditions are settled. This, therefore, greatly underlines the need for establishing a baseline data based on the protocol which may yield data on "unstressed/ normal" conditions to arrive at meaningful conclusions. In the present study, we were seized of the fact that evei a simp:e act of netting the catfish from the aquarium for sampling purpose may prove to be a stressful situation which might obscure the ultimate objective of the study. Hence, the twin objectives of the present chapter were. (i) to establish whetherihe sampling protocol followed by different investigators to study the mucous cells in fishes riot affect the normal histological and physiological status of these cells? and (ii) secondly, whether the mucous cells are uniformly distributed all over the skin with no region or gill - number based variations.
Our findings have shown that routine experimental handling of catfishes has caused total disappearance of mucous cells in the skin of both the catfishes which, however, vies on somewhat expected lines. But what surprised us most was the acuteness of response which seems to have been more accentuated since both these catfishes are devoid of scales on the skin surface and, therefore, were in direct contact with the handling implement i.e. the nylon net which was used to take out these fishes from aquarium during the experimental sampling. However, the possible underlying mechanism which brings about such compete disappearance of mucous cells following handling stress in both catfishes has not been investigated because it is not order the focal theme of the present study. Based on our long experience of having worked with these fishes, we found both these catfishes are extreme:y hardy and quite capable of withstanding simultaneously performed surgical manipulations like hypophysectomy, gonadectomy and pioealectomy an the same individual with almost negligible mortality despite such heavy surgical trauma. Also, in one of our earlier studies, we have established conclusively that the repeated handling stress caused in the present protocol does not activate HPI axis since plasma cortisol levels do riot show any significant increase in H. fossil's. What then, is causing the complete disappearance of skin mucous cells is an interesting proposition for further investigation. Another observation in the present study is that gill mucous cells seem unaffected by the same handling stress which brings about the complete disappearance of skin mucous cells. To the best of our knowledge, there is no other report in literature on any fish species where such varied response to the same stimulus and in the same species has been observed. At this point, it is believed that the reason of variations in such a response could he physical rather than metabolic. Hence, the skin of both these catfishes which are subjected to touch stimuli have responded by sudden and complete exudation of mucus and the ultimate loss of its mucous cells. However, since gills are internalized by opercular covering and hence, quite unlike skin, not in direct physical touch with the implement of netting, have shown the normal distribution of mucous cells all over gill epithelium even following the handling stress. The observation in the present study that anesthesia MS222 (Tricaine methanesulphonate) provide the complete resting i basal level conditions as reflected in the normal histopalhology of skin mucous cells and obviate stress-induced changes is interesting. Anaesthesia has been frequently applied in aquaculture and live animals experimentation to subdue neuromotor control. Hence, this approach is ideally suited to collect samples from unstressed fish and to generate baseline data.
We have also observed highly significant region-based variations in the density of Inteaumentary mucous cells In both catfishes. Interestingly, while the profiles of region based variations are different in both catfishes but the highest abundance of mucous cells in both these catfshes is recorded in the same region i.e. below the dorsal fin. Further, the degree of inter-region variations is more marked in C. hatrachus which has recorded a near total absence of mucous cells in the caudal region whereas in H. fossils these cells are present in good noticeable number even in the anal region which recorded the minimum population of these cells in H. fossilis The possible reasons of the maximum abundance of mucous cells in and around dorsal fin region in both the catfishes could possibly be due to its protective strategy against predators. In both these catishes, external body contours and undulating swimming movements are more or less identical and dorsal region below the pectoral fin is not as much subjected to gyrating body movements as the other parts of the body and hence may serve as the ideal site for the predator for grasping. Abundant mucus covering over this region due to greater number of mucous cells may well be an adaptational response against such adversities. The total lack distributional vacations in the abundance of mucous cells populations in different gills located antero-pesteriorly in both catfishes is exactly the same as observed in the handling response to gills in both these catfishes. At this point, we may not be able to offer any other plausible explanation for such a lack of distributional variation in gills except to reiterate the same logic of the internalized existence of girls unlike integument and complete lack of any chance of tactile stimulus acting directly on the gill surface,
In conclusion, it may he stated that the findings of the present chapter have demonstrated comprehensively that the handling stress brings about the total disappearance of mucous cells in the skin but not in gills and that there is a region-based distributional variation of mucous cells in the integument but not the gills of both catshes. These findings may be of practical significance to those involved in the study of fish integumentary biology and fish toxicology using histology and histcpathology as the end markers.
4 (Chapter II) Integument:
This chapter deals with H. fossils and C. batrachus integument which, unlike mammals, is one of the major organs of mucus secretion. It forms an all-round covering over the body surface and serves as the direct interface between animal and its environment. Fish integument is essentially based on generalized mammalian pattern and performs majority of the functions associated with vertebrate skin. However, in certain ways, teleost skin is unique and histologically diverse since it reflects adaptation of the teleost to the challenges posed by aquatic environment. In this chapter, three important aspects of catfish integument have been dealt with. The first aspect relates to general morphology and histology including ultrastructural studies and also the qualitative and quantitative aspects of glycoconjugate rroieties in the skin mucous cells of H. fossilis and C. batrachus. The other two aspects describe (a) salinity and seasonality induced-changes in the skin mucous cells number and size and (b) circannel (seasonal) changes in the sulphomucin contents of skin mucous cells of H. fossiiis. In the present study, the histological studies have shown that all the three classical lavers viz. epidermis, dermis and hypodermis including that of basement membrane are clearly demarcated in both the catfish species i.e. H. fossils and C. batrachus. However, there are finer structural differences between the two species. In H. foss/Us, dermis is twice of the epidermis whereas in C, batrachus it is four times. In epidermis, the abundantly present spherical shaped mucous cells are largely confined to outer most layer in C. batrachus but are present both in the outer as well as middle layer in H. fossil/s. These mucous cells show variations in shape, size, degree of granulation and vacuolation during normal seasonal variations and also under salinity stimulation. Mucous cells do not get stained with hemetexylin and eosin but are positively stained with PAS and AB singly or in combination, The middle layer of epidermis of both the catfishes is overwhelmingly occupied by large size club cells, which are characterized by cap like vacuclar area, eosinophilic cytoplasm. These cells are elongated and columnar with multiple nuclei in C. batrachus and round or spherical with 1-2 centrally placed nuclei in H. fossilis. Other cell types such as sacciform cells, swollen cells, ionocytes, cuticle-secreling cells and merkel cells as observed in some other teleosts do not most in both these catfishes. The last layer of epidermis, stratum germinativum is a single-layered not too well-demarcated layer and unl~Ce the mammals, is not the only multiplying layer in fishes but ail the epithelial cells overlying stratum germinafivum are metabolically active. There is no evidence of any upper most keratlnized layer or cuticle covering area of the epidermis in these catfishes. The lymphatic spaces containing lymphocytes provide protection against microorganism and nutrition to stratum germinativum. In addition their features like taste buds, apical pit are also observed. Just beneath the basement membrane lies a continues layer of chromalophores, a characteristics of poikiotherms which also exist sporadically in the subcutis region of the skin of those catfishes. The dermis of both the catfishes correspond to the typical teleostean plan of being formed by an upper loosely arranged stratum spongiosum and lower compactly packed stratum compactum which are well demarcated in H. fossifis and somewhat diffused in C. batrachus. Both these dermal layers are made up of collagen fibers which do not get stained with PAS and AB (2.5) & (1.0) but lake up distinct stain with Masson Trichrome. The dermis of these catfishes is dchly invested with blood vessels, nerve ending and sense organs. A well-defined hypedermis or subcutis, characterized by large number of adipose cells with in- between empty spaces with sporadic blood supply and nerve endings and some chromatophoresis also observed in catfishes. It is generally assumed that the amphibious fishes which are capable of cutaneous respiration are characteized by the thick epidermis due to partial 02 uptake. However, the critical evaluation of available data on epidermal thickness in air-breathing and non-breathing fishes including that of the present study on both catfishes do not support such an assumption. Interestingly. the club cells in C balrachus are much taller, than those of H. fossills but epidermis thickness remains the same This situation is explained in the catfishes by the fact that the area of club cells and mucous cells follow an inverse relationship leaving the effective thickness of the epidermis not being altered due to this compensation. The above findings clearly show that the histological features of both these catfishes conform to the basic architecture of a typical tolcostear species with interesting deviation from broad generalization which makes this study significant.
The ultrastructural feature of H. fassilis integument have also been studied employing both SEM and TEN approaches. The SEM study of catfish H. foss/ifs integument shows the overwhelming presence of pavement cells which border the outermost epithelial layer. These cells are characterized by conspicuous concentrically arranged microridges with a single layer border. Al the junctions of these PVCs, there are occasional openings presumably of the MCs which lie buried under these PVCs. These openings are clear, unblocked and are the exit points of the We for secretion of mucus which extensively fills the space between the microridges. Since the skin of this catfish is scale less and is also devoid of any other epidermal or dermal derivatives which may project out of the skin, the entire integument surface under SEM view shows the predominance of only PVCs and the opening of the MCs. The SEM study of the catfish integument has not revealed any other cellular or other structura'. details. The perusal of literature related to the GEM study on the integumentary surface structures show relatively fewer studies as compared to TEM studies on the same structures v hich may be largely due to the fact that SEM studies have the drawback of revealing only the limited information. Any environmentally altered condition such as ambient salinity, sudden change in pH or temperature leads to copious exudation of mucus which covers the pavement cells of skin. A similar effect has been observed in the present study when the catfish has been transferred to 25% SW which has resulted in the copious secretion of mucus which filled the microridges to the extent of blurring its contours.
Under TEM observations of the skin, almost all the different cellular components can be observed quite unlike the SEM study which largely captures the view of surface lined pavement cells and the orifices of the MCs. Understandably, this is because of the plane of visualization. However, even under TEM studies, the predominant cells are MCs and pavement cells, which are universally present in the skin of all teleost fishes. The other cell-types which have been described under TEM integument studies on other teleosts are filament cells, cuticle-secreting cells, undifferentiated cells, market cells and club cdls. Chloride cells, in addition to gill epithelium, have also been found to occur in the skin of large number of euryhaline teleosts but are mostly absent in stenohalire fishes. Rodlet cells which are likely to farm non-specific defense mechanism of the skin have also been reported in epidermis of the fish exposed to stressors. In our study on H. foss/Us, we have clearly deciphered the presence cf NICs, pavement cells and the club cells within the limited observations made by us. Mucous cells clearly show the intermingled mucosomes of electron lucent (pate) and electron dense vesicles in the tap water maintained catfish. Interestingly, there is a well-marked regional aggregation of the mucosomes into distinct patches of electron dense and electron light granules. While we have observed a large number of chloride cells in the gills of both FW & 25% SW maintained H. fossilis but these cells are conspicuously absent in the all of this fish. A similar absence of these cells has been reported in the skin of C. carpio even though chlodde cells have been reported in large number in euryhaline teleosts. Based on the above observations, it is tempting to suggest that the sterohalirity of the teleost may perhaps be in some way related to the absence of the chloride cells in the skin which will limit the ability of these fishes to make forays into divergent salinities.
The present study has also elaborately described the distribution of glycoprotein (GPs) moieties in the mucous cells. The H. fossi/is exhibits the predominance of neutral GP whereas those in the C. bafrachrrs, it is acidic GP. This study has also shown tissue specific GPs variations in the same species as seen in H. fossilis where skin mucous cells contain neutral GP but it is the mixture of all GPs in the gill mucous cells of same fish. The observations of present study, as also of others, have clearly established that the distribution of various types of GPs in skin mucous cells does not conform to any specific pattern of either phylogenetic kinship, salinity based habitat i,e, FW, SW or brackish water or any other morphological or structural demand of a particular species. If one probes the functional role of various types of GPs, a fair degree of apparent overlap is clearly noticed. The protection against the invasion of pathogenic microorganism has been associated with both sulpho- as well as sialomucin and similarly thick viscosity imparted by acidic mucin and somewhat more fluidic mucus produced by neutral mucin has been associated with an over all lubrication function in teleasts. Interestingly, sulphomucin has been shown to facilitate the osmoregulatory homeostasis in freshwater teleosts but not all freshwater teleosts have predominance of sulphomucin. The present study has clearly established that it is not prudent to associate the occurrence of any specific GPs moiety in skin mucous cells to only one specific function since each such function is governed by multiple type of GPs.
The salinity- induced changes on the number and size of the skin mucous cells in H- fossilis show an interesting btnhasic response. A significant hyperplasia and hypertrophy is observed fallowing transfer of this catfish to 25% and 30% SW which is followed by a sudden and significant decrease in 35% SW. Our present observation on skin mucous cells are in complete contrast with those observed 'n gill mucous cells where no changes are observed in mucous cells area and number upto 30 % SW but a significant increase is registered in 35% SW. In literature, there are conflicting reports where in certain studies a sudden decline in mucous cell number and size has been reported whereas in equally good number of species a significant increase is recorded- These apparently conflicting observations have been explained by certain authors based on the following reasoning. A changes in salinity or parasitic or any other stresses causes an initial release of mucus on the skin surface as a combative action which leads to exhaustion of mucous cells causing the reducteon in their number and size. This phase is, however, followed by the generation of new skin mucous cells which requires varying time in each species and ultimately causes the increase in mucous cells number and size. It is thus clear that a clear-cut biphasic response occurs following transfer of fish to altered salinity. Hence, the conflicting reports on the apparent anomaly in mucous cells dynamics following salinity or other stimuli may possibly be due to random sampling at varying time whereas it does underline the need for a well spread the course study. But in H. fossils the graded increase in Z% and 30% SW and a substantial decline in 35% SW with uniform period of acclimation points to the role of mucous cells in osmoregulatory homeostasis of this catfish.
Another interesting aspect of this study is the circannual variations recorded on monthly basis. In the size, number and sulphomucin content of skin mucous cells of H fossilis. The results clearly show ar almost parallel profile change in number and size of mucous cells which are marked by two major peaks-ore spring peak during Apri: and the other winter peak during December- January. The sulphomucin contents also register high values during March and June to February with highest levels during January but a near total absence during April and May. There are hardly any studies on the seasonal variations in mucous cells dynamics largely due to its arduous execution and more importantly due to muldplieity of factors operating in natural habitat and it becomes virtually impossible to quantify all of them. In the instant study, it becomes difficult to predict whether the observed changes are the result of single factor or the collective response of consortium of these factors, However, the available data suggest that the seasonal temperature variations of both high as well as low magnitude are governing the hypertrophy and hyperplasia corresponding to one peak at the onset of high temperature during Apnl and the some peak is evident at the falling temperature during December and January. The observation on sulphomucin profile clearly suggests that this SF moiety is not required during April and May when its contents in mucous cells are virtually nil whereas it is maximally required during January when temperature is extremely low. Interestingly, while in the present observation a sulphomucin seasonality does not ft into any of its documented roles but our data do suggest its intimate involvement with low temperature adaptation. This is an important lead to pursue this newly postulated role of sulphomucin which needs to be probed in other teleost species.
(Chapter III)
Gills: The gills are the major respiratory and csmoregulatcry organs of aquatic vertebrates including fishes and have functional comparison with the lungs which are the counterpart respiratory organ of terrestrial vertebrates. The significance of gills as an organ with multifunctional entities for successful adaptation of fish in diverse environment cannot be overemphasized. Hence, chapter III of this thesis deals with the study of its general morpho-anatomy, and more importantly, the role of various epithelial ce:l types which constitute this organ. The other important aspects include (i) qualitative and quantitafive assessment of the different types of glycoprotein moieties in IvICs of the gills of H. fossilis and C. bafrachus in fresh water. (ii) circannual profile (based on monthly sampling) of the changes in The glycoprotein moiety, number and size of the MCs in the gill of the catfish H. fossilis. (iii) changes in glycoconjugate contents, number and size of MCs in the gill of the catfishes H. fossils, following gradual transfer of the catfish from TW to higher salinities i.e. 25%, 30% and 35% SW. (v) localize the Ccs with the help of light microscopy in the TW maintained catfish H. fossilis gill using variety of hisiochemical stains and to quantify the changes in number and size following sequential transfer to 25% and 30% SW. (v) immunohistochemistry and elector microscopy (TEM & SEM) to further confirm the cellular entity of Cc in the gills of H. fossils and to study their structural alterations followhg transfer to higher salinity i.e. 25% SW.
The studies on the gross moroho-histology on catfishes C. batrachus and H. fossils reveal that these catfishes have five pairs of gdl arches where four pairs are complete gills (holobranch) and the fifth gill arch is provided only with the gilt rackers. There is a distinct difference in the cartilaginous component of the gill apparatus in both the catfishes. In H. fossilis, gill arch is rod like cartilaginous structure with two rows of relative:y longer and spiny gill rackets whereas in C batrachus, the gill arch is extremely sturdy and thick and gill rackers are short and fused with each other. In both the catfishes H. fossiris & C. batrachus, a complete gill called holobranch is made up of hundreds of filaments or primary lamellae which are also called the unit of gills. The filaments of H. foss/ifs are spiny or conical towards the to side but in C. batrachus these are blunt, We have clearly demonstrated the presence of pillar cells along with its vascular components in both the catfishes. The filaments of both these catfishes bear a series of two parallely arranged rows of secondary lamellae with numerous blood capillaries which originate from the branchial filamentous arteries. These blood capillaries form central blood channels which contain different type of blood cells. Such blood channels are alternated by the presence of pillar cells which extend their flanges sideways and cover the blood channel from either side. These pillar cells along with their flanges provide the cartilaginous support and also ensure uninterrupted blood supply to the lamellae which is covered by the basement membrane. Generally, gill arch, filaments and secondary lamellae are covered with the branchial epithelium which is mullilayered on gill arch and filaments and bilayered on secondary lamellae. In the present study, both the catfishes i.e. H. fossils and C. batrachus display more or less similar pattern which has also been described in many other teleosts. However, secondary lamellae do exhibit different features e.g. the epithelium in C. batrachus is thick and its size is reduced whereas these are long and pointed in H. toss/Ifs.
The cellular features of the teleost gill epithelium clearly show that it is made up of largely three different cells types ie. pavement cells (PVCs), chloride cells (Ccs) and mucous cells (MCs) and the occurrence of other cell types such as rodlet cells and undifferentiated cells (UCs) is very rare and reported in very limited fish species. Our study on both catfishes shows the occurrence of only three above-mentioned cell types and ro rarely occurring cells such as radlet cells, undifferentiated cells etc. have been observed under light microscopy. In the present study, there is an overwhelming number of PVCs followed by MCs and then Ccs. However, we have not been able to calculate the exact quantitative proportions of each of these cell types. We have clearly localized the Ccs in H. fossilis gills with add hematein and they are present in the interlamellar and lamellar region. But in C. batrachus gill, the above stain has not been tried even though the presumptive Cos based location by HIE staining do seem to exist though in reduced numbers. The occurrence and distribution of MCs in the gill filaments of H. fossilis and C. batrachus are largely simlar to those found in the gill epithelium of other teleosts. In both the caffishes, MCs are mostly present in interlamellar and occasionally on secondary lamellae. The predominant presence of MCs in the intorlamellar region and their relatively reduced number in the secondary lamellae is intriguing. It is likely that the abundance of PACs in the interlamellar region may serve as an immediate source to provide mucus for lubrication function whereas their reduced number in the secondary lamellae causing less mucus production may facilitate more efficient respiratory function performed at the level of secondary lamellae.
The second aspect deals with glycoprotein distribution in mucous cells of H. fessilis and C. batrachus. Gycoconjugates are the main moiety of the MCs present in the different organ system including gills. Broadly, two major type i.e. neutral and acidic types of GPs are found and the latter type is made up of either carboxylated or sulphated or both types. Each component of the glycoconjugates has been associated with multifunctional roles and their occurrence and abundance has keen correlated with the different type of ecological niche which the animal occupies. Such information becomes more critical in case of aquatic fauna particularly fishes which occupies environment of divergent salinities. In catfish H. fossilis, it seems evident that each MC contains the small moiety of each different type of GPs i.e. neutral, sulphated and carboxylated and that no MC has been found to exist with only one type of glycomo'xety. Our observations on the catfish H. fos&lls dearly suggest that each gill MCs contain all the different types of GPs in relatively different proportions and that no individual MC contains any specific type of exclusive moiety. However, in the present investigation on another catfish Glades batrachus, glywconjugate profile is gdte different from the H. fossils. In C. batrachus, unlike, H. fossilfs sulphated and carboxylated acidic GPs existed as pine moieties and latter was relatively more abundant than the former and the two together almost equal with that of the total acidic GPs quantified by AB (pH 2.5) staining. The relative proportion of the acidic, neutral and mixed glycoconjugate was found to be almost equal to each other. Any instance of glycacanjugate qualitative and quantitative abundance similar to that of C. batrachus in the present study is not documented in literature. Interestingly, both these catfishes are phylogenetically quite akin and occupy similar kind of habitat but their glycoconjugate profiles seem vastly different from each other. These findings led to the conclusion that similarities in ancestry, in social habits and habitats of different species may not necessarily generate the same mucus GP profile. The relative predominance of one GP moiety of the teleost can be explained on the basis of greater predominance of one function over the other. However, the most interesting situation is observed in the present study with regard H. fossil/s gill MCs where each MC shows heterogeneity of moiety in each cell. This has prompted some workers to suggest that gill mucus in these fishes is involved in multifarious furotiocs of gas exchange, osmoregulation, pathogenic protection as well as binding and uptake of xenobiotics. It is likely that the heterogeneity ocwrr ng in the glycoconjugates in H. fossils
10 ~y~ branchial MCs would contribute to various functional roles quite unlike G batrachus where such functional diversity may be limited to only restricted functions.
The other aspect relates to salinity induced changes in alycoconiuoate moiety in MCs In H. fossils. The available information suggests that, by and large, neutral GP is more predominant in freshwater telecst whereas acidic GP particularly sulphated type is abundant in seawater. The present study on two catfishes shows that each MC contains all types of glycomoieties in H. fossilis where acidic moiety almost equals with that of neutral type of GPs in TW fishes. However, in another catfish C. batrachus, the profile of mucous cells GP is quite different and we do encounter MCs with exclusively sulphated and carbaxylated gill MCs where carboxylated are predominant over the sulphated containing MCs. The total neutral GPs are slightly more than the total acidic counterpart. If one considers the specific functional role of various type GPs, there is a considerable overlapping in the reported functions of each GP. The occurrence of heterogeneity in the gill mucus GPs in H. fossilis suggests that the protection against pathogenic microorganisms accorded generally by acidic GP and the lubrication of gill epithe:ium provided by neutral GP may be of crucial significance to this cai'ish. Hoviever, the relative abundance of neutral GP and exclusive presence cf carhoxylaled and sulphated moieties in gill MCs of C. batrachus may conform to conventional pattern observed in other FW tefeosts. Admittedly, while the avaiVabfe data in the present study on these two catfishes is strongly suggestive of the role of various GPs, more experimental evidence needs to be adduced to establish more definite functional roles of the GPs in the catfishes. There is clearly ro change in the GP moiety profile in H. fossils following its transfer to 25%, 30% and 35% SW. This is in contrast to several reports on variety of teleosts particularty those on euryhatine fishes where a distinct shift from neutral mucin in FW to acidic particularly sulphomucin in SW has been reported. This lack of response is understandable due to the fad that H. fossilis is a stenohaline fish and fails to osmoregulate beyond 10% SW and the survival up to 35% SW is through passive tissue tolerance. Since the su!phomucins which is said to increase the mucus viscosity which, in turn, facilitates SW- adaptalion is not present in predominant proportion, the catfish presumably fails to osmoregulate or conversely since this catCsh is stenohaline due to the lack of other euryhalinity features, the additional production of sulphomucin may simply not be required.
However, there are significant changes in the size and number of MCs profile of the H. fossilis upon transfer to higher salinities. In 25% SW, there is distinct swelling of MCs which disappears in 30% and 35% SW. Similarly, the density of MCs counted as the numbers per unit area registers significant increase in 25°I SW, followed by a significant dip in 30% SW and then the MCs density becomes normal i.e. equivalent to TW control in 35% SW. The size of MCs in this catfish in higher salinities shows a progressive increase i.e. noticeable increase in 25% which becomes statistically significant in 3g% and 35% SW. What could then be the reason of enhanced mucus secretion, even though temporarily, following transfer to higher salinities both in stenohaline as well as euryteline teleosts. It is also interesting to rote that even the transfer from SW to FW evokes increase in MCs density. It is tempting to suggest that it may simply be a stress-induced response to allow the fish to adapt to the changed environment and as soon as adaptation process is completed, the mucus secretion is returned to normal levels. The above suggestion is based on the observations that in most of the
11 fishes, the same response of increased mucus secretion is reported to both from FW to SW and vice- versa transfer. The variation in response pattern in different teleosts could be attributed tc species- specific variations where environmental factors may not play a very overriding ro:e. The multifunctional entity of mucus with specific focus on its osmoregulatory participation is a fascinating area which needs to be probed further to elicitmore precise answers.
Another important area covered under the Present study Is the study of the seasonal variations in mucous cells population of gill in H. fossilis. The catfish H. fossi6s Inhabits various freshwater ecosystem mostly confined to muddy and derelict ponds with low levels of water and dissolved oxygen. These water bodies will be subjected to a cyclic trans-evaporation during elevated temperature leading to increase in salt concedration followed by its dilution during July-August due to influx of rainwater into these water bodies. It is, therefore, likely that the population of gill MCs and its glycomoiety may also exhibit some kind of seasonality in its population pattern and chemical moiety of its glycoconjugates. The present study on H. fossils on this aspect has shown interesting results. We used month of March as a control when all environmental parameters particularly temperature is very moderate (20-25°C) the MCs present a normal appearance in term of their shape which is ovoid and regular, granulation status, cell number and size which, on average, is 59 ± 2.5 and 46.7 ± 5.6 pmt/unit area respectively. However, their numbers record a sudden spurt from March to April nearly doubling during this phase and similar surges in number are also recorded from August to September and November to December. Interestingly, these three surges correspond with three different events i.e. a sudden rise In environmental temperatire from March to April, the onset of monsoon rain from August to September and a precipitous decline in environmental temperature from November to December. The month of May registered the least number of MCs population whereas during June, July and February, the MCs population is the same as recorded during March, the reference month of this study. There are no changes in the major environmental parameters during the months when the MCs population was similar to its reference month i.e. March. The seasonal profile in the MCs area is quite different with that of its seasonal population density profile. There is a consistent increase of varying degree in MC area noticed from May up to January registering highest average size during December. The months of March, April and February showed, more or less, similar MCs area It is evident from seasonal profiles that the number and size of MCs do not have a clear cut correlatior. However, few interesting facts NO worth noting. The number and the size of the MCs are maximum during December which is followed by a sudden decline to the next month i.e. January. Further, one of the common spurts both in the number and the size of the MCs, occurs from November to December. Also, there is an interesting inverse pattern of response in he size and the number of MCs during May when the number decreases whereas the size registers a significant increase. In a multifunctional environment where several environmental factors influence the physiology of an animal, it becomes often difficult to correlate all these changes on the L2sis of few variables. For instance, in the present study, the focus is mainly on the chances related to osmoionic balance vis-a-vis MCs seasonal profile. However, other concurrent factors such as reproductive seasonality linked with the homenstztic balance of an animal may also influence MCs seasonal population. The mixed glycoproteins would also have sizeable proportion of neutral moiety to provide an overall balanced function associated with
12 both acidic and neutral GPs. However, in a multifactorial ecosystem in which H. fossilis lives, it may be difficult to pin point the role of a single factor as being responsible for the observed seasonal variations in the GP contents of gill We of catfish H. fossil/s. The foregoing discussion of the seasonal variations in the density, area and GP content of the gill MGs of H fossilis suggest that these variations are governed by more than one environmental factors and the mechanism by which these environmental factor evoke seasonality in MCs profile in H. fossilis is still not clearly known.
Definitive identification of chloride cells in H. fossilisthrough various approaches and to study their physiological role is another important aspect of this study. The Ccs are one of the important cellular components of the branchial epithelium and have been extensively implicated in the processes of ionoregulatory adjustment in fishes. Hence, one of the important pre-requisites b study the role of Cc involves its definite Identification amongst the different cellular components of gill epithelium. Such a task hecomes even more challenging due to the fact that the Cos, unlike PVCs, are not present in overwhelming number.
In our own study on H. foss/is, we have used hemotoxylin and eosin but with lim.led success since the presumptive Ccs stained with hematoxylln and eosin were much fewer in number than those observed with more definitive histochemical techniques. Hence, a large number of workers have used lipid/ 3hospholipid based specific hlstcchemical techniques such as sudan black B, acid hematein and Ohampy Maillers fluid (OZI). All these are based on the principle that the Ccs have a predominant presence of lipid) phospholipid which are selectively stained with the above techniques. We have got excellent results with the use of acid hematein to localize the Cos in catfish H fossilis. However, it is surprising that despite such great versatility of this method, there are very few reports where this method has been used. One of the most extensively used techniques to selectively localize Ccs under the light microscopy is Champy-Maillet technieue, also called Osmium Zinc Iodide (OZI), The Ccs of large number of teleosts including H. fossiks has yielded positive results with OZI. However, one of the disadvantages of this technique is the appearance of uniformly black stained Ccs with no visible granulation thus severely limiting interpretation based on the physiological status of Ccs. This is quite in contrast to the Ccs stained with acid hematein in the present study where the degree of granulation in the Ccs are quite elaborately visible. Hence, if one has to base observations on the charge in size and number of Ccs following experimental treatment, OZ: is the most suited technique. In H. fossilis, there seems to be consistency in shape of Ccs which are round or oval both at intedamellar and also lamellar location. In our study on H. fossils, the Ccs were largely confined to interlamellar or the lip region either singly or in cluster of 2-4 cells with varying size. Very few cells were observed in the lamellar region.
A unique feature in our observation on H. fossilis is significant increase in the number and size of Ccs in 30% SW which is preceded by a substantial decrease in the number of these cells in 25% SW. It seems that H. fossilis up to 25%SW is behaving like a typical stenohaline fish registering a decline in the Ccs number. But in 30% SW, it is presenting a pattern of euryhaline fish which, by and large, registers significant increase in the number of Ccs in higher salinities. This, however, is somewhat
13
THESIS knotty situation since H. fossilis does not suggest any indication of active osmeregulation at 30% SW which is evident from the elevated plasma osmolarity and unchanged levels of Na• /KtATPase in gills.
For immunolocalization of catfish Ccs in gill epithelium, we have used commercially available antibody from the Developmental Studies Hybridoma Bank. This monoclonal antibody (IgG a-5) developed by D. M. Fambrough against the catalytic a subunit of chicken NrIK'-ATPase binds specifically to the cytosolic epitopes and recognizes all three isoforrns (a1, a2 & a3) of Na•/K-ATPase in vertebrates and several invertebrates epithelia. The immunoreacfive cells observed in our study designated as Ccs and localized by immunostaining against a subunit of NrIK*-ATPase were Found to be varying in shape ranging from ovoid to polygonal which are typically of the same shape as reported in many other teleosts. We observed the presence of filament & lamellar Ccs both in TAN & 25%SW maintained fish. However, there is no difference in their shape and size except that the number of lamellar cells appeared to be fewer. Further, there does not seem to be any remarkable change in the distribution pattern of these two differentially located cells In TW & 25% SW.
Electron microscopy studies of the Ces and MCs: (i) Under the scanning electron microscopy (SEM) study of gill of H. fossilis, each holobranch gill arch, the typically arranged primary filament giving rise to leaf-like secondary lamel]ae on either side are visible. These lamellae are wrinkled and backwardly curved close to apical end. At higher magnification in the gill surface ultrastructure (SEM), the most visible feature is the presence of large number of pavement cells (PVCs) characterized by the presence of concentrically arranged micoridges. We also observed the presence of taste buds located at the apical surface protuberance epithelial cells in gill arch. These taste buds corespond to the type 1 of the taste buds- An extensive mucus covering either in the form of sheet or the spherical flocculent droplets of varying size lying close to MCs opening have been observed in the catfish gill surface structure.
A close comparison of our results on the type of Ccs observed under SEM and TEM in H. fossrris may appear anomalous since we have identified two types of Ccs by TEM whereas only one types is described under SEM. This apparent paradox is largely based on the fact that the criteria of differentiating two types of Ccs under TEM studies is based on the mitochondrial differentiation and the intensity of electron density in the cytoplasm which cannot be visualised by the surface study under SEM where only one type is visible. The Ccs observed in the present study on H. fossifis are somewhat unique and of single type characterized by the elevated swelled structure with the presence of faint r:ngs resembling with those of the microridges of PVCs and'Mth a very small inconspicuous side opening. To the best of our knowledge, such architecture pattern of Ccs have not been documented in literature for any other teleost fish. The only significant change in the salinity transfer of catfish to 25% SW is that the number of MCs openings show a remarkable increase. This observation is further corroborated by our light microscopy studies where the transfer to higher salinities have registered a considerable increase in the number of the We while under SEM study only the opening of these MCs can be visualised. (ii) Transmission electron microscopy (TEM): In the present study on the ultrastructure of gill epithelium of catfish H. fossilis by TEM clearly
14 established the presence of typical Ccs along wiihthe PVCs, accessory cells (ACs) in higher salinities and the pillar cells (PCs) which are confined to secondary lamellae in close association with blood channels. A few undiferentialed cells (UC) fired along the basement membrane of secondary lamellas have also been observed. The Ccs are quite abundant in number mostly ovoid in shape and display atl characteristic features of Cc such as high mitochondrial density, extensively amplified basolateral tubular system and vesiculotubular system confined mostly at the apical surface. Like a typical FW stenohaline teleost, the apical membrane of the Cc of H. fossilis is somewhat Flat or at places slightly convex, mostly devoid of any microvdli projections. Such characteristic feature of the apical membrane have also been observed in few other FIN stenohaline fishes. We also observed two types of Ccs differentiated on the basis of electron dense (osmophilic) Cr dark Ccs and electron lucent (pale) light Ccs designated as Ccl and Coll respectively. These two types are also differentiated on the basis of the shape of mitochondria which in the case of the former have only round shape mitochondria whereas in the latter type both round as well as elongated mitochondria were seen. In the present study, we also observed a tubulovesicular system (TVS) located at the subapical cytoplasm comprising of numerous vesicles and tubules. It is generally assumed that the function of this TVS is to transport numerous vesicles and fuse them with the apical membrane. In H. foss/Us a large number of PVC characterized by the presence of microvilli and microplicae with cytoplasmic vesicle, distinct golgi apparatus, lysosomes and comparatively fewer mitochondria have been observed. The occurrence of PVCs in conjunction with Ccs both in freshwater and marine teleosts i.e. stenohaline or euryhaine in nature have been observed. In the present study an H. fossilis, the PVCs form an overlying extended covering on the Cc and finally joined at the apical surface side by side. Interestingly, junctions between the Cos and PVCs have been multistranded indicating thereby that these junctions are not leaky and do not allow the passage of ions to external environment which is a physiological advantage to a FW teleost like H. fossilrs. We have been able to detect the presence of few accessory cells (AC) in H. foss'is maintained in TW which differs with the observations on many other FW stendhallneieleosis.
In our observation on catfish, the greater abundance of ACs in higher salinity may simply be an attempt to acquire osmoregulatory capability which, however, does not seem to succeed as may be in the case of goldfish and juvenile rainbow trout which show poor adaptability in SW despite the presence of AC. Following transfer of H. fossilis to higher salinity i.e. 25% SW, we have observed the appearance of apical pit and enhanced number of lamellar Cos but there is also a concomitant reduction in the number of mitochondria and much less invagination in the basolateral system 'n Cos which may largely account fora poor adaptability of this fish in higher salinities.
(Chapter IV)
Gastrointestinal tract: The gastrointestinal tract (GIT) in all vertebrates, with mouth at one and and anus at the other, is essential for procuring food from external to the internal environment. From the anterior mouth, food passes posteriorly into successively arranged regions such as the oesophagus, stomach and finally
is into the intestine. There are large differences in the morphology and physiology of the gastrointestinal regions between vertebrate groups and also between different feeding strategies within the same vertebrate group. The digestive tract wall of various vertebrates has remarkable similarity in terms of gross function as well as histological organization. The GIT, other than mouth and pharynx, is characterized by four layers i.e. tunica mucosa, tunica submucosa, tunica muscularis and the outermost tunica serosa.
The survey of literature reveals that a large cumber of teleost fishes of Indian subcontinent have been studied. However, no systematic study has been carried out to study the gross morpho-histology, the qualitagve and quantitative distribution of glycoconjugates in various segments of GIT of H. fossilis. Further, no data is available on the effect of higher salinity on the changes of glycoconjugate moiety and the population of mucous cells in the gastrointestinal tract where they are speculated to play an important role in fresh and salt water osmoregulation. In the present chapter, an attempt has been made to document the detailed histomorphology, distribution of glycoproteins (GPs) component in the GIT and qualitative and quantitative changes of GPs following transfer of H. fossilis to higher salinities.
The first aspect which deals with gross moroholoaical structure of digestive tract of H. Fossils which essentially shows such structural features which are similar to those found in other teleost species. Cesophagus, the first segment of the GIT is a short passage anteriorly attached with mouth and posteriorly with stomach. Most of the workers have divided the stomach into either two or three distinct regions, In carnivores and omnivores fishes, the stomach is mainly Y & U shaped because it is used as a reservoir and can be easily differentiated in three parts when it is filed with food. In some herbivorous fishes, due to the absence of stomach, oesophagus is directly connected with intestine and its anterior portion which becomes bulky or swelled is called the intestinal bulb which is presumably used as a storage organ of the ingested food. It is apparent from the literature that there is considerable variation in the nomenclature to demarcate different regions of intestine. Some workers have divided intestine into tvo portions i.e. small and large or anterior and posterior without any specific criterion. The internal histology of gastrointestinal tract of different fish species, like other vertebrates, show the similar arrangement of the gut wall layers as seen in catfish H. fossilis where it is composed of four layers i.e. mucosa, submucosa, muscularis and serosa. In H. fossiis the oesophagus mucosal layer shows deep longitudinally arranged folds which have also been reported in the fishes inhabiting divergent environment such as in marine teleost, eel Anguilla anguilia and freshwater species, Silverside Odontesthes bonadensis. The anterior most covering i.e. the mucosal epithelium in H. fossilis is structurally uniform, stratified and made up of basal cuboidal cells: intermediate columnar mucous cells and superficial layer of flattened cells. It has been observed in the present study that the catfish mucosal epithelium also contains large number of columnar mucous cells which secrete abundant quantity of mucus which is spread over epithelium. Based on the above, it is logical to presume that the mucus layer over this region of GIT is performing the function of protection and helping in the process of swallowing of food during ingestion. The muscularis mucosa consisting of stratified muscular fibers and usually helping in the movement of folds has been found
16 absent in the catfish H. fossilis and other teleosts species. Similarly, the taste buds are also found to be absent in catfish H. fossils like in many other species. The muscularis, typically, consists of inner longitudinal and outer circular muscles and both are smcoth muscles and a similar arrangement is observed also in the catfish oesophagus where the longitudinal muscles are arranged in isolated bundles extending Into the submucosa. In the catfish, the serosa which fs continuously present externally and is made up of endothelial cells is known to reduce the friction and to protect the surrounding organs. Like oesophagus, the structure of the walls of all regions of stomach of H. fcssilis resembles with those of other fishes where it is composed of hi-layered muscularis, the connective tissue of submucosa, the muscularis mucosa, lamina propria of connective fibers with gastric glands and inner epithelial layer of mucosa. Externally, the stomach of catfish H. fossilis is dvided into three regions i.e. cardiac, fundic and pyloric where the gastric glands are present in the cardiac stomach which increase in fundic region but are completely absent in the pyloric region. The cardiac and fundic regions of the stomach of catfish H. fossilis are characterized by the presence of compound tubular type gastric glands but these glands are totally absent in the pyloric region. The muscularis mucosa is well developed in the pyloric stomach of H. fcssrlis whereas it is poody developed in cardiac and fundic regions. This may possibly indicate that the functional significance of combination of well developed musculature and the absence of gastric glands in pyloric region is to mix and push the food distally. The mucosel epithelial cells o` H. fossills stomach which lines the lumen and pits, are tall :olumnar cells with basally placed oval nucleus and numerous apically concentrated granules. These secretory granules secrete mucus which stains positive with PAS! AB(pH 2.5) and may serve to protect the stomach epithelium from the auto digestion caused by enzyme and acid. Interestingly, in contrast to oesophagus, the arrangement of muscularis of catfish in stomach is reversed with the existence of thin outer longitudinal and slightly thick inner circular smooth muscles towards the external serosa. One of the unique features observed in the fundin region of stomach is a highly significant reduction in the thickness of muscularis layer, perhaps not reported in any other teleost which is concomitant with highly developed gastric glands occupying most of the thickness of GIT in this region. Such a histological feature seems to have functional significance since the ma or load of digestion is tang place in this region for which extensively developed gastric glands are extremely important. Simultaneous reduction of the muscularis is possibly because of the lack of any significant function as well as to keep the thickness of entire GIT in a reasonable limit. Ore is also tempted to suggest the respiratory role due to sudden increase in the vascularisalion of this region of GIT. However, concrete evidence needs to be adduced to confirm this assumption. The circular muscles in the pyloric region of catfish tend to be more developed than in cardiac region, assuming the appearance of globular or spindle shape gizzard found in birds. Interestingy, in pyloric region of catfish H. fosslfrs, the musculads layer Is arranged into inner oblique followed by circular and longitudinal muscles, In the intestine of the catfish., a well developed serosa slmilar to that found in the stomach region, is present and is made up of loose connective tissue with blood cells.
The second aspect of this chapter deals with studies on the function and identification of GPs in the catfish where such studies in other teleosts are very limited. However, the fact remains that GPs plays an important role in the gastrointestinal wail viz ubricatfon, protection of mucosa layer
17 against chemicals, hypertonic media and acidity in the stomach, defense against parasites, and formation of a diffusion harder for various ions and possibly role in transepithelial ion transport in intestine, The catfish H. fossilis is omnivcmus and the GPs which are present in the whole gastrointestinal tract seem to be both neutral and addle types whereas in oesophagus and terminal pad of stomach these are predominantly neutral. Interestngly, the sulphated glycoconjugates have not been observed in the gut mucosal lining of the catfish H. fossils. The absence of sulphomuch in the GIT of catfish suggests that such a functional necessity in catfish may either be diminished or not required at all. The oesophagus mucosa lining of catfish H. fossilis, shows the predominant presence of neutral GP even though acid GPs is also present in small measures. The qualitative differences in the glycocenjugate moiety present in the mucus of oesophagus may he associated with the different functions. The absence of salivary glands in fishes may be duly compensated by the lubrication function provided by the mucus secreted by the oesophagus mucous cells. However, the predominance of neutral GP as observed in catfish may further aid in lubrication function whereas the acidic glycoprotein may protect the mucosal lining from the invasion of various pathogens which may enter with the swallowed food. Significant variations in glycoconjugate composition in stomach have been described in many teleost species. In the present study, the occurrence of GPs on the surface epithelium of stomach which stains positive with PAS and AB (pH 2.5) indicate the mixture of neutral and acidic GP. Similar high quantities of both neutral and acidic glycoconjugates in the stomach mucosa have been reported in the several teleost species. In the present study, the sulphated GP has been found to be absent in the gastric mucosa of H. fossilis even though it has been shown to be present in many other different fishes which may probably be related to diet. An Interesting observation of the present study relates to a sudden transition of neutral GP present in the sphincter region of the stomach leading to the duodenum into predominantly acidic moiety. Although no evidence is adduced but it is likely that this transition may be useful to kill any possible pathogen occurrence in the ingested diet of the catfish.
The intestine of H. fossilis shows the uniform distribution of GPs in all portions i.e. duodenum, mid intestine and terminal part ,rectum) which are made up of neutral and acidic GPs. In H. fossilis. the number of mucous cells increase towards the distal part (Le. rectum) which may be related to an enhanced need for lubrication for the smooth ejection of faecos as reported in other fish species. Among the functional multiplicity of mucus, acid GPs have been implicated in the increased viscosity of mucus in thefsh alimentary tract which lubricates the undigested materials for onward progression into the rectum. Neutral mucosubstance are is said to be involved in the enzymatic digestion of food transforming it into chime and also facilitating absorptive function.
The third aspect of this chapter deals with osmoreeulatory functions. if any, of the catfish GIT. In a classical osmoregulatory pattern, gastrointestinal tract (GIT) of a freshwater teleost, unlike its marine counterpart, plays a minimal role. Such a role is largely confined to intestinal uptake of Na' and CI- from dietary source to compensate diffusional loss of these ions across the gill to an extremely hyposaline external medium. The catfish H. fossils survives only up to ac extremely limited salinity range through the process of passive tissue tolerance. Any substantial role of GIT in higher salinities
18 up to 35% SW as an osmoreguatory organ is cearly not expected. The observations of the changes in GIT internal histology corroborate this assumption. However, there is an overwhelming evidence to show that while there is no significant qualitative and quantitative change on the GPs moiety of the GIT mucosal lining of various segments, there are highly noticeable disruptive charges in the internal mucosal lining and even on the underlying layers of various segments. These changes are in the form of extensively damaged stratified epithelium, necrosis in submucosa and lamina propria, profound vacuolation of submucosa of oesophagus, elongation of gastric glands, hemorrhagic changes and enhanced mucus production. These changes become more pronounced as the salinity increases from 25% to 35% SW. In this study on catfish H. fossL s, which has barely 35% SW tolerance limit, none of the changes documented for euryhalflne fishes were observed here giving credence to our assumption that GIT is not playing any active osmoregulatory role in catfish in higher salinities. However, the fact of the matter is that there are highly significant degenerative changes in catfish GiTin higher salinities which may have been caused by the process other than osmoregulation. In the present study, a careful analysis reveals that the changes observed in the catfish GIT in higher salinities quite closely resemble to the changes observed in those fish species which have been exposed to various pollutional load. This leads to most likely conclusion that the histopatholugical changes seen in catfish GIT following transfer to highaIr salinities are more of a manifestalien of salinity induced stress response rather than an osmoregulafory l adaptation which is quite extensively reported in euryhaine teleosts transferred to marine environment.
Hence this chapter has provided significant data on the histological organization of various segments of GIT of the catfish H. fossf's. its glycoconjugate distribution and their possible physiological role in various GIT segments and effects of enhanced salinity on the GI7 organization.
Concluding Remarks
The information thus generated tin the above study has built up a comprehensive overview of better and more precise understanding' of the morphology, histology and cellular composition of skin, gill and Cl tract, the important osmoregulatory target organs in fishes. It has also provided an insight into the biochemical composition of mucus glycoprateins, a majorcomponent of overlying mucus on the above organs, its seasonal profile and salinity and stress induced changes in these catfishes. The consortium of such an information in con;'unctlon with the pre-existing documented information on these aspects will greatly enhance our current understanding of osmoregulatory adjustment in teleost in general and in tropical freshwater stenohalinefishes in particular S ROLE AND DISTRIBUTION OF SELECTIVE CELLULAR OMPONENTS OF OSMOREGULATORY TARGET ORGANS IN SOME FRESHWATER TELEOST FISHES
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
F, P
BY SUBUHI ABIDI
DEPERTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA) 2013 00161 IIIIIIIIVINIVIIIIIIIIIIIINInII
fLC& AG:N Q L
4';r -
".&r ,1 fit''1 ': '' a
11
NY.
DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002
Certificate
I certify that the work presented in the thesis entitled "ROLE AND DISTRIBUTION OF SELECTIVE CELLULAR COMPONENTS OF OSMOREGULATORY TARGET ORGANS IN SOME FRESHWATER TELEOST FISHES" has been carried out by Subuhi Abidi under my supervision and it is suitable for the award of Ph.D. Degree in Zoology of Aligarh Muslim University, Aligarh
bal Panvez ADepartment of Zoology A.M.U., Aligarh
iJ CONTENTS
i. Acknowledgement ...... I ii. Details of figures ...... I1-Vt III. Introductory Preface and Objective of Study ...... 1-7 iv. Materials and Methods A. Collection and care of fish ...... 8 B. Artificial seawater ...... 9
C. Tissue collection ------..._9
D. Histology of tissues ...... _...... 9 -10 E. Staining procedure ...... 10-15 F. Immunohistochemistry. _ ...... 15.16 G. Visualization of sections ...... 16 H. Electron microscopy ...... 16 -17 Chapter I. Handling stress and mucous cells region-based distribution
1. Introduction ...... 18-19 2, Experimental protocol ...... 19 3. Observations ...... 19-20 4. Discussion ...... 20-24 Chapter II. Integument 1. Introduction ...... 25-40 2. Experimental protocol ...... 41 3, Observations ...... 42-47 4. Discussion ...... 47-61 Chapter III. Gills 1. Introduction ...... 62-74 2. Experimental protocol ...... 74-75 3. Observations ...... 75-83 4. Discussion ...... 83-106 Chapter IV. GIT 1. Introduction ...... 107-127 2. Experimental protocol ...... 127 3. Observations ...... 128-132 4. Discussion ...... _._ ...... 132-143 v. References...... _ ...... _...... _...... 144-178 vi. Appendix ...... VII-X Acbowfe4ments
First and Foremost I want to pay my gratitude to most magnificent God who enlightens my heart with spiritual light to find the true path oflife.
Prof Igbal Parwez, my supervisor, gave his kind supervision to me for the present work As he is an extraordinary teacher of an ordinary student like me, Ifeel highly privileged to work with him, His strict discipline and great vision not only told me research methodology but taught me the art of living. His constant encouragement and rewarding appreciation always energized me to think novel. He moulds me into a new sculpture, which is more capable to accept challenges. In return to him for the little knowledge which I got from him, Ican only offer my respectful obeisance to him.
I am greatly indebted to Prof. Irfan Ahmed, Chairman, Department of Zoology for providing me infrastructure support.
I am highly grateful to Dr. Sher Ali, R7t, New Delhi, Prof. Apurna Dixit, School of Life Science, J.N. U., New Delhi and Prof Jamal Ahmed, IN. Medical college A.M. U for their concern and timely contribution.
I am obliged to Dr. Aysha Qarnar who boosted me whenever Igot disappointed and Dr. Fauzia Anwar Sherwani for keeping my moral high and maintaining my level of patience. I am also grateful to Dr. ( Mrs) Hina Parwez for her help, support and encouragement.
Thanks of also due to my lab mates i.e.. Dr. Arif Ahmed, Dr. Muslufa Arif Kama! and Imran Mustufa Malik and friends for their suggestions and cooperation.
Whatever we do in this world reflects the image of our parents. If today I'll be able to achieve anything whether socially or scientifically, its whole credit solely goes to the blessings of my great parents and other family members particularly my Bajjo, Mrs. Rehana Kazmi who provided me rock-like support during my acute moments of crisis. For now ,Thanks everybody.
Subuhiyl6 LIST OF FIGURES
Chapter L•Stress and Locational distribution
Fig. No. Legend of Figures Pg. No. Fig-l.1 Effect of stressed and non-stressed handling on skin and gill mucous cells of 20(i) catfish Fl ossi(is. Fig-1.2 Effect of stressed and non-stressed handling on number of mucous cells in the 20(] gill epithelium of catfish H. fossilis. Fig-13 Effect of stressed and non-stressed handling on skin and gill mucous cells of 20(ii) catfish C batrachas. FU- l.4 Effect of stressed and non-stressed handling on number of mucous cells in the 20(it) ill c itheliumofcatfish Cbatrachus Fig-1.5 Mucous cells abundance in tour different regions of the integument of 20(iii) catfish H. fussilis showing samples taken from below the anal, caudal, dorsal and pectoral This respectively. Fig-1.6 Number of mucous cells in four different regions of the integument of catfish 20(iii) H. fossilis. Fig-1.7 Mucous cells abundance in four different gills of catfish H. foscilis taken 20(iv) from anteriortu poMcrTor side. Flg-1.8 Number of mucous cells in four different gills of catfish H. fossi(ts taken from 20(iv) the antero- osterlor side Fig-1.9 Mucous cells abundance in catfish C batrochus integument, sampled from 20(v) below the anal, caudal, dorsal and ectoml fms. Fig-i.to Number of Mucous cells in four different regions of the integument of catfish 20(v) C. batrachus. Fig-1.11 Mucous cells abundance in four different gills of catfish C. babachus taken 20(vi) from anterior to osterior side. Fig-1.12 Number of Mucous cells in four different gills of catfish C. batrachis taken 20(vi) From the nntero- osterior side.
Chapter II: Inteounfent
Fig-2.1 Gross structure of integument of H. foul/As. 47(i) Fig-2.2 Gross structure of integument ef C. bafrachus 47(11) Flg•2.3 Length of epidermis and dermis of the integruneni of catfish H. fassills 47(iil) & C. bntrachta. Fig-2.4 Area of mucous cells and club cells of the integument of catfish H. fossi its 47(iii) & C. batrachus. Fig-2S Different types of glycoprotein moieties in mucous cells of the integument of 47(iv) H. fassilis maintained ilTW. Fig-2.6 Different types of glycoprotein moieties in mucous cells of integument of 47(v) C'. ban-achus maintained in TW. Fig-2.7 Per cent distribution of glyceproteins in mucous cells of the integument of 47(vi) catfish 11. fo..viIir. Fig-1.8 Per cent distribution of glycoproteins in mucous cells of the integument of 47(vi) catfish C. batrachuv. Fig-2.9 Relative per cent distribution of various types of glycopmtein-containing 47(vii) mucous cells in the integuments of H fossils & C. bafrachus. Flg-2.I0 Different types of glycoproteins moieties in mucous cells of integument of 47(viii) catfish H. fassilis after tmnsfcx to 25^/ 6W showing hypertrophy and itureace in cell number. Fig. No. Legend of Figures Pg. No. Fig-2.1i Different types of glycoproteins moieties in mucous cells of integument of 47(ix) catfish H fossil is after transfer to 30% SW showing hypertrophy and increase in cell number. Fig-2.12 Different types of glycoprotcins moieties in mucous cells of integument of 47(x) catfish H. fossilis after transfer to 35°%SW showing hypertmphy and decrease in cell number. Fig-2.13 Area of mucous cells in the integument of catfish H. fossilis following transfer 47(xi) to 25%, 30% & 35% seawater. Fig-2.14 Number of mucous cells in the integument of catfish H. fossilis roliowirig 47(xii) transfer to 25%, 30% & 35% seawater. Fig-2.75 Area &Abrmdanee of mucous calls of the integument (upper panel) and gill 47(xiii) (lower panel) of catfish H. fossilis following transfer to 25%, 30% & 35% seawater. Fig-2.16.1 Seasonal variations in the abundance and size of mucous cells in integument of 47(xiv) catfish H. fossilis during March to August represented by panels A - E respectively. Fig-2.16.2 Seasonal variations in the abundance and size of mucous cells of integument of 47(xv) catfish H fossil's during September to February represented by panels F - 7 res ectivel . Fig-2.17 Snasunal variations (March to February) in the area of mucous cells of the 47(xvi) integument of catfi5ll H. ossills Fig-2.18 Seasonal variations (March to February) in the abundance of mucous cells of 47(xvii) the int umcnt of catfish H. fossiliN. Fig-2.19 Seasonal variations on the number and area of integwnentary (upper panel) and 47(xviii) branchial (lower panel) mucous cells of catfish II.. fossil's. Fig-220 Seasonal variations In the abundance of sulphated glycoprotein in mucous cells 47(t) in integument. Panels A -3 represent March to Fehmery respectively. Fig-2.2i Seasonal variations in the abundance ofsnlphated glycoprotein in mucous cells 4700x) of the integument of catfish 11. fossilis. Fig-222 Scanning electron photomicro raph of skin of catfish, H. fossilis maintained in 47(noci) tap water. Fig-2.23 Scanning electron photomicro raph of skin of catfish H fossils maintained in 47(xxii) 25% sea water. Fig-2.24 Transmission electron micrograph of skin of catfish H. friosd.1 maininined in 47(xiii) tap water. Fig-z.25 Transmission elecnon micrograph of skin of catfish H. fossil's maintained in 47(xxiii) 25% sea water.
Chapter III: Gills
Fig- A Schematic teleost fish ll 63 Fig- B Structural features ofsin Ic gill lamellae 64 Fig- C Arterio-arterial blood supply in bronchial and head region 65 Fig-fl Schematic di am showing blood flow in lamellar re ion 66 Fi -3.1 External features of Hetern neustesfossil's. 83(i) Fig-3.2 (A) (lead of Hereropuerestas fossilis showing the arrangement barbels, eyes 83(i) and pectoral fm with spine (B) In situ p silion of ll in IC fogsills. Fig-3.3 (A) A complete holobranch) gill of H. fossilis, (B) Magnified view of a 83(i) complete gill showing filaments with lamellae and gill rockers attached with gill arch and gill septum oft!. ffacsilis.
Ill Fig. No. Legend of Figures Pg. No. Fit-3.4 t Hinology of gill of H fossilia showing lamellae and secondary lamellae. Panels 83(ii) (A) - (G Fig-3.4.2 The general histology of gill of H. fossilis showing gill arch, gill makers and 83(iii) taste buds. Panels (II) -(K). F¼-33 External features of Clarins bafrachtrs. 83 iv Fig-3.6 (A) Head of C. 8arrachas showing the arrangement barbels, eyes and pectoral 33(iv) fin with spine and) In silo position of gill, Fi -3.7 A complete hotohmnch) gill of CC batrachus. 33(iv) Fig-3.8.1 The general histology of gill of C. balrachus showing lamellae and secondmy 83(v) lamellae. Panels (A) - (G). Fig-3.a2 The general histology of gill of C. batrachvs showing gill arch, gill rockers and 83(vi) taste buds. Panods (H) - (K). Fig-3.9 Different types of glycoprotein moieties in mucous cells of bronchial 83(vii) e ilhe5um of K assNis maintained in T W. Fig-3.10 Dilierent types of glycoprotein moieties in mucous cells of branchial 83(viii) e ithelium of C. bahochm maintained in TM. Fig-3.11 Percentage of glycoprotein in mucous cells of the bronchial epithelium of 83(ix) catfish H fossdis. Fig-3.12 Percentage of glycoprotein in mucous cells of the bronchial epithelium of 83(ix) catfish C batrachus Fig-3.13 Relative per cent distribution of various type of glycopreteins - containing 83(x) mucous cells in N. jossilis & C. batrrachus. Fig-3.14 Different types of glycoprotein moieties in mucous cells of branchial 83(xi) epithelium of Fl. ossilis maintained in 25% SW. Fig-3.15 Different types of glycoprotein moieties in mucous cells of branchial 83(xii) epithelium ofH.furxilis maintained in 30% SW. Fig-3.16 Different types of glycoprotein moieties in mucous cells of bronchial 83(xiii) epithelium ofH. fossil is maintained in 35% SW. Fig-3.17 Abundance of mucous calls of the gills (upper panel) and integument (lower 83(xiv) panel) of catfish H. fossiiis following transfer tc 25%, 30%x id 35%n SW. Fig-3.18 Number and size of bronchial and integumentary mucous cells of catfish FL 83(xv) fr~xsilis fallowing transfer to 25%, 30 ;'o and 35% SW. Fig-3.19.1 Seasonal variations in the abundance and size of mucous cells in gills during 83(xvi) March to June represented by panels A-D respectively. Magnified view of mucous cells of the above panels re resented by panel a-d res ectivcly. Fig-3.19.2 Seasonal variations in the abundance and size of mucous cells in gills during 83(xvii) July to December represented by panels E-H respectively. Magnified view of mucous cells orthe above anels represented by panel c-h respectively. Fig-3.19.3 Seasonal variations in the abundance and size of mucous cells in gills during 83(xviii) January and February represented by panels I &J respectively. Magnified view of mucous cells of the above pmels rwresented by anels i &' respectively. Fig-3.20 Seasonal variations in number (upper panel) and area (lower panel) of 83(xix) branchial mucous cells of catfish H. fossilic. Fig-3.21 Seasonal variations in number and area of bronchial (upper panel) end 83(xx) imc omen (lower ne1) mucous cells of catfish H. fossil is. Fig-3.22 Histclo®lizatian of chloride cells in the sagittal sections of gill epithelium of 83(xxi) TW maintained catfish H. fan ilE. Chloride cells are present in the interlamellar and lamellar regions. Fig-3.23 Histolocalization of chloride cells in the sagittal section of gill epithelium of 83(xxii) catfish H. foss//is maintained in 25% SW. Chloride cells are present in the interlamellar and lamellar re ion. Fig-3.24 Histolocalization of chloride cells in the sagittal section of gill epithelium of 83(xxiu) catfish N fossils maintained in 30% SW and stained. Chloride cells are present in the inicrlamellar and lamellar region.
IV Fig. No. l.egend of Figures Pg. No. Fig-3.25 Histolocali2ation of chloride cells in the sagittal section of gill epithelium of g3(xxiv) catfish H. fossilis localized with Champy Maillet's fixative) OZI, maintained in TW (panel A-C) and 25% SW (panel D-F) Chloride cells are present in the interlamellar and lamellarre ion. Fig-3.26 Abundance (upper pane.) and size (lower panel) of chloride cells of the gills of 83( Fig-3.28 humunolocalization of Nit, K`-ATPase rich cells (chloride cells) in the 83(xxvii) seeittal sections of gill epithelium of TW maintained catfish H. fousifis. Immunoreactive cells arc preseiit in the inlerrlamellar and lamellarregion. Fig-3.29 Immunolocalization of Na, K`-ATPase rich cells (chloride cells) in the 83(xievi'.i) sagittal sections of gill epithelium of 253'a SW maintained catfish H. fossils, Immunoreactive cells are resent in the interlamellar and lamellar re ion. Fig-320.1 Sanning electron microscopy of gill of catfish H. fossilis maintained in TW 83(xxix) showing gross view. Panels (A) - (F). Fig-331L2 Scantling clecnon micruscupy of gill of calfth K fisailis maintained in TW 83(xxx) showing magnified view. Panels (G; - (L). Fig-3.31.1 Scanning electron microscopy of gill of catfish H fossils maintained in 25% 83(xxxi) SW showing gross view. Panels (A) - (F). Fig-3.31.2 Scanning electon microscopy of gill of catfish H. fossilis maintained in 25% 83(xxxii) SW showing magnified view. Panels (G) - (L). Fig-3.32.I Trdnsrnission electron micrograph of catfish H. fossilis gill ephhelimn 83(xxniii) maintained in TW. Fig-3.32.2 High magnification transmission electron micro aph view of Chloride cells of 83(xwxiv) catfish H. fossilis gill epithelium maintained in TW Fig-3.33.1 Transmission electron micrograph of gill epithelium of catfish H. fossilis 83(xcxv) maintained in 25% SW (gross view). Fig-3.33.2 Transmission electron micrograph of gill epithelium of catfish H fossils 83(xxxvi) maintained in 25% SW (magnified view). Chapter IV: Gastrointestinal tract Fig- 4.1 In situ GestreintestinaL tract ((lIT) of IL fassifis 132(i) Fig- 4.2 Dissected out GIT of H.fossilis from oesophagus to rectum 132(i) Fig- 43 Magnified view of anterior GET. Roxes indicate various stomach regions 132(L) employed for sampling of tissue. Fig- 4.4 Crass & Longitudinal sections of oesophagus of H foss/Us showing tour layers 132(ii) i.e. serosa, muscularis, submuccsa and mucosa. Fig-43 Cross section of cardiac stomach of Hfossilis. 132(iii) Fig-4.6 Cross section offundiac stomach of H. fossilis. 132(iv) Fig-4.7 Cross section of pyloric stomach ofH. fossilis. 132(v) Fig-4.8 Cross & Longitudinal sections showing the duodenum of H. fossflis with Cent 132(vi) visible layers i.e. serosa, nmsc&aris (Longitudinal and circular), submucesa and mucosa. Fig-4. 9 Cross &Longitudinal sections of mid intestine showing four visible layers of 132(vii) H. nssifir. Fig-4.10 Cross &Longitudinal sections of rectum of H. fossilis with lavers i.e, serosa, 132(viii) muscularis (Longitudinal and circular), submucosa and mucosa. Fig-4.I1 Cross sections of oesophagus of caltlsh H. fossilis maintained in TW showing a 132(ix) great predominance of neutral glycoprolein. Fig. No. Legend of Figures Pg. No. }5g-4.12 The cross sections of cardiac & fundic stomach stomach of catfish H. foss ills 132(x) maintained in TW showing neutral, acidic and mixture of neutral and acidic lyco rotein res ectivcly. SIg-4.13 The cross swfion of Pyloric stomach & duodenum of catfish fl. fossils 132(xi) maintained in TW showing neutral, acidic and mixture of neutral and acidic 1 co rotein respectively. Fig- 4.14 The cross section of mid intcsline & rectum of catfish FL fossilis maintained in 132(xii) TW. Fig-4.15 The gastrointestinal tract of catfish H fossitis maintained in 25% SW. 132(xiit) Fig-4.16 The gastrointestinal tract of catfish H. fusrdis matntained in 30% SW. 132(xiii) Fig-4.17 The gastrointestinal tract of catfish ff fossilis maintained in 35"%SW. I32(xiv) VI Introductory Preface and objective of study Introductory Preface Fishes constitute a major vertebrate group and a predominant component of aquatic fauna. The body surface of all multicellular organisms including fishes is protected by epithelial lining which serves as a physical barrier between the internal milieu and the external environment. Integument, gill and GI tract constitute a large surface area to serve as contact surface for the passage of solute, water and also possible invasion of pathogens. The occurrence of a thick and prominent mucus layer over the epithelial surface of these organs, in addition to others, contribute a great deal to lubrication for smooth passage, interface for the passage of gases and ions, defense against predators, protection against any frictional and mechanical injury and invasion of the environmental pathogens. It is largely because of the latter reason that the anatomical location of mucosa-associated lymphoid tissue (MALT) has been subdivided into gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT) and gill-associated lymphoid tissue (GIALT) (Esteban 2012). Hence, mucus, an innocuous but complex slimy secretion contained predominantly in the cells called goblet cells and spread over various epithelial surfaces has assumed considerable biological significance due to its wide distribution in variety of vital organs. In view of its diverse functional nature, the study on mucus has of late, gained considerable momentum amongst clinicians, biochemists, histologists, physical chemists and biologists. This prompted Sir Frances Avery Jones and Reid (1978) highlighting the significance of mucus to observe that it is a field of research that offers great potential to clinicians and biologists because "we cannot do without mucus, irritating though it may be if there is too much or too little and predictably fatal if absent altogether in many system of the body where it plays a vital role". It is, therefore, essential to have an overview of the physic-chemical and biological characteristic features of mucus. 1. Detailed Account of Mucus: a. Nomenclature: There has been great deal of confusion over the nomenclature with reference to mucus amongst the clinicians and physiologists with the vague and indeterminate terminologies such as mucoid, mucoprotein, mucosubstance, mucopolysaccharides, mucin etc. which seems to have been used rather interchangeably (Starts. et at 200 1).The term mucin has also been used very widely but inconsistently which according to some authors may refer to glyceproteins which have been assigned to MVC gene family (Lai et al 20(J9). In addition to mucin, mucus secretion consists of lipids, salts, proteins, macromolecules and cellular debris (Lai et at 2009). It has been documented in the literature that mucus layer is in a dynamic process of being continuously secreted, shed and discarded or digested and this recycling spans from minute to hours. The mucus flow rate is about 5 mm/min and the mucus layer in renewed roughly every 20 minutes (Ali and Pearson 2007). Continuous secretion and clearance by peristaltic forces lead to quick turnover times which vary from organ to organ. The viscosity of mucus which is 1000-10,000 times greater than that of water ensures that neither viruses nor hydrophilic macromolecules are capable of penetrating the fluid of this viscosity. The characteristic slipperiness or stickiness of the mucus is largely due to its high water content coupled with the presence of high-molecular weight macromolecules which have gel- forming ability which may vary in different organisms. For example, in most vertebrates including fishes, it is glycoprotein (mucins), polysaccharides In algae and higher plants and mucopolysaccharides in invertebrates. All these macromolecules in mucus have the ability to swell in water, interact and provide three dimensional configuration to water. b. Broad Composition: The initial efforts to isolate and characterize the constituents of mucus proved quite difficult largely due to inability to obtain mucus in a tolerably pure state. The conventional electrophoresis and ion-exchange methods remained unsuccessful because the mucus due to its biocciloidal and gel-like structure forming visco-elastic network did not lend itself to electrophoretic separation. However, with the emergence of gas-liquid chromatography, it became possible to isolate the components of mucus and subject it to detailed study (Schrager 1970). Broadly mucus consists of 95% water and remaining 5% is made up of organic and inorganic constituents. The organic constituents arc typically made-up of carbohydrates, proteins and lipids either in free or conjugated forms called glycoconjugates. The relative proportion of the various organic and inorganic constituents of mucus show considerable variations amongst the total 5% proportion, of which glycoconjugate constitute the major chunk 0.5 — 5% followed by 1% inorganic salts, 0.5 — 1% is free protein and lipids. The inorganic constituents are salts and other dialysable components. Among the free components lipids are fatty acids, phospholipids and cholesterol whereas the proteins include innate immune components such as complement, c-reactive proteins, antibodies, immunoglobulins, antimicrobial peptides (AMP), enzymes like lysozymes, acid and alkaline phosphatases, proteases, hemalysin, agglutinin and lectins. Reid and Clamp (1978) have described glycoconjugate as the polymeric substance consisting of carbohydrate which is covalently linked to materials, usually lipid or protein (but not nucleic acid). It has been further elaborated that the glycoconjugate linkage formed between carbohydrate and protein is of two types i.e. proteoglycane and glyeoproteins and it is the latter which forms the principal constituent of mucus and imparts its typical physiochemical characteristics. The difference between the two protein glycoconjugates is 2 that the proteoglycans is made u? long unbranched carbohydrate chains (glycosaminoglycan) most of which have a repeating disaccharide structure and hence contain stretches consisting of hexuronic acid. The mucus glycopreteins, by contrast, are made up of relatively small carbohydrate units (about 10 monosaccharide residues hence called oligosaccharide units), usually branded, little or no repeating structure and without hexuronic acid. The mucus glycoprotein is of extremely high molecular weight and more than 50% of this molecule weight consists of carbohydrate. The structural organization is such that a polypeptide chain forms a central care to which oligosaccharide units are embedded along its length attached to almost every third amino acid. This projected oligosaccharide unit frum the polypeptide chain gives the appearance of a porcupine or a lamp or a test tube brush. The acid groupssialie acid and sulphate are attached terminally to these bristles which protect the polypeptide core from the action of proteolytic enzymes. Such glycoprotcins are of high molecular weight (0.5 — 20 lvlDa) have protein backbone to which carbohydrate side chains are attached which make them highly glycosylated consisting of 80% carbohydrate moiety. The carbohydrate component of mucus g)ycoprotein is largely made up of galactose, fucose, N-acetylglucosamine, N-acetylgalacrosamine, sialic acid (N-acetylneurarninic acid) and traces of mannose and sulphate (Bansil, and Turner 2006). The protein core of the mucin comprising remaining 20% (— 200 — 500 Kda) is largely made up of serine, threonine and proline (STP) which constitute nearly 60% of the amino acid and the rest such as cysteine are derived from globular proteins. The linkage between the carbohydrate and protein in mucus glycoretein is termed `O-glycosidic' because the linkage is through an oxygen atom. This type of linkage is susceptible to cleavage by alkali except when the amino acid involved is terminal. Physicochemical properties of mucus may certainly be dependent on both protein as well as carbohydrate component of the mucus glycoprotein. However, since glycoproteins are largely composed of carbohydrate which is in immediate wutact with the environment, the glycoprotein chemistry will be greatly dependent on the chemistry of oligosaccharide units particularly the peripheral sugar which are in terminal position. It is important to mention here that several other types of glycoproteins other than mucus glycoprotein may be found in the sites such as blood which is termed plasma glvcoproteins. However, these glycoproteins will be different from the mucus glycopretein in as much as that it may have less than 25% carbohydrate component, have N-glycosidic linkage and may not have the predominance of serine, threonine and protein (STP) as found in mucus glycoproteins. c. Sourcc(s) of mucus secretion: As stated earlier, mucus is largely produced by goblet cells which are abundantly present in all epidermal surfaces but other cell types such as s wciferm cells (Sadovy et al 2005) and acidophilic granular cells/ serous goblet cells (Whitear 1986, Pickering and Fletcher 1987) may also contribute to mucus secretion. d. Variations in mucus composition: Considerable variations have been reported in mucus composition from the generalized picture described above. For instance the mucus in leech skin is highly glycosylated containing 80% carbohydrate and other 20% protein which in addition to typical threonine and serine may have glycine in place of proline. Similarly, there may be additional presence of glucose and mannose which are generally not reported in the carbohydrate moiety of other mucus. Similarly, certain fishes like H. fossilis, C. barrachus and mullets have been found to have high proportion of lipids in mucus (Lewis .970, Venkaiah and Lakshmipathi 2000). e. Acidic and neutral glycoproteins: The glycoproteins (GPs) elaborated by the bronchial mucous cells have been studied by a range of histochemical procedures. Broadly, there are two types of glycoproteins present in the mucous cells, one is neutral and the other is acidic glycoconjugate and the latter is either carboxylated or sulphated type also referred to as sialo- and sulphomucin respectively. These different chemical moieties in mucus glycoproteins can be identified histochemically using PAS which stains neutral glycoprotein with characteristic magenta shade and alcian blue pH 2.5 (AB 2.5) which stains both acidic glycoproteins as blue. Exclusive sulphated moiety of acid glycoprotein can be identified by alcian blue at pH 1.0 (AB 1.0) which excludes the staining of carboxylated acid glycoproteins. Both acid moieties in the same section / cell can be visualized distinctly histochernically by the combined staining with aldehyde fuchsin (AF) —AB (pH 2.S) which stain carboxylated acid glycoproteins purple and sulphated moiety as blue. This can also be done by another alternative approach i.e. methylation followed by AS 2.5 stain which suppresses the carboxylated component and specifical[y stains sulphated moiety as blue. However, when methylation is followed by saponification and stain with AB (pH 2.5), the carboxylated moiety is stained positive whereas sulphated component remains suppressed. The histochemical techniques have been very extensively used by biologists to localize neutral and acidic glycoprotein in mucous cells. Some workers have further characterized the GPs with oxidisable vicinal dials by further confirming with the acetylation before PAS to block the oxidation of 1, 2 glycol groups by the periodic acid which would yield the negative result with PAS i.e. no magenta colour will appear. This is further confirmed by the sequential process of acetylation, saponification, PAS to restore 1,2 glycol groups which react with periodic acid and yield magenta colour. Also, it is established by a amylase digestion before PAS reactions which yields magenta colour . For the selective visualization of static acid (carboxylated GP) and some of their chain variants at Cl and for C9, a technique named PA*S (selective periodic acid Schiff reaction) which involves oxidation with 0.4 mirl periodic acid in 1M IICI for 1h at 4°C yielding magenta colour is used. The selectivity of this reaction is based on the fact that there is an increase in the rate of oxidation of sialic acid residues together with the decrease in the rate of the oxidation of neutral sugurs. Further, to localize sialic acid with vicinal diets i.e. carboxylated and neutral GPs, PAPS (periodic acid oxidation- phenylhydrazine-Schiff) involving oxidation with 1% periodic acid for2h at RT followed by treatment with 0.5 % phenvlhydrazine for 2h and incubation with pararosanitine Schiff for 4h to yield magenta colour which confirms the presence of both these GPs together. For the differentiation of mucins that allows single section identification of side chain 0-acylated, and nonacylated, sialic acids in contrasting colors, KOH/PA*S method (saponification- selective periodic acid Schiff reaction) has been used which involves saponification with 0.5% in 70% ethanol for 30 min at RT to deacetylatesialic acid residues followed by PA*S method yielding contrasting colour. Saponification-selective periodic acid- borohydride reduction-periodic Schiff reaction (KOHiPA*Bh/PAS) method has been used to characterize the neutral sugar whereas another method named PA.BhJK01-UPAS (Periodic acid- borohydride reduction-saponification-periodic Schiff reaction) is employed to visualize sialic acids with 0-acyl substituents at C7, C8 and C9 and 0-acyl sugars which yield PAS positive results. Lectins are carbohydrates binding proteins other than enzymes and proteins and are considered reliable tools to characterize glycoconjugates in tissues. A battery of several lectias have beer used to study the binding on the tissue sections to characterize glycocanjugatcs in gills, skin, Gil and other tissues. Hence, histochemical methods have proved to be valuable tools for localization and characterization of high molecular weight glycoproteins in leleosteanmucus which may reflect functional characteristics. 2. Functional Overview of Mucus with Specific Reference to Osmoregulation: The trident mucus functions include lubrication, water proofing and indeed protection. Even though mucus overlays the epithelial surfaces of gill, skin and GIT but it plays a more critical role in case of integument and gills which is the first line of defense against the microbial exposure. In case of GIT, mucus functions as the `skin of the gut' where it acts more effectively than the skin of the body to provide protection to gastric mucosa against powerful acid and proteolytic enzymes. Fish skin mucus serves as a repository of a variety of biologically active substances. Due to relatively impernmant nature of mucus to pathogens, it traps and immobilizes these microbes which get constantly removed due to flowing water current thus preventing their stable colonization. In fish mucus, the various active immune components and defense molecules with biocidal activity include variety of proteolytic enzymes, complement, C-reactive proteins, immunoglobutins, criaotoxins, pheromones and lectins. Among the important enzymes are lysozyme, acid and alkaline phosphalase, cathepsins, superoxide dismutase (SOD), estrases, trypsin and metalloproteases. Thu documented antimicrobial peptides in fish mucus are the alpha-helical amphipathetic peptide which is very common in addition to others such as pleurocidins, pisadins, pardoxin, hipposin, oncorhyncin III & 1I. Among the commonly occurring proteins are lectoferrins, histories and ribosomal proteins whereas the important immunoglobules include igM molecule as a predominant isotype in addition to polymeric immunoglobulin receptor. The fish lectin, agglutinins may confer a limited amount of immunity against irfectinn by pathogens. Compliment brings about cell lysis of activation of immunoglobulins and other materials. Interestingly, mucus provides a renewable surface and whatever is attached to mucus is rapidly lost. The stress-mediated release of mucus due to heavy metals presence helps to bind these heavy metals to mucus which is removed as shed mucus to prevent its entry into the fish. The involvement of mucus in osmoregulation has been suggested in some earlier experiments in eel Anguilla anuilla where wiping of fish skin mucus compromised the osmoregulatory ability (Baldwin 1948, Negus 1963). Hypophysectomy of these fishes reduced the number of goblet cells and replacement with prolactin, an important osmoregulatory hormone in fresh water fish, reversed this effect. Abundance of goblet cells on fish integument and gills may correlate with environmental salinity (Johnson 1973 and Laurent 1984). That sulpho- and sialomucin of fish mucus have been very intimately linked with the maintenance of homeostatic balance in divergent salinities (Clamp et al 1978). Hence, there is strongly suggestive evidence that fish mucus in integument, gill and GI]' plays an important role in osmoregulatory adjustment of telcosts. 3. Rationale of the Present Study: The fresh water stenohaline catfish H fossilis and C. batrachusare important components of capture and culture fishery of the Indian subcontinent and are preferred consumer's choice. These catfishes survive up to a salinity of 35% seawater or 1,1% NaCI (Parwez et al 1979 and Parwez et al, 2001). They actively osmoregulate up to 10% SW and their survival up to its maximum salinity tolerance limit is achieved through passive tissue tolerance. The basic osmoregulatory mechanism of H. jossilis in high salinities as well as in deionized water involves the coordinated participation of the major target organs i.e. gill, skin, gut and kidney (Goswami eta] 1983, Panvez et at 1984, Sherwani and Panvez 2008). The actions of these target organs are hormonally-mediated process with prolactin from adenohypophysis and cortisol and arginine vasotocin (AVT) from adrenal cortex and pars nervosa respectively playing the major regulatory roles (Panvez and Goswami 1985, Sherwani and Parwez 2008). However, despite the fact that mucus covers at least three major osmoregulatory target organs i.e. skin, gills and gut and is known to be a multifunctional entity, its biochemical nature and possible role in these osmoregulatory target organs have not been studied. Moreover, no detailed account with reference to cellular and other histological features of the above three important osmoregulatory target organs i.e, skin, gill and gut is documented in literature. Clearly, such an account is extremely important to understand the role of mucus vis-à-vis thcsc thrcc target organs in the overall perspective of osmoregulatory adjustment. Hence, the objectives of the present study are as follows: 4. Objectives of the Present Study: (1) To study the detailed organizational arrangement with particular reference to morphology, histology and cellular details of skin, gill and GI tract of catfish, 11 fossilis and also C. bcarachus on selected target organs. (II) To map the relative abundance of mucous cells in different regions of integument and antero-posterior gill lamellae of H. fossilis and C. batrachus. Also, to study the effects of handling stress on the population of mucous cells in the above two catfishes. (111) To localize and characterize the glycoprotein moieties of mucous cells in all the three target organs using a wide array of histochemical stains. (1\7To study the monthly and annual variations and salinity induced changes in the abundance and size of:rucus-secreting cells with particular reference to the changes in its glycoconjugate moiety and to explore the physiological significance of the specific glycoproteins. (V) To identify and localize the chloride cells on the branchial epithelium by light microscopy, histochemically, immuno-histochemically and through ultrastructural studies using SEM and TEM approaches and to study salinity induced changes in chloride cells population. 5. Expected Outcome of the Study: The information thus generated will help us to build up a comprehensive overview of better and more precise understanding of the morphology, histology and cellular composition of skin, gill and Gl tract, the important nsmoregulatory target organs in fishes. It would also provide an insight into the biochemical composition of mucus glycoptoteins, a major component of overlying mucus on the above organs, its seasonal profile and salinity and stress induced changes in these catfishes. The consortium of such an information in conjunction with the pre-existing documented information on these aspects will greatly enhance our current understanding of osmoregulatory adjustment in telcost in general and in tropical freshwater stenohaline fishes in particular. I Materials and Methods Materials and Methods A. Collection and Care of Fish: Live specimens of catfishes Hewropenc'~lus /o.S.cili.s (local name Sin~uhi) and ('lurius hairadiuv (local name '1agur). eighing heteen 35 - 40 gin (approxirnatclv 6 cm Length) %%ere obtained from local fish market of Aligarh. The fish %%ere kept under laboratory conditions in glass aquaria (7 x 33 x 22 cm) containing stored dechiorinated tap %cater. The %%crc acclimatircd fur IS - 20 da%s in the lahorator% prior to the initiation of experiments. During this period the \\ere fed daily .-hi lihini n \%ith minced meat and \\ater in aquaria 'as changed daily b siphoning ofl and replenishing simultaneously ith fresh\\ater adjusted to lahorator' temperature. External features of Heteropneustes fossi/is External features of Clarias batrachus 8 B. Artificial Sea Water: Artificial sea water (SW) was prepared in dechlorinated TW according to Goswamie! al (1983). Briefly, to prepare full strength SW (I00%), the following salts (gm) were dissolved in I liter dechnorinatedTW; NaCI, 400.8; KCI, 9.8; CaCl2 (fused), 10.1; MgC12-6112O, 52.7; Na2SO4, 27.8; NaHCO3, 2.5; and NaBr, 0.6. Further dilution (25% SW in the present study) was prepared by diluting 100% SW with dechlorinated TW. C. Tissue Collection: (a) Skin- A small skin piece just below the dorsal fin of approximately lcm2was excised along with some underlying muscles. (b) Gills- All the gills of both sides along with gill arches from anterior to posterior side were taken out. (c) Gastrointestinal Tract (GIT)- The entire GJTwas removed and small segments were cut from oesophagus, stomach (anterior, middle and posterior), intestine (duodenum, mid intestine and rectum). D. Histology of Tissues: (Fixation, rinsing, dehydration, clearing, infiltration, embedding and sectioning). I. The skin and all gills were fixed in Camoy's and aqueous Bouin's fluid (Appendix-I) for 2 and 24 hrs keeping them completely immersed in these fixatives. 2. After the optimum fixation, the tissues were rinsed with distilled water several times and then dehydrated with ascending alcohol grades of 30%, 50%e, 70%, 90% and up to 100%, keeping in each grade for 1-2 his and then transferred to xylene for clearing with 2-3 intermittent changes, until the tissues became completely transparent. 3. For wax infiltration, the dchydrated and cleared tissues were transferred in a mixture of xylene and wax (50% xytene + 50% wax) for 10 min in an oven maintained at. 65°C to allow the xylene to come out of the tissue and the wax getting infiltrated into it followed by transfer to pure molten wax and kept in an oven for appropriate time depending upon the tissue giving two changes of fresh wax. 4, For block making, the molten paraffin wax (Qualigens, melting point 60-62°C) was poured into the cavity block whichwas smeared with glycerine and the tissue was embedded into it at the desired orientation. 5. When the wax solidified completely (approx. 30 min), the blocks were easily removed from the cavity block and trimmed according to the desired plane of sectioning. E 6. Five micron thick sections were cut with the help of micrototue (Leica RM 2125 RTS), stretched at a temperature of 40-45°C over the egg albumin coated slides to ensure sticking of the sections. E. Staning Procedure: (a) General Histology: S.N Stains Interpretation of staining reaction References 1. Harris Nuclei (blue), cytoplasm (pink) Humason Hematoxylini (1967) eosin 2. MassonsTrichrome Nuclei (black), cytoplasm (red), Masson Collagen (blue) (1929) (i) Harris Hematoxylinand Eosin, (Il.umason 1967) 1. The slides were deparaffinized in xylene and hydrated through progressively decreasing alcoholic grades and transferred into distilled water. 2. The sections were stained with Harris alum Hematoxylin (Appendix-I) for 30 min, rinsed with DDW and destained in 0.1% HCI for 20 to 30 seconds. 3. This followed the extensive washing of the sections with running tap water until blue colour appeared. 4. The sections were dehydrate inascending alcohol grades (30% to 95%) and transferred in 0.2% eosin in 95% alcohol for counterstaining followed quickly by dehydration in 95% and I00% alcohol, cleared in xylene and mounted with DPX. (ii) Massons Trichrome, (Masson 1929) I . The deparaffinized slides were hydrated by the same steps as described above. 2. The sections were mordant in Bouin's solution for 1 hr at 56°C, cooled and washed in running water until yellow color stops appearing and rinsed in DDW. 3. Now the sections were immersed in Weigert's iron hematoxylin solution (Appendix- 1) followed by DDW fur 10 min each and again stained in Biebrich scarlet-acid fuchsin solution (Appendix-I) for 1 D-I5 min and finally washed with DDW. 4. The sections were now put in Phosphomolybdic-phospholungstic acid solution (Appendix-I) for 15 min and then stained with Aniline blue solution (Appendix-I) for 5 min. 10 5. Finally, these sections were rinsed with DOW and transferred in Glacial acetic acid for 5 min, dehydrated in alcohol grade, cleared in xylene and mounted with DPX. (b) Glveohisloehemical stains: B.N. Staining Interpretation of staining reaction References Techniques 1. AB (2.5) Glycoconjugates with acidic groups Mowry (1963) (carboxyleted and sulphated) 2. AB (1.0) Glycoconjugates with 0- Lev and Spicer sulphateesters (1964) 3. Active Methylation Glycoconjugates with 0-sulphate Spicer and (AM)/AB(2.5) esters Warren (1960) 4. AM/Saponification Glycoconjugates with carboxylated Spicer and Lillie (K011)/AB(2.5) moiety (1960) 5. PAS Glycoconjugates with neutral moiety McManus(1948) 6. PAS.tAB (2.5) Glycoconjugates with neutral and Mowry(1963) acidic moiety 7 PAS/AD (L.0) Glycoconjugates with neutral and Spicer etal. sulphated moiety (1967) 8. AF/ AB(2.5) Glycoconj ugates with carboxylated Cameron and and sulphated moiety Steele (1959) (i). Alcian Blue (pH 2.5) / AR (2.5), (Mowry 1963 see Pearse 1968) 1. The slides containing tissues were deparaffinized in xylene, hydrated in down grade alcohol series i.e. 100 to 30% and finally brought to water. 2. The slide were stained with 1% AB 8GX (Sigma) in 3% acetic acid for 30 min. 3. The sectionswcre washed in running water for 5 min, dehydrated with ascending alcohol grades i.e. 30% to 100%, cleared in xylene and mounted in DPX. (ii). Alcian Blue (pH 1.0)1 AB (1.0), (Lev and Spicer 1964) I. The slides were hydrated and transferred to 1.0% alcian Blue SOX in 0,1 N-HCl for 30 min. 2. These slides were blotted dry with fine filter paper, dehydrated in alcohol, cleared in xylene and mounted in DPX. 11 (iii). Active Methylation, AMJAB (pH 2.5), (Spicer and Warren 1960) 1. Brought the sections up to hydration and coated with a film of 0.5% aelloidin and kept in pre-hested 08: 99.2 mixture of Con.HCI and Methanol for 5 hrs at 60°C toactivate Methylaticn. 2. These slides were then washed in running water, rinsed in absolute ethanol and kept in equal part of ethanol and diethyl ether to remove celloidin filar and washed again with water. The sections were stained with 1% AB 9GX (pH 2.5) for 30 min, washed with running water for 5 min, dehydrated with alcohol ascending series, cleared in xylene and mounted in DPX. (iv). AM/Saponircation, KOH / AB (pH 2.5), (Spicer and Lillie 1960) I. After methylation as described above, the slides were placed in 70% ethanol. 2. For saponification, the slides were kept in 1% KOH made in 70% ethanol for 30 min. and treated with ethanol and diethyl ether in equal part for removal of the celloidin film. 3. Then these sections were washed with running water and stained with 1% AB 8GX (pH 2.05) for 30 min,again washed with running water, dehydrated in alcohol and finally mounted in DPX. (v). Periodic acid Schiff's (PAS) Method (pH 2.5), (McManus 1948, sec Pcarsc 1968) 1. The slides were brought hydrated and transferred to 1% aqueous periodic acid for 15 min for oxidation, washed with running water for 5 min and stained with Schiffs reagent (Appendix -1) for 15 min. 2. Then the tissue were washed with running tap water for 5 min, dehydrated in upgrade alcohol series i.e. 30 to 100°%alcohol, cleared in xylene and mounted in DPX. (vi). Alcian blue (p112.5) —Periodic acid Schift's, Al) (2.5)-PAS, (Mowry 1963) 1. The sections were brought up to water and rinsed briefly with 3% aqueous acetic acid. 2. The sections were stained with 1% Alcian Blue RGX (pH 2.5) made in 3% acetic acid for 2 hrs, rinsed briefly in water and then in 3% acetic acid, washed in running water and distilled water quickly. 3. These sections were then transferred for oxidation for 15 min in 1% periodic acid solution at room temperature, washed in running water for 5 min and subjected to the same steps as described above in para (v) for PAS staining. 12 (vii). Alcian blue(plf 1.0) —Periodic acid Schiff 's, PAS-AB (1.0), (Spicer et al 1967) 1. Step I is the same as described in AB,PAS pH 2.5 above. 2. The sections were stained for 2hrs in 1% AB RGX in 0.1 N-HCI. 3. The tissues were blotted dry with fire filter paper, then oxidized with 1% periodic acid for 15 minand subjected to the same steps as described above in pora (v) for PAS staining. (viii). AldehydeFuchsin /Alcian Blue, AF/AB(2.5),(Camcron and Steele 1959) 1. Deparaffinized and hydrated the sections, transferred in Alcian blue (pH 2.5) as described inpara (i) above. 2. Then the slides were oxidizedin Gomori's solution(Appendix - I) for about I min, rinsed with distilled water for 5 min, bleached in sodium bisulphite solution until the permanganate colour is completely removed. 3. These slides were washed in running water for 5 min, transferred to 70% alcohol for 2 min and stained in Aldehyde fuchsin (Appendix 1) reagent for 10 min at room temperature. 4. The back of slides were wiped off and rinsed in 95% alcohol until no more Aldehyde fuchsin stain comes out of the sections. 5. The slides were washed in absolute alcohol two times for 5 min each, cleared in xylene and mounted in DPX. (c) Staining Techniques used for localization of Chloride cells; S.N Stains Interpretation of staining References reaction 1. Acid Hematein Nucleuprotein and Phospholipids Baker(1946) ( blue) 2. Sudan Black B Phospholipids ( black) McManus (1946) _9. Tnh,idine Blue Metachrnmatic glycoprotein Belanger and Hartnett (brown), Phospholipids (red) (1960) 4. ChampyMaillet's Phospholopids (black) Garcia-Romeo and fixative/ OZ] Masoni (1970) 13 (i) Acid Hematein Method, (Baker 1946 see Pearse 1968) I. The gillsof H. fosrilis were fixed in Formal calcium (Appendix-I) for 18 hrs and then transferred to dichromate calcium (Appendix-I) for 18 hrs at 22°C and then into a fresh dichromate calcium for 25 hrs at 60°C. 2. The tissues were washed with distilled water and paraffin blocks were made as described earlier. 3. Five micron thick sections were cut, stretched and stuck on the slide; deparaffinized and brought to water. 4. These section were transferred imo mordant (dichromaw-calcium) for I hr at 60'C, washed with DDW thoroughly, and transferred to acid hematein solution (Appendix-I) for 5 hrs at 37°C. 5, The sections were then rinsed with DDW, transferred to borax- ferricyanidedifferentiator (Appendix-I) for 18 hrs at 37°C, washed with DDW, dehydrated, cleared and mounted in DPX. (ii) Sudan Black B, (MeManus 1946, see Pearse 1968) 1. The tissues were fixed in a solution containing 1 gm cobalt nitrate dissolved in Wool DDW with I Oml of 10% CaCI, and lOml of 40% formalin for S-weeks and paraffin blocks were made as described earlier. 2. Five micron thick sections mounted on slide were brought to 70% alcohol, transferred in saturated Sudan Black B madein 70% alcohol for 30 min at room temperature. 3. They were then washed with 70% alcohol, quickly for removing the excess dye and dehydrated with ascending series of alcohol, cleared in xylene and mounted in DPX. (iii) Toluidine Blue Method, (Belanger and Hartnett I960see Pearse 1968) Brought deparaffinized sections up to 30% alcohol and stained with 0.1% toluidine blue in 30% ethanol for 30 min, rinsed with 70% and 95% alcohol thoroughly, transferred in absolute alcohol for dehydration, cleared in xylene and mounted in DPX. (iv) OZI Staining Method, (Garcia-Romeu and Masoni 1970) 1. The gills were excised from all fishes, cut in small pieces of 2 mm size and immersed in freshly mixed ChampyMaillet's fixative(Appendix-l) for 16 hrs at room temperature. 2. These small pieces of tissues were washed with DDW for 5 - IC times to remove the excess fixative, dehydrated with a series of ethanol and processed for making paraffin blocks as described earlier. 14 3. Five microuthick sections were cut, taken on slides, deparaffinized and mounted in DPX. F. Immunoh istochemist ty: a. Tissue fixation The gill tissues were fixed for 8-l0 hrs in 4% paraformaldehyde (Sigma) (Appendix-I) prepared in (JIM phosphate butler saline (PBS) (Appendix-I) for 5-10 hr. The tissues were sequentially transferred to PBS containing 10% and 20% sucrose for 2hrs each and finally to 30% sucrose where they could be stored up to 72 hrs at 4°C. These processed tissues were embedded in tissue-tek OCT compound (Sakura USA) and the blocks were sectioned at 15 pm thickess at —25°C on Cryostat (Leica CM-18.50) and the sections were mounted on the slides coated with poly-L-lysin (Sigma P 8920) and stored at -20°C overnight or until use. The slides coated with poly-L-lysin were prepared by dispensing 25 tl of poly-L-Iysin on a slide and spreading it evenly with finger. b. Antibodies: For immunolocalization of gill Na'-K'-ATPase, a monoclonal antibody specific for the .-subunit of chicken Na /K'-ATPase ay was used (Takeyasu et at 1988). The antibody (u5) developed by D.M. Pambrough (Johns Hopkins University MD, USA) was obtained from Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Science, Iowa City, iA 52242, USA, developed under the auspices or the National Institute of Child Health and Development (NICHD). The antibody was purchased in the form of cell culture concentrate (0.1 ml) which is now in routine use for identification of chloride cells by way of their high Na`fK -ATPase levels (Witters et al 1996 and Wilson et at 2000). The secondary antibody was generated against IgG of mouse in rabbit (Department of Zoology, Nagpur University campus, Nagpur, India). c. Staining technique of immunohistochemistry: Before use, the poly-L-lysincoated slides stored at —20°C were brought to room temperature for I hrs, a well around the sections was drawn with PAP pen and transferred to the humidity chamber. These tissues were then rinsed with PBS three time for 5 min each to wash out the fixative and were finally washed again with triton X 100(Appendix-I) for 20 minwhich is mild detergent, used to render the antigen accessible to the antibodies and also helps to expose the antigen sites and facilitates antigen-antibody reaction and greater signal to noise ratio. The slides were then transferred in blocking solution (50% PBS + 50% BSA) for 30 rain, which is used to prevent non specific cross-reactivity of both primary and secondary antibody with the tissue. The slides were incubated in primary antibody at the antibody dilution of 1:1000 15 in PBS for 2hrs at room temperature. After incubation the slides were rinsed 3 times with PBS for 5 min each and exposed to secondary antibody (1:100 in blocking solution) (Appendix-I) at room temperature for 1 hr and then rinsed 3 times with PBS. The sections were now incubated in streptavidinperoxidse (Appendix-I) for I hr at room temperature and washed 2 times with PBS and DDW- Following this, DAB (Appendix- I)was applied on the sections for 5-7 min until reddish brown colour appears and washed the slides property with DDW and mounted in glycerol gelatin at 60°C and visualized under microscope (Carl Zeiss model Axioskop 40 FL). G. Visualization of Sections: The sections were visualizedunder Zeiss microscope (Carl Zeiss model Axioskop 40 FL) at the suitable magnification and images were captured with Axiocam lCc3 camera and 4 sectionscach from Sfishes were quantified with Auto measure software (Re]. 4.8). For statistical analysis, the data were expressed as mean } S.E. Analysis of data was carried out using GraphPadlnStat software and one-way analysis of variance (ANOVA) followed by Tukey's test to calculate the statistical significanceofthe data. H. Electron. Microscopy: (i) Scanning Electron Microscopy (SEM) Anesthetized fishes with MS222, were sacrificed and gills and skin were excised as detailed in section D. Dissected tissues (size 1cm) were prefixed in a Kamovasky's fixative (2.5% Glutaraldehyde and 2% paraformaldehvde (Sigma) dissolved in 0.1 M PBS (pH 7.2)(Appendix-1) at 4"C for 10 hrs, then rinsed three Limes with 0. IM PBS and postfixed in 1% osmium tetroxide (Sigma) in O.1M PBS (pH 7.2) for 2 hr at 4°C. The tissues were washed with buffer 3 times for 15 min each at 4°C followed by dehydration through graded series of methanol at 4°C (25%, 50%, 70%, 80%, 90%, 95% and 100%), air dried and finally transferred in hexamethyldisilazane (FIMDS) for 15 minat room temperature. Then specimens were critical-point dried using liquid CO2 in a critical-point drier (Hitachi HCP-2), mounted on the aluminium stub and sputter coated with a gold palladium complex of 35 nm thick film, kept for 4 min in a vacuum evaporator (Electron microscopy sciences EMS15CR) and examined under SEM (Zeiss EVO KD25). (ii) Transmission Electron Microscopy (TEEM) The gills and skin (size- 1mm) were fixed in Karnovsky's fixative (2.5% Glutaraldehvde and 2% paraformaldehyde dissolved in 0.1 ht PBS pH 7.2) for IC his at 4°C, washed 3-4 times with 0.1 M PBS and postfixed in 1% osmium tetraoxide (Sigma) in 0.1 M PBS for 3 his at 4°C. The tissues were rinsed thoroughly with PBS buffer to 16 remove excess fixative, followed by dehydration through a graded series of ethanol (10%, 25%, 50%, 75%, 90%. 95%) at 4°C for 10 min each and then allowed to warm to room temperature. This was followed by four changes of absolute ethanol for 15 min each, cleared in toluene for 30 min and embedded in Epon 812 and polymerized at 60'C overnight. Ultrathin sections (1 pm) were cut with diamond knife using ultratome (Leica EM UC6), mounted on grids, double stained with 2% uranyl acetate in 50% ethanol for 10-15 min followed by lead citrate for 5-10 min and examined at 75 kV under Transmission Electron Microscope (JEOL JEM-2100F). Chapter 1: Handling stress and mucous cells region-based distribution Chapter-I (Handling stress and mucous cells region-based distribution) I. Introduction: This chapter, not initially intended to he written and added, is an outcome of a simple curiosity which ultimately became a scientific necessity. Hence, this chapter is the shortest of all, barely qualifying into the status of an independent chapter in length but logically placed first due to strategic significance of its contents. As would be evident from the `Introductory Preface' of this thesis that the focal theme of the present investigation is to explore the role of overlying mucus layer in three important osmoregulatory organs i.e. skin, gills and the GIT. This mucus is being secreted chiefly by the goblet cells / mucous cells in these organs. The sampling protocol, normally followed, is to act out fishes from aquaria, sacrifice them and excise the target tissues for histological and other investigations_ However, considering the basic tenets of stress physiology, one wonders if this simple innocuous protocol of sampling is not exerting any stress onto the fishes and particularly on the mucous cells which are known to play a significant role in defense and protection of fishes against unfavourable conditions and invading pathogens. The curiosity was whether netting of fishes for sampling purpose for these target tissues not exerting stressful conditions on the fishes in general and the catfishes, under present study, in particular. If so, then, any data generated based on the above protocol of sampling will be 'grossly suspect' until the issue of perceived stressful conditions due to aforesaid sampling procedure is settled. This, therefore, greatly underlines the need for establishing a baseline data based on the protocol which may yield data on "unstressed/ normal" conditions to arrive at meaningful conclusions. Our concerns in this study were the following: (a) Does the sampling protocol normally followed by different investigators to study the mucous cells in fishes not affect the normal histological and physiological status of these cells? (b) If the above apprehension is correct, what, then. is the alternate approach to collect samples to generate a truly baseline data generated under normal' unstressful conditions in fishes. (c) Generally, the samples of skin and gills mucous cell studies are collected arbitrarily from one specific region of body for skin and any gill randomly. This is based on a very liberal and definitely unscientific assumption that mucous cells are uniformly distributed all over the skin with no region or gill - number based variations. It is, however, extremely important to systematically study the validity or otherwise of such assumptions. (d) Based on the outcome of para (c) above, it is proposed to chalk out a robust and scientifically sustainable sampling protocol for the present study on mucous cells population in both the catfishes. II. Experiment Protocol: All the Experiments were performed on acclimatized catfishes H. fossilis and C. barrachus under controlled laboratory conditions as detailed in Materials and Methods of this thesis. Exoeriment Protocol 1: The catfish H. fossilis and C batrachus were allocated in four groups. Five fishes each of H foss//is were allocated to Group I and II and similar number of C. batrachus were transferred to Group III and IV. The fishes of group I and III were subjected to handling stress and group II and IV were left unstressed to serve as control. The physical stress was caused through repeated handling with not followed by sampling of skin piece from below the dorsal fin and gills ftotn branchial apparatus. For unstressed control group, the level of the water in the aquarium was gradually reduced by siphoning off so as not to disturb these fishes. Then appropriate quantity of MS222 (FINQUEL) wns dissolved in small quantities of water and gradually added to aquarium water. The fish got unconscious within 3-5 minutes which were gently taken out by net and kept on the wet napkin and the skin and gill samples were taken as in the other gioups. These tissues were fixed in Camoy's and Bouin's fluid and processed for histological observations as per details given in Materials and Methods. Experiment Protocol 11: The acclimatized catfish H fossiiir and C. batrachus were allocated in two groups. Five fishes of H fuss ills were allocated to Grouu I and C. batrachus were transferred to Groin II. After anaesthetization the fishes from each group were sacrificed and small pieces of skin of four different regions (i.e. below dorsal, caudal, pectoral and anal fin) and four pair of gills from anterior to posterior were excised and fixed in Carnoy's and Bouin's fluid for histological processing following the same procedures as described in Experiment I above. III. Observations: Experiment 1: Effects of handling stress on mucous cells (MCs) population on the skin and the gills of the catfish H. fossilis & C. batrachus. It is evident from the perusal of Fig. 1.1 - 1.4 that the handling stress causes a dramatic effect on the populations of integument MCs in IL fossilis and C. bap achus to the extent that they altogether disappear from the entire epidermis of both these catfishes (Fig. 1.1 A and 1.3 A) leaving no traces whatsoever of any stained mucous cells. On the contrary, 19 the corresponding control groups of both catfishes where the sampling was done after subiecting the fishes to anesthesia MS222, the integument showed completely normal, intact well distributed mucous cells all over the epidermis of both catfishes (Fig. 1.1 B and 1.3 B). In H. fossilis the differential staining with PAS and AB (pH 2.5) clearly show the mucous cells with different UP contents (Fig 1.1 B) which, however, are not so clearly visible in C. batrachus integument (Fig. 1.3 B). Interestingly, there are no perceptible changes in the population of gill mucous cells following handling stress in both the catfishes (Fig 1.1 C, D and 1.3 C, D). The quantification of gill mucous cells number per unit area in both the catfishes do not show any significant differences between handled and control groups (Fig. 1.2 and 1.4). Experiment H: Mapping of mucous cells abundance in different regions of the integument and different gills arranged antero-nosterinrly in H fossilis & C. batrachus. The population of mucous cells have been mapped in four different regions of integument viz. just below anal, caudal, dorsal and pectoral fms in both catfishes. The data on H. fossilis reveals that the highest abundance of mucous cells is found in the region below dorsal fin and the least below the anal fn and pectoral and caudal region show progressive decrease compared to dorsal region (Fig. 1.5 and 1.6). The difference between the lowest (anal region) to the other regions i.e. caudal, dorsal and pectoral is statistically highly significant (P<0.001). However, C batrachus presents an altugether different profile of region-based abundance of mucous cells in its integument. It resembles H. fossil is in as much as the highest abundance is found in the region below dorsal fin but the least abundance in C. batrachur is recorded in caudal region where the mucous cells are very few (Fig. 1.9 D) followed by pectoral and anal region in increasing order. Quite unlike the H foss/Ifs where the population variations in at least three regions viz caudal, dorsal and pectoral was not very drastic, in C. bairachxs, these inter-region variations are quite substantial (c.f. fig. 1.6 and 1.10). Similarly, following the pattern of absence of any effect on handling stress on gill mucous cells population, no differences were observed in the inter gill population abundance of mucous cells in both the catfishes (Fig. 1.7, 1.8, 1.11 and 1.12). IV. Discussion: Hans Selye, while observing the effects of noxious stimuli on laboratory rats, coined the term 'stress' and defined it as "the non-spec(c response of [he body to any demand for change' (Selye 1936, 1973). Subsequently, several alternate definitions were proposed to convey the notion of unease, distress, discomfort or disturbance caused by certain stimuli which were designated as "stress" to cover the stressful stimuli. It is well established that interplay between stressors and stress responses is highly complex and some stress responses may themselves function ns stressors and vice-versa (Harper and Wolf 2009). It is observed that the term `stress' has been used rather loosely to include almost any type of adverse condition and its consequent outcome that a fish might 20 ... '~ i~/ I I I •. PC Dermis ~ ~ • .' Ep1de,mis Muscles i Dermas 50x L AS B ' •' , >, .. ; Mujous CHs "~, . • Mucofrs Cells 5 1.. S •.• •.. , .•••. Fig-1.1 Effect of stressed (panel A & C) and nonstressed handling (panel B & D) on skin and gill mucous cells of catfish H. foss//is (PC = Pigment cells). Note the complete absence of mucous cells in the skin of stressed catfish (panel A). 16 A 14 12 NS to U g O 6 w O aL E 2 2 z 0 Nonstressed Stressed Fig-1.2 Effect of stressed and nonstressed handling on number of mucous 10 cells in the gill epithelium of catfish H. foss//is. Significance calculated with reference to nonstressed catfish. NS = Not Significant. 20(i) ' v r - 5,. fI •.. - Dermi4 b a'. ` Dermis T.... Mus ' - y T Muscles 50x ♦- A SOx ~ ~• - e R •,' Mucous Cells . 50x ~ .ti ;' • "I, Fig-1.3 Effect of stressed (panel A & C) and nonstressed handling (panel B & D) on skin and gill mucous cells of catfish C. batrachus (PC = Pigment cells). Note the complete absence of mucous cells in the skin of stressed catfish (panel A). Fig-1.4 Effect of stressed and nonstressed handling on number of mucous cells in the gill epithelium of catfish C. batrachus. Significance calculated with reference to nonstressed catfish. NS = Not Significant. 20 (ii) - Mucous Mucous cells 4.l9 . j cells - '~!~ - „ifs. sox y..~ Mucous N Mucous • ~ cells •~ ~ M cells III ~ ~ r 51 r D Fig-1.5 Mucous cells abundance in four different regions of the integument of catfish H. fossil/s. Panels A-D showing samples taken from below the anal, caudal, dorsal and pectoral fins respectively. 180 E5 16O- * * *** 140 120-i *** 100 I 2 80 60 o v 40; 20 - z 0 Anal Caudal Dorsal Pectoral Fig-1.6 Number of mucous cells in four different regions of the integument of catfish H. fossil/s. Each bar represents mean ± SEM for five fish, **'kP<0.001 (significance calculated with reference to anal fin region versus rest other regions). 20 (iii) I- - /~ \ • • MU COUS • • , Mucous ., cells ~ ~ • • _I • '• cells, z - •~ '.` ;• = •A 5 - ` /Mucous-' . Mucqu• • cells ' ' .• '-• •fie{{s .. `••., -i '. * ' •: Fig-1.7 Mucous cells abundance in four different gills of catfish H. fossilis taken from anterior to posterior side shown in panels A-D respectively. 45 C5 1 40 NS NS T =~ 35 I NS 30 w 25 I 20 - 6 15 10 E z 5 0 Gill-1 Gill-2 Gill-3 Gill-4 Fig-1.8 Number of mucous cells in four different gills of catfish H. foss//is taken from the antero-posterior side. Each bar represents mean ± SEM for five fish. Significance calculated with reference to gill-1. NS = Not Significant. 20 (iv) Mucous Mucous '►mowcell cells . • 1 - SOx A 50x B *\ S r cells cellsv I ~. ~ fir• ~. • 50x ' • ! 0 50x D Fig-1.9 Mucous cells abundance in catfish C. batrachus integument, sampled from below the anal, caudal, dorsal and pectoral fins and represented by panels A-D respectively. 25 , 0 *** 20 15 w f~' C L ~ 5 NS E Z _ 0 Anal Caudal Dorsal Pectoral Fig-1.10 Number of Mucous cells in four different regions of the integument of catfish C. batrachus. Each bar represents mean ± SEM for five fish, *P<0.05, ***P<0.001, NS= Not Significant (significance calculated with reference to caudal fin region versus rest of the regions). 20 (v) Mucous - ft cells - ~~ Mucous cells 50hx - A F . Mucous cells .. Mucous . -~ • cells 50x .'. 0 50x.. Fig-1.11 Mucous cells abundance in four different gills of catfish C. batrachus taken from anterior to posterior side shown in panels A-D respectively. is NS 16 14- NS NS 12 - W 10 Cf g - 3 O 6- s- 4 0 6- E 3 0 2 Gill I Gill 2 Gill 3 Gill 4 Fig-1.12 Number of Mucous cells in four different gills of catfish C. batrachus taken from the antero-posterior side. Each bar represents mean ± SEM for five fish. Significance calculated with reference to gill-1. NS = Not Significant. 20 (vi) encounter if even it is not necessarily mediated by stress hormones. For example, the exposure to pollutants may be stressful but the tissue damage due to toxicity may not he mediated or caused due to failure of physiological adaptation. The fish and other aquatic vertebrates are subjected to greater variety of stressurs compared to their terrestrial counterparts. This is because they are more intimately dependent on the ambient environment and the changes in aquatic environment like salinity, pIl, alkalinity, dissolved solids, water borne toxicants and pathogens and nitrogenous waste accumulation will directly affect their homeostatic mechanism. Several excellent reviews are available on piscine stress physiology which include Pickering (1981), Gratzek and Reinert (1984), Barton (2002) and [wawa et at (2004). The response to stress in fish i.e. primary, secondary and tertiary is characterized by a cascade of three-tier responses depending on the severity and the duration of stressor exposure. Under the conditions of stress, the immediate primary response is based on two major axes i.e. brain-sympathetic-chromaffrn cell axis and brain-pituitary internal (HPI) axis, resulting in the release of cateeholamines (epinephrine and nor-epinephrine) and corticosteroids principally cortisol. Second tier responses are a series of interconnected metabolic and physiological changes at blood and tissue levels, including mobilization of energy substrates to yield energy to cope with stress. This entire metnhnlic pathway produces a burst of energy to prepare fish to overcome and meet energy demand. This is simultaneously facilitated by cardiovascular system by on hancing heart rate, gill blood flow and metabolic rule as well as decrease in plasma chloride, sodium and potassium ions (Portz eta] 2006). Hence, stress end point chemical indicators of fast tier response are catecholamine and cortisol and second tier indicator is chiefly blood glucose though plasma lactate levels increase have also served stress indicator in the fish subjected to hypoxia (Thomes et al 1999). Tertiary level response is manifested at the level of whole organism andl or population in the form of inhibition of growth, reduced reproductive capacity and altered behaviour etc. Davis (2006) has categorized stressors of fish into acute (short-term) and chronic (long- term) stressors. Acute stressors may include handling, confinement, abrupt changes in water quality and improper acclimation whereas chronic stressors include extended period of poor water quality, improper diets and stocking density which might ultimately lead to mortality. Accurate assessment of stress response is strongly dependent on establishment of resting or basal levels of physiological parameters designed to define the stress response. Barton (2002) has observed that response to a stressor is a dynamic and time- course dependent process which may provide the stress quantum only of that `instant stage' analogous to a 'snap shot' at that point of time. One may catch the peak level if one continuously monitors the values from an initial perception of stressor by CNS, reaching peak and ultimately reverting back to basal levels. Further, each stress indicator may show varying degree of response in the same species when compared with another stress indicator since various genetic, developmental and environmental factor may have modifying effect. 21 In experimental research involving live animals, it is important to recognize the different procedure steps which may be stressful to the experimental animal and may confound the results of experiment. Failure of the adherence to such practice may give rise to erroneous conclusions which may be misleading to future researches. The stress associated with physical capture and handling is often overlooked. However, the handling of the live fish with nets is almost inevitable during experimentation and will definitely alter the normal physiological response of the !isles. There is no substantial data available on the quantification of physiological response followed by handling during experimentation which may also culminate into liistopatbological changes. But the data on the capturing and handling prior to transport of fishes which may be the major cause of mortality is extensive (Grutter and Pankhust 2000, Barton 2002, Biswas et at 2006 and Harmon 2009) In literature, we have not come across any report in any other fish species where the effect of experimental handling on fish mucous cell population in skin and gill has been described even though these two target organs have been extensively studied with regard to mucous cell population and other related aspects. Most of the studies deal with the handling stress as a part of commercial netting, stocking and transport to the utilization site and the effects of such handling stress has been monitored at two levels. Broadly primary stress indicators i.e. plasma cortisol levels have been monitored in pre- and post stressed situations in Atlantic char Salvelinus alpinus (Lyytikainen et at 2002), brook trout Salvelinus fontlnalis (Benfey and Biron 2000), pallid sturgeon Scaphirhynchus alhaz.c (Barton et al 2000) and rainbow front Onchnrhynchur myki.rc (Renfey and Biron 2000). Similarly, plasma glucose levels as secondary indicator of stress have been documented in channel catfish Ictalurus purrctatus (Welker et at 2007), Eurasian pcn;h Perca Jluviatilu5 (Ientoft et al 2005), moe-turaI fish jundia Rhamdia quelen and nile tilapia Oreochromis niloticus (Barcellos et al 2011). An interesting phenomenon of habituation to the repeated stressor has been observed in juvenile rainbow trout (Barton et at 1997) which entails that much too frequent exposure to mild stressors result in desensitization of fish and attenuation to ncuroemdoerine and metabolic response. In the present study, we were seized of the fact that even a simple act of netting the catfish from the aquarium for sampling purpose may prove to be a stressful situation which might obscure the ultimate objective of the study. Our findings that there was total disappearance of mucous cells in both physically handled catfishes was indeed on somewhat expected lines. But what surprised us most was the acuteness of response which seems to hnve beer more accentuated since both these catfishes are devoid of scales on the skin surface and, therefore, were in direct contact with the handling implement i.e. the nylon net which was used to take out these fishes from aquarium during the experimental sampling. However, the possible underlying mechanism which brings about such complete disappearance of mucous cells following handling stress in both catfishes has not been investigated because it is not under the focal theme of the present study. Based on our long experience of having worked with this fish, we found both these catfishes are extremely hardy and quite capable of withstanding 22 simultaneously performed surgical manipulations like hypophysectomy, gonadectomy and pinealectomy on the same individual with almost negligible mortality despita such heavy surgical trauma. Also, we have established conclusively that the repeated handling stress caused in the present protocol does not activate HPI axis since plasma cortisol levels do not show any significant increase in H. foss ills (Slierwani and Parwez 2000). What, then, is causing the complete disappearance of skin mucous cells is an interesting proposition for further investigation. Another observation in the present study is that gill mucous cells seem unaffected by the same handling stress which brings about the complete disappearance of skin mucous cells. To the best of our knowledge, there is no other report in literature on any fish species where such varied response to the same stimulus and in the same species has been observed. At this point, it is believed that the reason of variations in such a response could be physical rather than metabolic. Hence, the skin of both these catfishes which are subjected to touch stimuli have responded by sudden and complete exudation of mucus and the ultimate loss of its mucous cells. However, since gills are internalized by opercular covering and hence, quite unlike skin, not in direct physical touch with the implement of netting, have shown the normal distribution of mucous cells all over gill epithelium even following the handling stress. The observation in the present study that anaesthesia MS222 (Tricainemethane sulphonate) provide the complete resting I basal level conditions as reflected in the normal histopathology of skin mucous cells and obviate stress-induced changes is interesting. Anaesthesia has been frequently applied in aquaculture and live animals experimentation to subdue neuromotor control. However, a number of parameters need to be considered in the choice of appropriate anaesthetic for fish. The anaesthetic to be used must have short induction and recovering lime, be harmless to user and environment, should show limited side effects and be economical to use. Another primary feature should be that it should mitigate the stress response and reduce or block HPI axis associated with handling stressors. Ortuno ei at (2002) tested four anaesthetics i.e. MS222, benzocaine, 2-phenoxytehanol and quinaldine in gilthead sea bream on basal glucose levels and concluded that MS222 was most suitable. Many other studies have concluded that MS222 was quite successful in both safety and efficacy. Another fish anaesthetic, clove oil, which is the herbaceous distillate of clove oil tree, also widely used in human dentistry, has been used in variety of fish species (Maricchiolo and Genovese 2011). The choice of the dose of anaesthetic is also critical and it should only the dose of sedation and not an overdose which may be detrimental. Hence, among wide array anaesthetics being used in fish, MS222 is the most preferred and is the only anaesthetic approved by the US Food and Drug Administration Centre f'or veterinary Medicine for use on food fish. Based an the foregoing information, it is evident that our choice of MS222 on the catfish to obtain baseline data is quite appropriate. Another significant observation of the present study is the highly significant region- based variations in the density of integumentary mucous cells in both catfishes. Interestingly, while the profiles of region based variations are different in both catfishes ~ut the highest abundance of mucous cells in both these catfishes is recorded in the same 23 region i.e. below the dorsal fin. Further, the degree of inter-region variations is more marked in C. batrachus which has recorded a near total absence of mucous cells in the caudal region (See fig. 1.10) whereas in H. fessilis these cells are present in good noticeable number even in the anal region which recorded the minimum population of these cells in H. fossilis (See Fig. 1.6). Bullock and Roberts (1974) have observed that density of mucous cells varies greatly in different fish species, region on the body and stage of growth. Some studies have demonstrated that density of mucous cells varies by sex and season as found in brown trout Salmo trutta (Pickering 1974, 1977). In a recent study on Japanese flounder Paralicmthys olivaceous, Yamamoto et al (2011) have studied the distribution of mucous cells on the body surface at six ocular and the corresponding six blind-side regions. The quantification of mucous cells number and size revealed that ocular-side of P. olivaceous had high number and larger cells than blind side and lower jaw skin of blind-side had very low numbers and the sizes of these cells (Yamamoto et al 2011). The results of this study clearly suggest that ocular side secretes more mucus than the blind side, is more exposed to water than blind side and the greater abundance of mucus on ocular side, which may be removed by water flow, may be advantageous for the fish. Low mucous density in lower jaw is explained by the elimination of slipperiness caused by excessive mucus secretion which may help to capture the prey effectively (Yamamoto et al 2011). In the present study, the reported observations in literature that there is a species — specific profile of mucous cell population is further strengthened by the present observation since the distributional profile in both these catfishes, though closely related phylogenetically, is quite different from each other. The possible reasons of the maximum abundance of mucous cells in and around dorsal fin region in both the catfishes could possibly be due to its protective strategy against predators. In both these catfishes, external body contours and undulating swimming movements are more or less identical and dorsal region below the pectoral fin is not as much subjected to gyrating body movements as the other parts of the body and hence may serve as the ideal site for the predator for grasping. Abundant mucus covering over this region due to greater number of mucous cells may well be an adaptational response against such adversities. The total lack distributional variations in the abundance of mucous cells populations in different gills located antero-posteriorly in both catfishes is exactly the same as observed in handling response to gills in both these catfishes. At this point, we may not be able to offer any other plausible cxptanation for such it lack of distributional variation in gills except to reiterate the same logic of the internalized existence of gills unlike integument and complete lack of any chance of tactile stimulus acting directly on the gill surface. In conclusion, it may be stated that the findings of the present study have demonstrated comprehensively that the handling stress brings about the total disappearance of raucous cells in the skin but not in gills and that there is a region-based distributional variation of mucous cells in the integument but not the gills of both catfishes. These findings may be of practical significance to those involved in the study of fish integumentary biology and fish toxicology using histology and histopathology as the end markers. 24 Chapter II: Integument Chapter-II (Integument) I. Introduction: Skin or Integument provides an all-round covering over the body surface and serves as the major interface between the animal and its environment. It is a multifunctional organ system which shows considerable variations depending on species, the specific region of the body and the type of environment inhabited by the animals. Amongst the broad functions associated with skin, the primary function is that of protection of (a) underlying organs against mechanical abrasion, (b) invasion of pathogen, (c) warding off the predaceous enemies by the presence of scales, (d) release of venom to ''kill the predator, (e) colour camouflaging to avoid being spotted and (f) release of fright pheromones to alert other or con-species. The other functions are thermoregulation particularly through sweat glands in mammals, cutaneous respiration in amphibians and larval fishes, friction-free locomotion facilitated by the slippery mucus covering and sensory perception due to the presence of taste buds, receptors for touch, pressure and temperature. Most of these integumentary functions can also be ascribed to fish integument in addition to the function of ion-regulation due to the presence of ionocytes in some teleost fishes. Further, the presence of bioluminescent organs or photophores in many mid water or bottom dwelling deep sea fishes or the multiple photophores spread over the body surface may serve the variety of functions of colour camouflage for protection, to illuminate dark waters or to attract the prey. The slippery mucus over the fish integument may aid friction-free locomotion at a great speed with less expenditure of energy. Some studies have also suggested that fish skin is an important site for ammonia excretion particularly in marine tcleosts. In short, fish integument performs majority of the functions associated with general vertebrate skin in addition to some of its own specialized functions due to its habitat need. A. Typical Vertebrate Integument: Based on the generalized pattern, it consists of three broad regions. An outer Epidermis derived from ectoderm, the next underlying layer Dermis or Corium derived from mesoderm which is followed by the third, the Subcutaneous Tissue which may also have a layer of fat called panniculus adiposus or subcutis being made up of several inches of thickness in case of obese individuals. In tetrapods, there may be a periodical shedding or sloughing of the epidermis which may either be partial or complete. The relevant details of each laver are as follows: 1. Epidermis: The epidermis of a typical vertebrate skin may have three distinct cellular regions which may either be single or multilaycred. These are the innermost stratum germinativum or malpighian layer resting on basement membrane, the middle multicellular transitional 25 laver and the outermost stratum corneum. In some cases, particularly in the skin of sole and palm of humans, the transitional layer has the additional layer of stratum granulosum and stratum lucidum. The stratum germinativum derives nourishment from the blood vessels of underlying dermis, divides continually to form new cells which are pushed upward to form multiIayered transitional layers. In most of the vertebrates, the cells of stratum germinativum are columnar whereas those in the transitional layer get gradually flattened and are of stratified squamous epithelial type. The outermost layer is made of flattened cells which have lost their nuclei, are dead and comified hence called stratum comeum wttich undergoes regular sloughing in many vertebrate groups. However, this may not he the universal feature in all the vertebrates. Majority of the skin derivatives such as feathers, Lair, horns, nail, claws and antlers are exclusively epidermal in origin. Some scales are epidermal whereas others are dermal in origin. Teeth are derived both from epidermis (enamel portion) and dermis (dentine). The epidermis may also have colour pigments which may either be contained in certain specialized pigment containing cells called ehromatophores such as in fishes, amphibians and reptiles or these pigments may be dispersedin the molpighian layer of the epidermis and the degree of pigmentation determines the skin colour. Interestingly, such vertebrate groups which are provided with fur, feather and scales, the pigmentation may be provided to these skin derivatives. The epidermis may also have soft integumentary derivatives in the form of glandular structures such as various kinds of glands e.g., mucous cellslgoblet glands, sacciform cells, glandular club cells etc, each with a specific secretion and specialized function. In addition, it may also have sensory structures such as taste buds and other sensory organs for perception of various kinds of stimuli. 2. Dermis: The dermis commonly has an outer vascular stratum spongiosum and a deeper thicker stratum compactum which ate merged with each other. The entire dermal layer is provided with an extensive network of loose connective tissue particularly collagen fibers which contains abundance of blood and lymph vessels, nerve endings, sense organs, and some assorted dermal derivatives. 3. Sabeutis: It is the last part of the integument which anchors with dermal collagenous fibers on one side and the underlying muscles- below. It is primarily made up of adipose cells with sporadic network of blood vessels, nerve fibers and occasionally chromatophores. B. A Brief Comparative Account Of Integument In Different Animal Groups: (i) Invertebrates: The invertebrate integument referred to as hypodcrmis does not correspond to the typical vertebrate integument organization. It has an outer noacellular layer called cuticle which is made up of chitin or calcareous material. This layer may undergo periodical sloughing called ecdysis. Below cuticular layer 26 lies a single layer of columnar cells called hypodermis which is essentially responsible for the secretion of chitin of the cuticular layer. Hence, in broad terms, the cuticular layer may be analogous to Stamm corneum though the latter is cellular and hypodermis may be equivalent to a single layer of stratum germinativum. (ii) The Amphioxus: Its integument partly resembles the invertebrates in as much as it has columnar epithelial cells secreting noncellular cuticle. But in addition, like typical vertebrates, it is made up of some sort of soft connective tissue forming dermis which, however, is without pigment. (iii) The cyclostomes: The integument has more advanced features with multilayered epidermis provided at outer surface with stratum corneum which, unlike most other vertebrates, are nucleated and living and secrete cuticular covering. The dermis is like that of typical vertebrates with extensive network of connective tissue, blood vessels, nerves, smooth muscles and chromataphores. (iv) The fishes: Here the integument is same as in cyclostomes with no dead corneal layer. The presence of keratin over the epidermis is reported in some fishes but the outer layer cells are net dead. In certain teLcosts, during breeding seasons certain comified areas in the form of pearl organ are visible. The detailed account of lish integument is described in the later part of this chapter. Fish integument resembles with the typical mammalian integument in as much as it has well-defined epidermis and dermis and the latter shows the distinct regions of stratum spongiosum and stratum compactunt. However, the epidermal layer shows the difference for not having the typical stratum gorminativum but all epidermis seems metabolically active. Further, fish scales, unlike reptiles, are dermal in origin which may project out through the epidermis. (v) Amphibians: The integumentary structure is typically that of vertebrates and is different from the lower groups to have dead stratum comeum which is more pronounced in the amphibians adapted to terrestrial life. Since amphibian skin also performs respiratory function, its dermis is richly supplied with large number of blood vessels. (vi) Reptiles; Due to the mostly dry habitat occupied by this group of animals, the reptilian skin is provided with the well-developed stratum comeum which is periodically shed off. The reptilian scales, unlike those in fishes, are derived from stratum comeum and are shed along with the outer layer. (vii) Birds: The body of the birds are mostly covered with feathers except in the region of beak, legs and feel. The feathers are the derivatives of stratum comeum. The bird skin which is normally thin becomes thick in the exposed region of legs and feet and may also be provided with protective scales. The extraordinary feature of bird skin is the abundance of muscle fibers to control the movement of feathers. The coloration 27 of the bird is generally provided by the pigment present in the feathers and scales which is not confined within a chromatophore. (viii) The mammalian integument has already been dcscribed in detail under the heading typical integumentary structure. C. Integumentary Derivatives: Certain structures which are derived from skin proper i.e. epidermis and dermis are called skin or integumentary derivatives which, based on their structural consistency, could be either soft or hard derivatives. The following are some of the generalized features of these integumentary derivatives. (i) In terms of their origin, an overwhelming number of these derivatives originate from the epidermis and very few such as fish scales originate from dermis. (ii) Some of these derivatives such as scales etc. may have wider occurrence in different vertebrate groups such as fish, reptiles, birds and mammals whereas others are group-specific such as feathers in birds and hair in mammals. (iii) Some of these derivatives may originate from both epidermis and dermis depending upon the group. For example, reptilian scales are epidermal whereas fish scales are dermal in origin. (iv) In certain instances, the same integumentary derivative may have dual origin i.e. partly dermal and partly epidermal such as in the case of teeth where enamel portion is derived from epidermis and dentine part from the dermis. (v) Various integumentary glands such as mucous, poison, sweat and sebaceous glands are basically soft and other structures e.g. scales, feathers, spines, horns, hoofs, antlers etc, are classified as hard integumentary derivatives. A brief description of a variety of integumentary derivatives in different groups of animals is given below: (i) Cyclostomes: There are no hard integumentary derivatives in cyclostomes. However, among soft derivatives are only unicellular mucous glands. Hagfishes have peculiar thread cells in epidermis projecting spirally coiled fine threads which entangle with mucus secretion tbrming protective slimy coat. In some cases, other unicellular mesocrine glands such as granular gland cells in lamprey and beaker cells have been described. (ii) Fishes The glandular derivatives include unicellular and multicellular glands which are as follows. OR. 1. Unicellular and multicellular mucous glands 2. Multiecllular poison gland 3. Luminescent photophores 4. Pterygopodialgland Both types of mucous glands- unicellular and multicellular are of simple saccular type. The mucus secreted in lungfishes such as South American lungfish, Lepidosiren and African lungfish, Protopterus fortes a dried-up protective cocoon during aestivation. The multicellular poison gland, associated with hollow groove on spines on the fin, mil and gill cover are analogous to hypodermic syringe, protect, from enemies. In one species, Synanceia from the Indian Ocean, the poison gland is particularly well-developed In some deep-sea dwelling elasmobranch and teleosts, an extraordinary light producing luminous gland called photophores is found which help these fishes to attract their prey. The structure of this gland has been studied in detail in Poriehthys in which the gland is shown to consist of a lens in the front and a pigmented reflector behind. In certain species of skates and rays, a special pterygopddial gland located on the clasper organ with little known function has been reported. Hard derivatives (scales: Among the hard derivatives, the most distinctive derivative in fishes are scales which may not be present in variety of fishes such as catfishes, eel etc. These scales predominantly develop from detmis though in few others, they develop from both layers of skin. Such scales are of four types. The placoid found in clasmcbranch are most primitive type. The ganoid have shiny coating of ganoin found in gar, the cosmoid and cycloid scales occur in many teleost fishes. In addition, fm rays which are dermal In origin found both in elasmobmnchs and teleosts are yet another hard derivatives in fishes. The modification of scales is seen in some fishes such as heavy fin spines in Squalas acanthias, the sting of Dasyatis and the heavy teeth on the rostrum of sawfish Prisfis. It is interesting to note that there has been a general trend in tetrapods to shift from dermal to epidermal origin of scale as has been seen in reptiles, birds and certain mammals. (iii) Amphibians: Glandular derivatives (Multicellular mucous & granular ;hands) There are abundant flask shaped multicellular simple alveolar mucous glands and granular glands or poison glands both opening at the surface through duct. The granular glands have granular cytoplasm and the secretion is like a milky fluid which in some instances is toxic to the predators. The granular glands are not as numerous as mucous glands and are located at the warts of the toad. The skin of the 29 living amphibians is smooth and lacks scales except in few toads and burrowing limbless caecilians. Hard derivatives (scales): (no had derivatives reported in amphibians) (iv) Reptiles, Soft derivatives : (Scent gland- only in few groups) Due to the terrestrial mode of life which gives rise to scaly and dried skin, the integument of the reptiles is virtually devoid of any gland. In only exceptional cases where these glands are present, these are basically for reproductive purposes and are called scent gland. The examples are two pairs in crocodile, one on the throat and the other on lower jaw. Some snakes may have these glands in cloaca) region and are for defense purpose. In certain turtle, these glands namely musk gland present in the lower jaw and below carapace may also he used for attracting opposite sex. Hard derivatives : (scales-both dermal & epidermal) The only hard integumentary derivatives in the reptilian skin are scales. However, there seems to be inconsistencies itt the literature regarding their origin but the overwhelming opinion is in favour of their exclusive epidermal origin. The reptilian scales are broadly put in two categories. One is represented by snakes and lizards in which these scales project backward and overlap the scale behind. Such scales exhibit complete periodic shedding, ecdysis and new set of scales is formed beneath the older ones. The other type of scales are represented by the turtles, crocodiles and alligators whete large scale cover the carapace and plastron. Beneath these scales lie the bony plates which may not conform to the pattern of scales. These scales may appear in the form of concentric rings indicating the number of growth period. These scales may get worn off and shed and new scales develop. In crocodiles, these scales on lateral, vertical and tail region may have a pit like depression which may indicate the location of sensory capsule below. Crocodilian scales do not exhibit ecdysis. In literature, dermal scales have been reported in some lizards, snakes and turtles. Modifications of epidermal scales have been observed in lizards & snakes such as the so-called horns of the homed lizard and the rattles of the rattle snake. (vi) Birds: Glandular derivatives: (Uropygial gland) The status of the glands in birds is somewhat similar to those of the reptiles siucc it does not require keeping the surface moist due to feather covering over the body. However, one gland, so named due to its location at the tail region, the uropygial is bibbed, has separate opening and secretes oil which is taken on beak or bill and is used to preen the feathers. This is to make them impervious and hence this gland is predominantly found in aquatic birds. 30 Hard derivatives: (Feathers, epidermal scales) The characteristic hard integumentary derivatives in birds are feathers which are exclusive to this group, epidermal in origin and are considered modified reptilian scales. Three main types of these feathers are classified on the basis of structure and distribution. These are hair feathers (filoplume), down feathers (plumulae) and contour feathers (plume). The beautiful colour of the feathers is due to specific pigment granules deposited during their development and the physical phenomenon such as refraction and interference of light rays. Replacement of feathers may occur throughout the life of the bird which may either be gradual throughout the year or seasonal called moulting. There may be presence of scales on the naked region of birds i.e. legs and toes. In some gallinaceous birds, a bony projection of the tarsometatarsus which is covered with horny scales, used for fighting, may be present. The web on the feet of aquatic birds i.e. geese, ducks and swans may also have the presence of scales. (vii) Mammals: Glandular derivatives (Mammary, Sweat, Sebaceous (Meibomian/Tarsa] glands to eyelids, Small glands of Zeis to eyelids, Scent glands, Modified sweat glands, Ceruminous or Wax producing gland, Glands of moll in the eyelids and Circumanal glands). Mammals have very extensive distribution of glands all over the body and some of them such as mammary, sweat and sebaceous glands are characteristic of the class mammals. However, some of the glands may be selectively absent in certain animals and some in certain part of the body. The broad features of these glands are as follows. Mammary gland: Characteristic to the class mammals, compound tubular or saccular in structure and apocrine in the nature, secretion in the form of milk for nutrition of young ones and restricted to specific region to the body. In Prototheria (e.g Platypus), these glands are compound tubular, without nipples and open directly on the skin surface. In Metatheria and Eutheria, the glands are compound saccular provided with nipples I teats which are always paired ranging from I to 11 pairs and proportional to the number of young ones to be delivered at birth. Interestingly, the nipples and the mammary tissue may be present in both sexes but they are functional only in lactating females although in some exceptional cases, the functional mammary glands have also been reported in males. Sweat glands: Also called sudoriparous glands present in majority of the mammals except few such as moles, cetaceans (aquatic mammal), scaly anteater, two-toed sloth. These glands are mostly distributed all over the body except the regions like lips, glans penis and nail bed. In some animals, they are confined to of specific areas such as in mice, rats and cats they are present in the underside of the paws, in rabbits around lips and in sheep, goats, cattle, pigs and dogs around the muzzle (snout 31 regien). The main function of these glands is temperature regulation through evaporation of sweat and excretion of metabolic waste. Sebaceous or oil glands Distributed all over except the palm and sole and mostly open into the hair follicle through the duct. Those not connected with hair follicle are present on corner of the mouth, lips, glans penis, labia minors and manunary papillae. The sebaceous gland connected with the eyelids are termed Meibomian gland where the secreted oily substance forms a laver or film between eyelid and eyeball. Some smaller glands with the same function are named as glands of Zeis. The main function of These glands is to keep the hair and skin soft and smooth. Scent Glands: They are either modified sweat or sebaceous gland and their distribution may vary considerably in different group of animals. Their secretion may either be attracting the same sex, or opposite sex or may serve to repel or frighten the predator. Modified Sweat elands: Ceruminous or wax-producing gland present in the external ear passages, structurally coiled hence resembling the sweat gland and secrete fatty wax-like substance called ear-wax. Similarly gland of Moll in the eyelid and ceruminous gland around the anal opening are modified sweat gland. Hard Derivatives: 1. Halts (Guard hair, under fur, long hair) 2. Head Projections (True horns, Antlers, Rhinoceros horns) 3. Foot Projections (Claws, Nails, Hooves) 4. Scales (Armadillo shell, Scales of Pangolin) 5. Baleen plates (Whales). Hair: Characteristic of the class mammalia and epidermal in origin. In some mammals, the whole integument may be covered with hair while in others such as whales it may be simply restticted to the snout region. The hair colour is primarily due to the pigment present therein. in some animals, there is periodic shedding of hair. Head Projection: These are mostly in the form of horns which are found only in class rnanuealia with the exception of one reptilian example of `horned toad" native to United States. Four different variations of these horns i.e. hollow horns in cattle, keratin fibres horns in rhinoceros, pronghorn in antelopes and antlers in deer family have been documented which serve as formidable weapon to fight with the adversary. Foot Projections: Claws, nails and hoofs are present in the distal end of the digits and grow parallel to the skin and are built upon the same basic plan. The claws in the cat family may be retractile and can be withdrawn back in the sheath when not in use The group of mammals possessing hoofs are called ungulates. 32 Scales: They may be present in some mammals and are mostly confined to tail and paw. However, in certain mammals, they extensively cover the body such as in scaly anteater where they are present all over the body except the ventral region. In armadillos scales fuse to form large plates over head, shoulder and ring-like band around mid-body region. D. Fish Integument: Fish integument, like other vertebrates, also consists of the three classical layers i.e. epidermis, dermis and hypodermis or subcutis below which lies the attached muscles. Essentially, epidermis is made up of epithelial cells, some of them may be secretory, dermis consists of collagen fibers and hypodermis is constituted by fat or adipose cells. However, there are some fundamental differences in the fish integument when compared to other vertebrate groups. 1. Epidermis: "three distinct regions i.e. upper, middle and lower can be identified on the basis of cell size, structure and function. In the mammalian system, the cells of the basal layer of the epidermis are columnar, actively dividing mitotically and pushing the cells upward forming a progressively flattening layer of cells. In teleost, on the contrary, all the cells in the epidermis are metabolically active and dividing and contribute to the overall growth of the epidermis even though the basal layer cells are more active. While in terrestrial vertebrates, the outermost layer is made up of dead, horny cells containing keratin, no such layer of dead cells occurs in fishes in general though few exceptions have been reported. Similarly, the term `cuticle' often referred to the top most layer in some fishes has been reported to be made up of thick mucus layer. Hence, even though the lower most layer of fish epidermis has often been referred to as `stratum germinativum' but it does not conform to the structural characters of this typical layer in mammals. Similarly, no distinct transitional layer of progressively reducing thickness showing typical stratum granulosum and stratum lucidum and uppermost stratum comeum is distinguishable in fish epidermis. Under the scanning electron microscope, the surface of the epithelium is characterized by the appearance of concentric ring like structures which often form finger print like pattern. These tnicroridges or micropapillae are attributed to perform mechanical protection or in holding the mucus secretion over the surface layer. The secretory cells in fish epidermis include goblet cells, sacciform cells, granular cells, club cells, merkel cells, venom cells and some non-secretory but functionally distinct cells such as ionocytes, intrusive cells, rodlet cells, luminescent organs may also he present. However, only some but not all, of these cells may be distributed in different fish species. Some workers have described the presence of lymphatic spaces which are located in the basal layer of epidermis and are characterized by the occurrence 1-2 lymphocytes which have deeply stained nuclei and narrow rim of cytoplasm. It has been shown that these lymphatic spaces show enlargement under pathological conditions and axe supposedly associated with protection against microorganisms. In certain instances, the specialized sensory organs such as taste buds more clearly seen if stained with Masson trichrome are also visible. Similarly, some other structures in the form of dermal invagination called pit organs are also occasionally observed. 2. Basement Membrane / Basal lamina: The last layer of epidermis finally rests on a basement membrane which separates epidermis from dermis. It is stained positive with PAS and acts as a filtration barrier and attachment site for epidermal epithelia] cells. 3. Dermis: In fishes, the dermis, like other typical vertebrates, broadly consists of two layers- the upper stratum spongiosuml laxum and the lower stratum compactum. However, in certain fishes this demarcation may not be clearly visible. Both these layers are made up of collagenous connective tissues with interspersed blood and lymph vessels, nerve ending, sense organs and assortment of epidermal and dermal derivatives. The upper spengiosum is so named due to loosely arranged connective tissues whereas the compactum consists of densely packed connective tissue fibers which give compact appearance to this layer. There are some vertically arranged strands present at regular intervals in this compactum layer which runs all along up to the end of this layer and are thought to provide reinforcement to this layer. An additional single layer called dermal epithelium consisting of modified fibrocytes located at the depth of stratum compactum has been reported by some workers which is presumed to function as regulatory barrier between stratum compactum and hypodermis. Below the basement membrane and above stratum spongioswn lies the distinct layer of chromatophores which imparts integumentary coloration to the fish. The thickness of the dermis varies from fish to fish and region to region including the variations in the width ratio of stratum cornpacmm and spongiosnm. 4. Hypodermic I Subeufis: it is regarded as the structural part of the integument proper which lies below dermis and above the body musculature. It is often provided with a deep chromatophore layer above and sporadically occurring blood vessels and nerve ending enclosed in perineurium. It is richly provided with Large number of adipose cells which allow considerable flexibility to the integument. Some workers have described the presence of myoseptum which differentiates this layer from underlying musculature. E. Secretory Cells in Fish Integumentary Epithelium: The description of secretary cells in the fish integument has given rise to great confusion particularly due to multiplicity of nomenclature of the same cell type. Ilene, mucous cells have also been called goblet cells, sacciform cells have been named granular cells, clear mucous cells, white cells etc. Similarly, club cells are also known by Giant cells, Leyding cells, goblet cells etc. This confusion further deepens when different investigators describe these cells with multiple names as distinct entities. Another interesting aspect is that each species may have only some of the secretory cells and not 34 all thus showing their restricted distribution in different species. A brief overview of the various types of secretary cells in fish integumentary epithelium highlighting their distribution, shape and function has been given in the following text. Broadly, these secretory cells may be classified into two categories one which includes those glands which are more widely distributed in different taxa and groups, such as goblet cells, club cells, saccifonn cells, ionocytes, market cells, epithelial cells and intrusive cells. The second category of the secretory structures are restricted only to specific taxa such as thread cells (only in hagfish), venom cells (spine-containing fishes) Luminescent organ (deep marine fishes) etc. a. Goblet cells / Mucous cells / Mucous glands These are most widely occurring glands/cells among fishes present in variety of organs principally, integument, gills and GI tract but have been reported to be absent in Polydon (Weisel 1957 and mudskipper Periophthalnrus (Suzuki 1992 and Park et at 2004). Structurally, these are either unicellular or in some instances multicellular, round to flask shaped, have basally placed nucleus and secrete mucus which is basophilic but remains unstained with routine histological stains such as hematoxylin-eosin (Blackstock and Pickering 1980). Ln integument, these are present either at the superficial or middle layer of the epidermis (Bullock and Roberts 1974). Since the mucus contains glycoprotein moiety the mucous cells are stained magenta with PAS and blue with Alcian blue indicating the presence of neutral and acidic glycoprolein. The varying shades of purple suggest the presence of mixture of neutral and acidic glycoproteins. It has been suggested that two types of goblet cells termed mucous goblet cells and serous goblet cells exist, the latter unlike the farmer exhibit acidoplulic staining and have been reported to occur in variety of fish species Like Salmonids, Uoby and Coelacanth (Whitear 1986). Their cytoplasm is made up of large sized granules and number of this type of cells increases with ectoparasite infestation. However, the occurrence of serous goblet cells is less common than mucous goblet cells and they are observed far less in number when both cell types are present together. The population or the number of mucous cells is highly variable which may include seasonal, temperature and reproductive stage or toxicity induced variations (Wilkinsand Jahesar 1979, Burton and Fletcher 1983, Quinious et al 1998. Zuchelowski at at 1981, Burton at at 1985, Tger et at 1988 and Berntssenl et al 1997). J. Sacciform Cells / Granular cells / white cells These cells have restricted distribution and have been found to occur iu cartilaginous fishes such as rays. Among telcosts, they are confined to gadiformes cods, catfish of the genus Corydoras and some salmonids (Mittal et at 1981, Pickering and Fletcher 1987, Fast et at 2002b and Sadovy at al 2005). The are, however, absent in jawless fishes and dipnoans (Whitear 1986). Under light microscopy, it often becomes difficult to distinguish these cells and is generally 35 characterized by clear to finely granular cytoplasm, basally located nuclei and predominantly proteinaceous secretion (Whitear 1986). in literature, these cells have often been referred by other names such as granular cells (Henrickson and Matoltsy 1968 and Sato 1978), white cells (Nisliioka of al 1985), clear mucous cells (Whitear 197] and Phrornsuthirak 1977). It is believed that these cells secrete `noxious substance' which repel or poison the infecting microorganism and predators (Elliott 2000). c. Club Cells Club cells, like many other integumentary cells, are also not universally distributed in all fishes and are predominantly confined to the fishes of superorder ostariophysi. This largely includes families like cypriniformes, siluriformes etc. which put altogether, cover nearly one quarter of known fish species and three- fourth of all freshwater species (Whitear 1986, Smith 1992 and Moyle and Cech 1996). Structurally, these cells are large, mostly oval or club shaped and contain 1- 2 nuclei, multilayered and mostly located in the middle portion of epidermis (Park et al 2004). The cytoplasm of the cells is eosinophilic which appears shrunk during the fixation of these cells thus leaving a clear vacuolar space mostly at the apical end (Park 2002). These cells secrete alarm pheromones which serve as anti predator defensive reaction. This secretion may also serve healing function in the event of damaged epithelia (Mittal and Garg 1994 and Elliott 2002). d. lonucytes lonocytes most widely occur in gill and opercular epithelia but have also been reported in the integument of some euryhaline fishes such as killifish, Fundulus heteroclitus, Ragarius bagarius, hagfishes, lamprey (called polyvillous cells) and in elasmobranebs (Zadunaisky 1984, Peek and Youson 1979, Whitear and Lane 1983 and Wtulear and Moate 1998). Their characteristic structural features are the same classical characters such as large number of mitochondria and highly branched tubular system. Functionally, they perform ion-absorption in FW and ion-excretion in SW including maintenance of acid-base regulation (Evans 1999). e. Merkel Cells Merkel cells as secretory cells of integumentary epithelia are not so widely distributed amongst teleost but have been reported in carp and minnow Phoxinus phoxinus (Whitear 1989 and Zaccone et al 1994) and also in lamprey, dipnoans not only in skin but also in oral epithelium. These cells can be recognized by relatively small ratio of cytoplasm to nucleus, presence of dense cytoplasmic granules and peripheral finger-like projections. These cells have been attributed multifunctional role such as Lactilc reception and responsive to cadmium toxicity as reported in carps. f. Intrusive Cells As the name indicates, it is a group of diverse cells which initially do not originate from fish epidermis but are subsequently lodged there. These include (i) chromatophores, (ii) neuromast cells, (iii) lcucocytos (iv) macrophages (v) various types of granulocytes (vi) protozoan parasites and (vii) rodlet cells. While the origin and functions of these cells is quite evident and need not be commented upon here, the last of the intrusive cell i.e. the rodlet cells have given rise to considerable controversy during the last several decades sharply dividing opinions into two schools. It has been viewed by some that these cells are nothing but a spotozoan parasite Rhabdospora thelohani which has got embedded into fish epithelium (Mayberry et al 1986). This assumption is based on the premise that (a) it is irregularly occurring within fish species (b) its association with certain pathogenic conditions and (c) its prolonged survival beyond the host cells death. However, others believe that these cells are the integral part of the host cells and cite the evidence of (1) wide distribution of these cells in different fish species inhabiting varying temperature and salinities (ii) the presence of these cells in larval and very young fishes (iii) lack of host inflatrunatoty response (iv) presence of desmosomes and functional complexes between these and adjacent cells and more importantly (v) the similarity of the DNA profile between host and these cells. Interestingly, due to overwhelming evidences for and against such assumption, the debate has still not settled with any conclusive view, The distribution of these cells have been reported both in freshwater and marine teleost species exhibiting pear or flask-shaped appearance with displaced nuclei towards wide end and membrane-bound retractile rodlets (Elliott 2000). Functionally, these cells are suggested to be involved in osmoregulation, sensory reception, lubrication and non-specific stress response. g. Thread cells These cells are exclusively restricted in hagfish and are characterized by elongated oval shape basal nuclei, a cluster of central mass of fine granules and peripheral thread like component. Upon being irritated the hagfish discharges copious amount of slime from the thread cells (Downing et al 1984). h. Venom cells These are assemblage of venom cells forming an encapsulated venom gland containing secretary granules and usually located at the base of spine. The discharge of the venom takes place when these glands are pressed and mptured and the venom passes through the hollow canal of the spine into the wound which is made by the spine itself. i. Luminescent organs I Photophnres These organs are well-developed and confined mostly in deep-sea marine fishes. The source of this bioluminescence could he either due to the presence of luminous bacteria in the luminescent organ or the light generated by self-luminous cells of the fish. These luminescent organs may be epidermal in origin and are made of 37 modified epidermal cells or these cells coupled with the reflector organ remain embedded in the dermis. STATUS OF STUDIES RELATED TO INTEGUMENT OF THE TELEOSTS OF INDIAN SUBCONTINENT: The work done in the Indian laboratories an various aspects of integumentary system on the fish fauna of Indian subcontinent has been quite overwhelming particularly on the catfishes, air-breathing fishes and carps. The literature survey reveals that nearly twenty five different species have been investigated on ten different aspects of integumentary system. Amongst the important carps are Indian major carp Calla calla, Labeo rohita, Cirrhinus tnrigala and Labeo calhasu and minor carp Cirrhinus reba, the common carp Cyprinus carpio, the exotic species. The major studied catfish species include H fosselis, C. batrachus, Bagarius hagarius, Rita rata, dlystus vittatus in addition to other air- breathing fishes such as Channa punclalus, Amphipnous cuckia, Mast acambelus pancalus, peppered loach Lepidocephalichthys guntea. The Indian hill stream fish Garra lamta has also been extensively studied due to its adaptation to life in Indian hill streams which is characterized by the presence of specialized adhesive pad which helps the fish in stone clinging' and `stone licking' habits. Among other sporadically investigated teleosts species from the Indian labs are Tetraodon ,fuviarilis, Barbus sophor, Nolopleruc notopterus, Ompok pabda, Colisa fasiata, Macrognathus acnleatum and Glossogohius giuris. The earlier studies on integumentary structures of teleosts of Indian subcontinent were on general histological organization of integument which understandably is the very basis for studies on other aspects. The earliest study recorded in the literature is that of Islam (1951) who reported the histology of skin of certain teleost fishes. This followed more such studies during 60's on Carla calla. Labeo calbasu, Rita rita andCirrhina reba (Kapoor 1966, Singh and Kapoor 1967, Mittal 1968, and Singh and Kapoor 1968). Subsequent studies in next few decades were predominantly carried out by Mittal and his co-workers who explored almost all possible aspects of integumentary system. The other important species studied for general histological organization of skin are Bagarius bagarius (Mittal and Munshi 1970), Channa striata, Tetraodon fluviatilis, (Mittal and Banerjee 1975, 1976), H. fossilis (Mittal and Munshi 1971), Clarias batrachav (Banerjce and Mittal 1976) and Indian major carps i.e. C. calla, L. rohita and C. mrigala and common carp C. carpiu (Singly and Mittal 1990). Integument main function relates to protection which is primarily achieved by overlying mucus layer which is exuded by underlying mucous cells of epidermis. This mucus is chiefly made up of glycoproteins (GPs) of the various types which is the main chemical moiety in the secreted mucus. Hence, a lot of subsequent studies have focused on the qualitative and quantitative analysis of the GPs content in the integumentary epidermal mucous cells mostly following the histochemical approaches. The Indian fish species studied for GPs contents in mucous cells are Channa striata (Mittal and Banerjee 1975), Monopterus cuchia (Mittal and Agarwai 1976, Mittal et at 1980 and Mittal et at 1994a), Barbus 38 sophor (Mittal et a] 1976a), C. barrachus (Banerjee and Mittal 1976), C. carpio, L. rohita, C. mrigala and C. calla (Singh and Mittal 1990), Maslacembelus pancalus (Mittal or al 1994b),Intereslingly, there have been some studies on the GPs content in specific organs such as operculum, lip and buccal epithelium of the fish species which included Labeo rohita, Cirrhina mrigala (Agarwal and MittaL 1992 a,b), Channa striata (Agrawal and Mittal 1994), Garra lamta (Mittal ct at 2002 and Pinky at at 2007), Rita rila (Yashpal at at 2007) and Lepidocephalichihys guntea (Mittal et at 2004). Some investigators have also characterized the antibacterial properties of mucus in Rita rita, Channa punciatus and Cirrhina mrigala (Kuppulakshmi et a1 2008 and Kumari et al 2011), Ompok pabda (Rajonsamantmy et al 2009). Nigain et at (2012), analyzed innate hmnune parameters such as lysozymcs, proteases, alkaline phosphatase, acid phosphalase, esterase and sialic acid in C. mrigala, L. rohita, C. calla, R. rita and C. punctatus. A comparative study on inmate immune parameters in the epidermal mucus of various fish species was carried out by Subramanian et al (2007).There are good numbers of studies on the fish skin surface lipids in C. calla, C. batrachus, C striates (Mittal and Nigam 1986), H. fossilis (Mittal ct all9766) and Llystus viltalus (Saxena and Kulshreshtha 1980). There are few reports on the histochemical localization of some enzymes such as alkaline phosphatase and acid phosphatase (Rai and Mittal 1983, 1991). Even though keratinization of fish integument is not of common occurrence but it has been described in at least four different teleost fishes of Indian subcontinent which include Notopterus noloplerus (Mittal and Muushi 1974), Bagarius bagurius (Miltal and Baneijee 1974a, Mittal and Whitear 1979), Burbus sophor (Mittal et al 1976a) and Garra lamta (Pinky et at 2004). ULtrastmetural studies on teleost skin are extremely limited and mostly limited to scanning electron microscopy which include teleosts like Mastacembelus pancalus and Bagarius bagrarius (Mittal et al 1980. 1995). Mittal et al (1995) studied carbohydrates in the mucous cells of the epidermis of bfastarembelur pancalus by means of electron-microscopic cytochemical methods using physical dovelopment procedures. This is quite an infonna6vc technique and gives vatuablc information on the in situ (iP contents at high magnification. Surface architecture studies by SEM have also been carried out on skin associated structures such as lips and operculum in C. batrachus (Garg at al 1995), Garra Iumia (Pinky et al 2002 and Mittal et at 2010) and Lepidocephalickthys guntea (Mittal et al 2004). Another important aspect relates to good number of studies done on the response of fish skin to certain ambient toxicants. Some of the earlier reported studies on this aspect were by Rajan and Bancrjee (1991 a, b) on mercuric chloride toxicity on H Jossilis epidemtis, mucocytes of accessory respiratory organs and skin and the mapping of protein, nucleic acid and calcium in the epidermis. Later on, Paul and Burerjee (I 996a, 1997) studied ammonium sulphate induced histopathological changes in the operculum lining of H. fossilis and its toxicity quantification on the same fish using mucocyte indexing. The other documented reports are those of Chandra and Banerjee (2003) on inorganic zinc chloride toxicity on the skin of C. striata and Singh and Banerjee (2008) on the sodium arsenate toxicity on skin epidermis of C batrachus. Overall, the toxicity related studies have been done only on few species like H fassilis, C. batrachus and C. striaia and the effects of only limited toxicants such as mercuric chloride, ammonium sulphate, zinc chloride and sodium 39 arsenate on the skin and operculum have been studied in the Indian laboratories. However, considering the remarkable species diversity in Indian subcontinent coupled with indiscriminate and injudicious use of toxicants in several spheres particularly industrial houses, more such studies on the effect of commonly discharged toxic effluents on larger number of teleost species of Indian subcontinent are warranted. Ambient salinity fluctuations may also have profound effect on the skin histo-physiology but studies on this important aspect are very few and reported only in H. fossilis mucous cells changes in relation to salinity and hyperosmotie stress-induced changes in the epidermis of Barbus sophor (Agarwal et al 1979 ab). Similarly, chloride cells which are regarded important cellular entity in osmoregulation have been reported to occur in skin of freshwater teleost Channa striata (Banerjee and Mittal 1975). Recently, Rai or al (2012) have studied the chromatophores in the relation to the healing of skin wound in Indian major carp Labeo robita. On the whole, several important aspects of integumentary system of the teleosts inhabiting Indian subcontinent waters have been studied and Mittal and his co-workers have made major seminal contribution in this field, OBJECTRES OF STUDY: It is evident from the foregoing description that while several aspects of the catfish H. fossilis and C. barrachus integumentary systems have been studied by some workers but many other important aspects such as ultrastructural studies, qualitative and quantitative studies on the GP moiety in the mucous cells have not been explored. Further, the effects of salinity stimulus and seasonality on the number, area, OP contents of skin mucous cells have not been studied in detail. Considering the over all significant role of mucus, such studies are of paramount importance. Hence, the present study has been designed to address the following objectives. i. To study the histological organization of integumentary systems and mmphometric analysis of its various zones and cellular components in the catfishes H. fossilis and C. batrachus using various histochemical staining procedures. ii. Ulbustnictural studies (TF,M & SElvl) of the integumentaty mucous cells (MCs), their different types and changes following transfer to higher salinity i.e. 25% SW. iii. Quantitative and qualitative localization of different types of glycoprotein moieties in the integumentary MCs of FL fossilis and C. batrachus iv. Seasonal variations (circannual and monthly) in the glycoprolein moiety, number and sine of integumentary MCs of H. fossils. v. Salinity- induced changes in the number and size of integumentary MCs of H. fossilis, following gradually transfer from TW to higher salinities i.e. 25%, 3C%and 35% SW. a9 II. Experimental Protocol: There are three main aspects of study on the skin of the catfish H. fossilis and C batrachus. The details of the experimental pxotoccl are described below. 1. The first aspect deals with the studies relating to general morphology and histolov and also the qualitative and quantitative analyses of glycoconiueate moieties of the skin mucous cells of H. fossilis and C. balrachus. To achieve this, seven individuals each of well-acclimated H. fossilis and C. balrachus were sacrificed, skin sample collected and fixed in Carney's fluid and processed for histology and histochemistry as per details given in Materials and Methods section of this thesis. The stains used for general histology are Harris hematoxylin/eosin (HIE), Massons Tzicluome and for glycoconjugatc histochemistry, Alcian blue (AB pH 2.5), AB (pH 1.0), Active methylation (AM)/fo[lowed by staining with AB(2.5), AM/saponifieation (KOH) followed by the staining with AB(2.5), Periodic acid schiff(PAS), PAS/AB (2.5), PAS/AB (1.0), Aldehyde fuchsin (AF)/ AB(2.5). The details of these staining techniques, quantification of various glycoconjugate moieties and the statistical analysis have been described in Materials and Methods. 2. The second aspect relates to study the salinity- induced changes iu the number and size of skin mucous cells of H. fossilis. Well-acclimated specimens of H. farrilis were sequentially transferred from TW to 25%, 30% and 35% SW after 7 days acclimation in each salinity. A group of seven fishes was maintained in TW to serve as control. The skin samples from 5-7 fishes from each salinity and TW maintained fishes were obtained and processed for the study of salinity induced qualitative and quantitative changes in the skin mucous cells of catfish as per details given above. Five specimens each from TW and 25°/&SW groups were sampled for ultrastrnetural studies by scanning and transmission electron microscopy of H fossilis skin as per details described in Materials and Methods. 3. The third aspect is Cooused on the quantitative seasonal changes in . the sulphomuein contents of integametary mucous cells of H. Jr..i is. For this, catfish H fossilis were obtained every month from the wild and acclimated in the laboratory for 3-4 days. Six to seven individuals of FL fossilis were anaesthetized and the skin samples were collected and processed to study seasonal changes in the size, area and sulphated Ul' of integumentary MCs of X. fossilis as described in Materials and Methods. 41 III. Observations: A. Histology of H. fossilis & C. batrachus Integument: All the three classical layers of the inte„.-umcnt viz. epidermis, dermis and hypodermis including that of basement membrane which is more clearly visible with Periodic Acid Schiff (PAS) staining are clearly demarcated in both the catfish species i.e. H fossilis and C. barrachus. Interestingly, the thickness of dermis in H. fossilis is about twice of the epidermis whereas in C. batrachus dermis is more predominant and is four times than that of epidermis (Fig. 2.3). Epidermis: Epidermis of both species has three distinct regions — a very thin layer of apical epithelial cells which have large number of mucus containing spherical shaped goblet cells which do not stain with hematoxylin and eosin (HIE) (Fig. 2.I(A & C), 2.2 (A & C)) but stain positively with PAS and Alcian blue (pH 2.5) (Fig.2.1 D; 2.2 D). In C. barrachus, the mucous cells (MCs) are by and large, confined to upper layer of epidermis with hardly any amidst the club cells (Fig.2.2 D) whereas in H jossi(is they are abundantly distributed in both upper epithelial cells and middle layer of club cells (Fig. 2.1 D). A very prominent layer of the chromatophcre is distinctly seen in both the species (Fig. 2.1 & 2.2) but there appears to be a layer of adipose cells right above chromatcphore layer in some instances in both species (Fig. 2.1 C and Fig. 22 C). In most instances, a thick mucus layer at the upper most surface which has often been referred to as cuticle by some workers is also visible (Fig.2.2 A). In H. fossilis and (2. batrachus epidermai thickness varies from region to region and ranges between 131 -176 µm (mean 155µm) and between L 13- 177 in (mean 157 um) respectively (Fig.2.3). The majority of the space of epidermis is occupied by multilayered club cells with 1-2 centrally placed nuclei which are predominantly columnar in C. batrachus and spherical in H fossilis (Fig. 2.1 & 2.2 C). The area occupied by club cells in C. batrachus is much greater than that in H. fossilis (Fig.2.4).The mean height and area of club cells is 85.22 un (range 66.2 - 114.7jam) and 2.47 rmnz (range 2.2 - 2.7 mm') in H. fossilis and 119.5prn ( range 72 - 151pm) and 4.6tnm' (range 4.4 -5.Smm2) in C. balrachus. These club cells have homogenous eosinophilic cytoplasm which seems to have shrunk during fixation thus having an almost consistent cap-like vacuolar area towards the apical side of each cell (Fig.2.1 & 2.2 C). Interestingly, the base of each cell has darkly stained eosinophilic area which is consistently visible in each club cell (Fig2.1 & 2.2 C). No distinctly different and well-delineated stratum genninativtun as described in other fishes is visible in both these species. Instead, the lower most layer is made up 1-2 layers of irregularly arranged epithelial cells with darkly stained prominent nuclei (Fig.2.1 & 2.2 C). Some structures resembling the feature of lymphatic spaces as described in other teleosts which are characterized by presence of 1-2 darkly stained small lymphocytes. 42 Dermis: A thin but distinct layer of basement membrane more clearly visible with PAS is seen which separates the epidermis with underlying dermis. Right beneath that is the continuous layer of black stained chromatophores (Fig. 2.1 & 2.2 D) The zonation of stratum spongiosum and stratum compactum is more clearly demarcated in C. batrachua compared to H fossilis when stained with H/E (c.f. Fig. 2.1 A and Fig. 2.2 A). In both species, dermis is much thicker compared to epidermis which is 2.1 and 4 times in H. fossilis and C. batrachus respectively (Fig.2.3). The spongiosum layer is somewhat lighter than compactum when stained with H/E which stains negative with PAS :AB (pH 2.5). When stained with Masson trichrome, spongiosum and compaztum are clearly demarcated and the former layer takes up greenish tinge whereas the latter shows an overall reddish colouration in both the species (Fig.2.1 & 2.2, B). The connective tissues constituting dermis may be collagenous or reticular which cannot be established with routine histological stain like IHH/L. However, we have established the existence of these fibers by using Masson trichrome technique which imparts specific colour to these fibers in both species (Fig. 2.1 & 2.2, A, B). In C. batractus, certain conical or pyramidical structures are seen mostly in the stratum spongiosum region which also penetrate into the compactum zone (Fig. 2.2 B) but no such structures have been observed in IL fosxilis. In ti-us fish. however, a very peculiar oval structure resembling with some embedded parasite in the dermal region is observed. However, the exact identity of this structure is not established. In both these species, under the used magnification, it has not been possible to establish the definite location of blood vessels, nerve fibers or any sensory structures including the taste buds with the routine histological stains. However, the taste buds and pit organs are clearly visible when stained with Masson trichome technique (Fig. 2.1, E; 2.2.E, F, G). Some instances of vertically arranged strands more conspicuously present in stratum compactum are observed but no distinct layer of dermal epithelium as reported in other teleosts is noticed here. The hypudermis or subcutis is clearly visible in both the species which is largely made up of adipose cells and is richly supplied with blood vessels (Fig.2.I & 2.2, A). This layer is, however, irregular in thickness though it runs all along the base of dermis. The hypodermis is closely attached with the muscles underneath and stains prominently with eosin and do show the myosepta which divides this muscular zone. B. Glyeoprotein Moieties in Skin Mucous Cells (MCs) of H. fossilis & C. batrachms: H. foss/its: Histochenucally, the skin mucous cells (MCs) show the presence of all the three types of glycoproteins (CYs) where neutral type which stains magenta with PAS and combinations of PAS/ AB (pH 2.5) and PAS/AB (pH 1.0) (Fig. 2.5, 0, E & F) is predominant (47%) followed by acidic GPs (41%) which takes deep blue stain with AR 43 at p1I 2.5 (Fig. 2.5 A & 2.7). Among the constituents of acidic GPs, carboxylated type is in relatively higher proportion (26%) than the other constituent i.e. sulphated type (14.9%). These are established by All pH (1.0) and methylation followed by AB (pH 2.5) staining type (Fig, 2.5 B & C) for sulphated moiety and active methylation followed by saponification and AB pH 2.5 staining (Fig. 2.5 D) for carboxylated GP. This is also confirmed with Aldehyde Fuchsia / AB (pH 2.5) staining , where dark purplish blue stained MCs show mixed moiety of sulphated and carboxylated and light blue MCs indicate only carboxylated glycoconjugates (Fig 2.5, H). A good number of MCs (27%) almost equal to carboxylated type contain the mixture of acidic and neutral moiety and stain purple with the combined staining of PAS! AB pH 2.5 and PAS/ AB pII 1.0 (Fig 25, P & F). C batrackus: The GPs of C. batrac$aus skin MCs present somewhat different profile where acidic GPs (27%) show the highest proportion (Fig. 2.6 A and 2.6) followed by mixed (acidic and neutral) moieties (23.6%), carboxylated (22.6%) (Fig. 2.6 D & 2.8) and neutral (21.7%) (Fig. 2.8). Sulphated GP, as in H. fassila, is the lowest (4.6%) (P<0.001) compared to acidic glycoprotein (Fig. 2.6 A-H & 2.8). What appears similar in the GP profile of skin MCs is that sulphated moiety is the lowest in both the catfishes and mixed GP moiety of acidic and neutral component is almost at the same proportion (Fig. 2.9). C. Salinity Induced Changes in the Number and Size of H. fossilis Skin Mucous Cells (MCs) Following Transfer To 25%, 30% and 35% SW: The perusal of Fig. 2.13 and 2.14 reveals that there is a significant increase both in the size as well as the number of MCs follow ng transfer to 25% and 30% SW. The increases in both these parameters of skin MCs is extremely significant (P<0.001) right from 25% SW (mean size 107.9 t 14.6 and number 30 f 4.2) when compared with TW catfish (mean she 50.2 f 14.9 and number 19.5 f 3.8) which gets further accentuated in 30% SW ((mean size 122.6 ± 17.8 and number 35 ± 3.1). However, in 35% SW, both size and the number of these skin MCs show a precipitous decline (mean size 85.2 f 6.2 and number 2233.3) compared to 25% and 3C% SW but MCs size still remains significantly higher (P<0.01) whereas number almost becomes equal to TW control. Hence, there is an excellent parallelism in the MCs size and number profile when transferred to 25%, 30% and 35% SW (Fig. 2.15, upper panel). However, the profile of the gill MCs in higher salinities is quite different from the skin MCs profile where thereare marginal increases in the size and number in 25% SW. But in 35% SW both the size as well as number of MCs show a dramatic increase (Fig. 2.15, lower panel) which is contrary to the profile of skin MCs where a significant decline in the size and number is observed in both these parameters (Fig. 2.15, upper panel). The histological appearance also corroborates the quantification data of size and number of skin MCs. In 25% SW maintained H. Jossilis, the MCs are found more prominently 44 into upper and lower rows where upper row has greater abundance of acidic GU's ( Fig. 2.10 A, E) and lower row is rich in neutral OP (Fig. 2.10 B, F and G). A thick mucus sheet is also apparent which maybe the discharged secretion of MCs. In 30% SW, there is comparatively greater degree of degranulation and the secreted mucus seems accumulated around the MCs (Fig. 2.11 A and G). Interestingly, the MCs show varying degree of degranulation but some of the MCs are fully laden with mucus secretion (refer Pig. 2.11 (3 upper row of MCs). The MCs in 35% SW maintained. fishes present a completely exhausted picture where oozed out mucus is iii the stage of migrating towards the periphery and a thick mucus layer is visible at the surface (refer Fig 2.11 A and G). D. Seasonal Variations in the Number and Size of Skin MCs of H. fossilis: A distinct seasonal cyclicity in the area and the number of skin MCs of Ti. fnssilis is observed (Fig. 2.17 and 2.18) which reveaLs an interesting parallelism in the profile of both these parameters (Fig. 2.19 upper panel). However, the quantum of variations in these parameters and a distinct time-lag specifically in the month of December and January is clearly visible. Considering March as reference mouth (Fig. 2.16.1 A) due to moderate environmental conditions particularly temperature in this part of North India, there appears to be a transient increase in the number and size of MCs during next month (i.e. April, Fig 116.1 B) which is followed by the decline in both these parameters during May and June and decline in the MCs number is even statistically significant during June (P 45 there are interesting, similarities and differences in seasonal profiles of skin and gill MCs in terms of changes in the cell area and numbers. While skin MCs show only a single peak during winter (December and January), the gill MCs show three peaks occurring during the months of April, September and December. Gill MCs profiles also show parallelism except in April where the response is somewhat inverse (Fig. 2.19, cf. upper and lower panel). E. Seasonal Variations in the Abundance of Sulphated Glycoprotein in the Skin MCs of H. fossilis: Even though sulphated GP is in The least proportion in skin MCs of both H. fossils and C. batrachus but this GP does play a very important role in adaptation and protection of teleost fishes against varying environmental challenges. The monthly seasonal variations in the abundance of the sulphated GP shows its total absence during the months of April and May, highest proportion during winter months (December — February) (sulphated GP cell numbers 16.7, 2.12 & 19.5 respectively) and somewhat moderate number during March, July/August, September and October/November (GP containing MCs number (13.5, 9, 8.2 & 6.2 respectively). These observations are further corroborated by monthly histological pictures using AB (pH 1.0) staining which specifically stains only sulphated GP moiety (Fig. 2.20). The increase of this GP during winter months of December to February also correlates with the overall increase in the number and size of MCs in H. fossilis (cf Figs 2.21 & 2.17, 2.18) during these winter months. Interestingly, during December, there was a mixed population of discharged and intact sulphated GP containing cells (Fig. 2.20 H) but during January all the above MCs appear degranulated and discharged (Fig. 2.201). F. Electron Microscopy Study of Skin: The ultrastructure studies of catfish H fossilis revealed interesting details which could not have been visualized under light microscopy. i. Scanning Electron Microscopy (SEM): Since it captures only the surface view, the skin of N. fossilis which is smooth, devoid of scales and has copious mucus covering over the integumentary surface, reveal only limited features. The most predominant structures are pavement cells (PVCs) which are characterized by very conspicuous concentrically arranged polygonal microridges which are whorl-like structures and appear like thumb prints (Figs 2.22 A-C). These PVCs are made up of single-ridged border and have intermittent pit-like openings located at the junctions of these cells. These openings are presumably those of MCs to exude mucus on the surface which seems to fill the space between the tniernridges. However, these openings are distinct and clear and not occluded with mucus droplets (Fig 2.22 A-C). Following transfer of H fossilis to 25% SW, a large amount of mucus is secreted and in the integument appearance at somewhat low magnification, the entire surface seems to m be covered with thick mucus sheet with scattered MCs openings and no whorl-like microridges are visible at this magnification (Fig, 2.23 A). However, at a still higher magnification (Fig. 2.23 B-C), the PVCs are visible but the microridges are not well- demarcated but blurred due to extensive mucus covering. No other cellular features could be visualized under SEM view in the present study. ii. Transmission Electron Microscopy (TEM): Under TEM, three distinct cell types i.e. PVC. MCs and Club cells (CC) could be observed (Figs 2.24 and 2.25). The PVCs are bordering the surface epithelium and distinct microridges at the periphery are clearly visible (Fig. 2.24 B), The CCs are characterized by uniform electron lucent vesicles giving an overall whitish appearance in the central space (Fig 2.24 A& B). The MCs are abundant and do show the distinct presence of electron dense and electron lucent vesicles occurring within the same MC either in the form of distinct regions (Fig. 2.25 B) or intermingled form (Fig 2.24 A). In some instances, discrete mucus granules with the MCs are also visible (Fig 2.25 C). No other cell types such as chloride cells, filament cells or rodlet cells are visible in the skin TEM pictures. No noticeable change is visible in the integument under TEM study in 25% SW maintained catfish except that the granules with different electron density get aggregated into specific regions (Fig 2.25 B) unlike in FW where they gives intermingled appearance (Fig 2.24 A). IV. Discussion: A. Gross Histology: The histological architecture of the skin of catfish H.fossilia and C. batrachus is fund amentally similar to those of other teleosts. The integumentary structure of these fishes is made up of three broad zones- the outer epidermis, the middle dennis and the innermost subeutis or hypodemtis which anchors with the underlying muscles. The epidermis is composed of three major segments- a thin flattened to euboidal multilayerd tiers of the epithelial cells which may have interspersed mucous cells (MCs) followed by a much thicker middle segment which is largely made up of secretary and non- secretary cells. MCs may be Inc type of secretory cells and the other type may vary from species to species and of those major types are club cells, sacciform cells, swollen cells or some unusual types limited to only few species. This laver contributes to major thickness of the epidermis. The last of the epidermis layer is stratum germinativum which is normally one layer thick, rests on the non-cellular basement membrane and may have the lymphatic spaces. 'The other inclusions in the epidermis may be taste buds and apical pit etc. The dermis is composed of the first strata of loose connective tissue immediately beneath basement membrane called stratum spongiosum or laxum followed by compactly arranged connective tissue and hence termed stratum compactum and boundary between two strata may not he well demarcated. The dermis has rich supply of nerves fibers, 47 Club cells Mucous c •'taste budsj P g~nt cells DerdaiS • .. Stratum spongium ems : . - a? `i► V j.4 . compactum '-e~~.~~` .i1-~ . ~~fr y` ~~s.,i~~'•• t ? _ 1 Mucous cells •. • 100X Pigment cells `4~~me • membrane Fig-2.1 Gross structure of integument of H. fossilis showing its various regions (Panel A & C stained with haematoxylin/eosin, B & E with Massons Trichrome and panel O with Alcian Blue, pH 2.5 / PAS). (A) Showing distinct regions of epidermis, dermis, hypodermis and underlying muscles, SOx. (B) Magnified view of epidermis and dermis showing mucous cells at the outer side and also interspersed between multiple layers of round shaped club cells which rest at the basement membrane. Distinct layer of chromatophores (pigment cells) separating epidermis from dermis is visible, 100x. (C) Further magnified view of club cells. Also note the round shaped mucous cells present between the club cells, 400x (D) Note large population of mucous cells present both at the outermost side which are mostly small and big sized lying below. Differently stained mucous cell types can be seen. Club cells though visible are unstained. A clearly stained basement membrane and chromatophore containing layer are also visible. 100x (E) Magnified view of taste bud, 400x. 47 (i ) Pigl~eat c Is JP till . o '4, spongium Dennis F -T " •' 4. ' t` Strat • • „~-~: -fir 5 ucous cells Club cells c y~ ,M asement membrdne 0 Fig-2.2 Gross structure of integument of C. batrachus showing its various regions (Panel A, & C stained with haematoxylin/eosin B, E, F, G with Massons Trichrome and panel D with Alcian Blue, pH 2.5/PAS). (A) Showing distinct regions of epidermis, dermis, hypodermis and underlying muscles, SOx. (B) Magnified view of epidermis and dermis showing mucous cells at the outer side and underlying large multilayer club cells resting at basement membrane. Distinct layer of chromatophores (pigment cells) separating epidermis from dermis is visible, 100x. (C) Further magnified view of club cells showing typical club shape and having mulitinuclei, 400x. (D) Abundantly present mucous cells at the periphery of epidermis. Club cells though visible are unstained. A clearly stained basement membrane and chromatophore containing layer is also visible, 100x. (E), (F) & (G) Show taste bud and pit, 10ox & 400x. 470i) 800 E 700 600 off! 500 N E 400 0D 1300 W 0 200 i10 H. fossilis C. batrachus Fig-2.3 Length of epidermis and dermis of the integument of catfish H. fossilis & C. batrochus. Each column represents mean i SEM for five fish.("`P<0.001 Epidermis versus Dermis). 6 N Ir 4 U w0 3 <2 1 0 Mucous cells Club cells Fig-2.4 Area of mucous cells and club cells of the integument of catfish H. fossilis & C. batrachus. Each column represents mean ± SEM for five fish. ("'P<0.001 H. fossilis versus C. batrochus ). 47 (iii) f ---Sul. Gly. i Aci. Glv. • 10ox Carbo. Gly. / 'O Sul. Gly. 1 tel •t $c,, 10ox Mix. Gly. Sul. Gly.. • •~. • 1 1 « S~*• 6 **All •$ Z:.. •~ S Neu. GIv. lWix. Gly. 100x Mix. Gly. • Neu, Glv. / ;j + "/P P'd4$I4sq •10ox • G~ 1x - --\ '` -.--' ! Fig-2.5 Different types of glycoprotein moieties in mucous cells of the integument of H. fossills maintained In TW, 100x. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moieties. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moieties. (C) Skin sections following active methylation and then stained with AB (pH 2.5) showing the sulphated glycoprotein. (D) Active methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylated glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour moieties indicating mixture of glycoproteins and magenta colour cells showing neutral glycoprotein. (F) Mucous cells stained with AB (pH 1.0)- PAS showing few purples cells which are mixture of sulphated and neutral glycoprotein and magenta cells showing neutral glycoprotein. (G) Pure neutral glycoprotein in mucous cells stained magenta colour with PAS. (H) Sulphated and carboxylated glycoprotein in mucous cells stained by AF- AB (pH 2.5), (Neu. Gly., Neutral glycoprotein; Aci. Gly., Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly., Mixed glycoprotein). 47 (iv) Aci. Glyn - Sul. Gly. ff ~l ~w 4► !100x • .~ Sul. Glyy Carbo. Gly. ~T 0 - ~M'►.+' mil - , ._4IFi... • _ 100x i S. Mix. Gly. Mix. Gly. (*1 1 -.~' ~..~., r} ► -,fir. •.I~..~- ~ ,, E 109sfr ., Neu. Gly. RM Fig-2.6 Different types of glycoprotein moieties in mucous cells of integument of C. botrachus maintained in T 100x.. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moieties. (E Mucous cells stained blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moieties. (C) Skin section following active methylation and then stained with AB (pH 2.S) showing the sulphated glycoprotein. (D) Actiu methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylate glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour moieties indicating mixture c glycoproteins and magenta colour cells showing neutral glycoprotein. (F) Mucous cells stained with AB (pH 1.0)-PA showing few purples cells which are mixture of sulphated and neutral glycoprotein and magenta cells showing neutr glycoprotein. (G) Pure neutral glycoprotein in mucous cells stained magenta colour with PAS. (H) carboxylate glycoprotein in mucous cells stained by AF- AB (pH 2.5), (Neu. Gly., Neutral glycoprotein; Aci. Gly. , Acidic glycoproteii Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly. , Mixed glycoprotein). 47 (v) ri so 10 a Acidic Sulphated Carboxylated Neutral Mixture Fig-2.7 Per cent distribution of glycoproteins in mucous cells of the integument of catfish H. fossilis . Each bar represents mean ± SEM, *** (P< 0.001) denote significant differences calculated with reference to neutral glycoprotein. 35 30 25 n20 i►% 5 0 Acidic Sulphated Carboxytated Neutral Mixture Fig-2.8 Per cent distribution of glycoproteins in mucous cells of the integument of catfish C. batrochus . Each column represent mean 2 SEM for five fish, *'* (Pc 0.0011 denote significant differences calculated with reference to acidic glycoprotein. 47 (vi) 60 50 C 0 4- 40 a+ N 30 C d u 20 10 0 Acidic Sulphated Carboxylated Neutral Mixture Fig.2.9 Relative per cent distribution of various types of glycoprotein-containing mucous cells in the integuments of H. fossi/is & C. batrachus. The values depict mean t SEM for five fish, * (P < 0.05), ** (P < 0.01), *** (P < 0.001) denotes significant differences calculated between the glycoproteins of H. foss//is and C. batrachus. 47 (vii) Aci. Gly. • . -- •Sul. . -- • .` A ~~0 e A • ~ B • Carbo. Gly. Sul. Gly. VA S.y X, 100x C. 100x `~ 4 0 Mix. Gly. ~ _ X11 •s't Mix. Gly. - 100x E 100x Y~ F Q^ Neu. Gly. Su I. Gly. • ~0 /"? a. Carbo. Gly. JIII i t I. Fig-2.1O Different types of glycoproteins moieties in mucous cells of integument of catfish H. jossilis after transfer 25% SW showing hypertrophy and increase in cell number, 100x . (A) Mucous cells stained deep blue with Alcian b (pH 2.5) showing acidic mucopolysaccharide moieties. (B) Mucous cells stained deep blue with Alcian blue (1.0 1 showing sulphated mucopolysaccharide moieties. (C) Skin sections following active methylation and then stained with (pH 2.5) showing the sulphated glycoprotein. (D) Active methylation followed by saponification (KOH) and then stair with AB (pH 2.5) showing the carboxylated glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing pur colour moieties indicating mixture of glycoproteins and magenta colour cells showing neutral glycoprotein. (F) Muu cells stained with AB (pH 1.0)-PAS showing few purple cells which are mixture of sulphated and neutral glycoprotein ; magenta cells showing neutral glycoprotein. (G) Pure neutral glycoprotein in mucous cells stained magenta colour N PAS. (H) Sulphated and carboxylated glycoproteins in mucous cells stained by AF- AB (pH 2.5), (Neu. Gly., Neu, glycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotf Mix. Gly. , Mixed glycoprotein). 47 (viii) Aci. Gly. Sul. Gly. W 100x VN a 1OOx 4, ' 1 Carbo. Gly. .r - •4 Su.l Gly. !~ c,~; 100x / ' Mi x. wa. Mix. Gly. Neu. Gly. Neu. 100x W. GI Neu. A1I4I ~• Carbar y. • - loox H Fig-2.11 Different types of glycoproteins moieties in mucous cells of integument of catfish H. fossilis after transfer to 30% SW showing hypertrophy and increase in cell number, 100x . (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moieties. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moieties. (C) Skin sections following active methylation and then stained with AS (pH 2.5) showing the sulphated glycoprotein. (D) Active methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylated glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour moieties indicating mixture of glycoproteins and magenta colour cells showing neutral glycoprotein. (F) Mucous cells stained with AB (pH 1.0)-PAS showing few purple cells which are mixture of sulphated and neutral glycoprotein and magenta cells showing neutral glycoprotein. (G) Pure neutral glycoprotein in mucous cells stained magenta colour with PAS. (H) Sulphated and carboxylated glycoproteins in mucous cells stained by AF- AB (pH 2.5), (Neu. Gly., Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Suiphated Glycoprotein; Mix. Gly., Mixed glycoprotein). 'r 47 (six) I ~ Ad Gly~~ Sul. Gly. • ~'~ ' Sul. Gly. .. M ~ i Car . G r' Mix. Gly. Mx• ly• Neu. • Neu. s + 1 ~ E box ' r F Carbo. Gly. .v. O Fig-2.12 Different types of glycoproteins moieties in mucous cells of integument of catfish H. fossilis after transfer to 35% SW hypertrophy and decrease in cell number, 100x . (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moieties. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moieties. (C) Skin sections following active methylation and then stained with AB (pH 2.5) showing the sulphated glycoprotein. (D) Active methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylated glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour moieties indicating mixture of glycoproteins and magenta colour cells showing neutral glycoprotein. (F) Mucous cells stained with AB (pH 1.0)-PAS showing few purple cells which are mixture of sulphated and neutral glycoprotein and magenta cells showing neutral glycoprotein. (G) Pure neutral glycoprotein in mucous cells stained magenta colour with PAS. (H) carboxylated glycoprotein in mucous cells stained by AF- AB (pH 2.5), (Neu. Gly., Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly. , Mixed glycoprotein). s 47(x) SIZE 140 120 E —100 C) 80 0 U 60 0 40 4J Q 20 C+7 140 120 E 100 ** C) o 80 tA 3 0 60 0 40 C) L < 20 a TW 25% SW 30 % SW 35% SW Fig-2.13 Area of mucous cells in the integument of catfish H. fossilis following transfer to 25, 30 & 35% seawater. Each bar represents mean ± SEM, ** (P< 0.01), (P< 0.001) denote significant difference calculated with reference to TW mucous cells. Upper panel represents the same results of the bar diagram given in the lower panel. 47 (xi) NUMBER 40 *** 35 ** Q 30 22 25 V 20 15 C 10 Z 5 [ol 40 a 35 4 ** E 30 a, 25 U 20 0 U 15 O d 10 E z3 5 0 TW 25% SW 30 % SW 35% SW Fig-2.14 Number of mucous cells in the integument of catfish H. fossilis following transfer to 25, 30, 35% seawater. Each bar represents mean ± SEM, " (P< 0.01), •** (P40.001) denote significant difference calculated with reference to TW mucous cells. Upper panel represents the same results of the bar diagram given in the lower panel. Note the significant increase in mucous cells number in 25 & 30% SW . 47 (xii) SKIN 140 —size ## NUMBER # 120 35 i 30 ._ 100 25 U 20 0 U 15 10 0 5 E 3 Z 0 0 TW 25% SW 30 % SW 35% SW GILL 140 120 MC size in gill #ti MC number in gil' t9 120 O *** 100 E C -;100 80 d 0 80 U 0 60 U 0 60 U 40 O ro 40 0 OJ aJ 20 20 E Z 0 0 TW 25% SW 30% SW 35% SW Fig-2.15 Area & Abundance of mucous cells (MC) of the integument (upper panel) and gill (lower panel) of catfish H. fossilis following transfer to 25, 30, 35% seawater. Each bar i represents mean ± SEM, * & # (P< 0.05), " & ## (P< 0.01), ' (P< 0.001) denote significant difference calculated with reference to TW mucous cells. 47 (xiii) MARCH Mucous cells .-..~ y Mucous cells 400X • 0, - IL ► APRIL - Mucous calls •/ : - ,, ' •* ,LAr 41 ~.+ Mucous cells Mucous cells ..I.I..*v. Org' •WA box C i —ca JUNE Mucous cells Mucous cells i t • VTO 0^ . *fto •. box 400x d JULY/ AUGUST • Mucous cells . 'iWi*óh Mucous cells r xJ 4 Fig-2.16.1 Seasonal variations in the abundance and size of mucous cells in integument of catfish H. fossilis during March to August represented by panels A-E respectively. Mucous cells stained with AB-PAS(pH 2.5), 100x. Magnified view of mucous cells of the above panels represented by panel a-e respectively, 400x. Note the significant decrease in number of mucous cells during June. 47 (xiv) SEPTEMBER ucous cells Mucous cells , hO box - r F 400X , OCTOBER-/NOVEMBER Mucous cells Mucous cells 4 `e G `... ___ DECEMBER "O ;~cous cells r. ~ ~. ••~' Mucous ceTfs ' 18. H 400X 10ox b -h JAUNARY Mucous cells • / Mucous cells ' ___ - - .. loox r ` ` 400X FEBRUARY • Mucous cells Mucous cells 1 • r' t 4 OX%% .~,$¼ J Fig-2.16.2 Seasonal variations in the abundance and size of mucous cells of integument of catfish H. fossils during September to February represented by panels F - J respectively. Mucous cells stained with AB-PAS (pH 2.5), 100x. Magnified view of mucous cells of the above panels represented by panel f - j respectively, 400x. Note the significant increase in number of mucous cells during December. 47 (xv) Size 100 N^ 90 E v 80 70 O 60 O 50 040 a 30 20 10 0 100 N E90 v p~ u 70 C] H 30 60 50 O 40 30 < 20 10 0 a~~~c pQi~ ~2~ Joe Jy` ,o¢~ ~o~ • ~¢~ aC1 l` \po S a\ , S Fig-2.17 Seasonal variations (March to February) in the area of mucous cells of the integument of catfish H. fossilis. Each bar represents mean ± SEM, * (P< 0.05), *** (P< 0.001) denote significant differences calculated with reference to the month of March. Upper panel represents the same results of the bar diagram given in the lower panel. 47 (xvi) Number 50 a 45 E 40 N 35 30 0 25 20 15 L Ol 10 .0 E5 Z 0 50 M 4S w 40 3 35 - 30 u H 7 25 0u 20 015 io E3 Z 5 [D7 ~~r ~~ aJ ce J5~ ~~~ Je• ~~~ all a(i ~ F '~~ a~~ h O~o~ c~ F Fig-2.18 Seasonal variations (March to February) in the abundance of mucous cells of the integument of catfish H. fossilis . Each bar represents mean ± SEMI * (P< 0.05), *** (P< 0.001) denote significant differences calculated with reference to the month of March. Upper panel represents the same results of the bar diagram given in the lower panel. 47 (xvii) Skin Sc 100 45 i0 a e 4C C 35 pi 3C U 2c 0 U 2C w a,O 15 E 1C 10 Q z z LO 0 , , J~~y~ ~~e~ ~e , c`, `,ate I ho GILLS 18C .80 16C .60 .40 .20 —^ U) L00 N 0 s0 i0 O 10 I-d 4 GO oJe. ,p~~ JaC1 JaC1 lJ\+\P~ S~Q`¢ce Oho* `45 ,ate Fig-2.19 Seasonal variations on the number and area of integumentary (upper panel) and branchial (lower panel) mucous cells of catfish H. fossilis. Each value represents the mean f SEM, ' (P < 0.05), " (P< 0.01), '"' (P< 0.001) denote significant difference with reference to month of March. Comparisons have been made amongst the values of the same parameter. 47 (xviii) Mucous cells Mucous cells / A .-..., ` loox MARCH PA 100x SEPTEMBER F ~,~,'~ , y Mucousceils r • Mucous ells 'r 41,,J r ~,~,,,..,• ,... . A .~.► .fir,.. --•~'`~ 100" APRtL 1OOp OCTOBER/ NOVEMBER G Mucous cells 1 • V if ►1',4" I'~ ar1 No .I 4 ioox C ,lox MAY DEDECEMBER H ,~~Mucous cells Mucous cells 100x J Oe'r p 100x JANUAR Mucous cells ~ pmt" Mucous cells '' ' ~ ioox~-,I lIJl"fiAUGUST E loox FEBRU~9% R,e Fig-2.20 Seasonal variations in the abundance of sulphated glycoprotein in mucous cells in integument stained with AB (pH 1.0), 100x. Panels A-J represent March to February respectively. Note the significant decrease in the number of mucous cells during April and May 47 (xix) Number 25 * 20 U 15 0 U 10 w 0 ES 2 ** 0 25 M co !• 20 C U 15 0 U 10 0 5 3 2 *** 0 `` Qc aJ Jce JS~ Oe~ JC ,oe~ acJ act P yeQ~~ oho. Ot~~ ~a ~~ Fig-2.21 Seasonal variations in the abundance of sulphated glycoprotein in mucous cells of the integument of catfish H. fossilis . Each bar represents mean ± SEM, ' (P< 0.05), *** (P < 0.001) denote significant differences calculated with reference to the month of March. Upper panel represents the same results of the bar diagram given in the lower panel. 47 (xx) Fig-2.22 Scanning electroi photomicrograph of skin c catfish, H. fossilis maintained ii tap water. (A) surface c epidermis. Pavement cells wit distinct boundaries (—s concentrically arrange microridges ( ) and th mucous cells opening (–+ 4230x. (B) Magnified view c Pavement cells with distine boundaries (- concentrically arrange microridges ( ), the mucoc cells opening (—), 6880x. (( Magnified view of Mucous cel 47 (xxi) 3-'J. t . i,* ?ir 'dt gj•... -a z 1 \ Fig-2.23 Scanning eiectron )otomicrograph of skin of catfish, fossilis maintained in 25% sea water. (A) surface of epidermis p - showing covered pavement cells m :] with mucus X 4230x. (B) Magnified view of Pavement cells with distinct boundaries ( —* ), and concentrically arranged microridges ( ), the w 1e_ i/;. 4r: mucous cells opening ( —► )X 11 6880x (C) X 12140x. Note '.• ~~ 1. extensive mucus covering all over the pavement cells. • ti L 2 ftW{ 47 (xxii) ;f• Micro ridges I. • • ht granule.. -~ I . rr l X70 a ,wI 2Sjj Fig- 2.24 Transmission electron micrograph of skin of catfish H. fossalrs maintained in tap water. (A) Mucous cells (MC) with electron dense and electron light granules and basally located nucleus (N), The MC are surrounded by pavement cells (PVC) and Club cells (CC) 6980x. (B) PVC with micro ridges at the peripheral end of the cell, 5820x. 2 micron A 2 micron • CC PVC CC •- Fig- 2.25 transmission electron micrograph of skin of catfish H. fossilis maintained in 25% sea water. (A) Mucous cells (MC) surrounded by pavement cells (PVC) and adjacent club cells (CC), 5820x. (B) Magnified view of MC with electron light and dense granules. 12000x. (C) Scattered mucous cells granules, 5820x. 47 (xxiii) blood capillaries and occasionally occurring dermal papilla. The last layer - subcutis essentially consists of large fat cells, nerve plexus and blood capillaries and is finally connected with thick layer of muscles. A distinct lining of chromatophores lying just below the basement membrane separates epidermis from dennis. Bath the catfishes under present study have all the above features but finer differences do exist. The outermost epithelial cells in both catfishes are made up of single layer of flattened cells also called pavement cells which have the abundant presence of MCs which in case of C. batrachas are largely confined to outermost layer but in H fossilis these are also present in the middle layer in good numbers. The MCs are mostly spherical but may undergo change in shape during different months or render salinity stimulus to assume irregular shape and varying degree of granulation. No distinct nucleus is visible and cytoplasm is either completely laden with mucus to give the opaque appearance or at times appear greatly vacuolated. The distributional pattern of MCs in the integument of different teleosts seems to vary in different species and fall into three distinct categories. One where MCs are confined only in the uppermost epidermal layer or pavement cells and this is represented by fish species such as C. batrachus (in the present study), Pleuronectes platessa and Platichthys fiesus (Pletcher et at 1976), Cyprinus carplo, Labeo rohita and Cirrhina mrigala (Singh and Mittal 1990) and Pseudobagrus fulvidraco (Park et al 2002). The other category where MCs occur both in the upper most and middle part of the epidermis such as in H fossilis (present study), Carla calla (Singh and Mitial 1990), in entire epidermal thickness in Callionymus rirso and C. decorates (Sadovy at al 2005) and in upper and middle layer of epidermis in Syachiropus splendidus, S pieturatus, S. ocellatus and Repomucenus richardsonii (Sadovy et at 2005). The third and somewhat rare situation is reported by Mastacembelus armalus where large MCs we present in stratum laxum (dermis) and small MCs in epithelium (Zaccone 1981b). The shape of the MCs is either spherical or flask-shaped occurring either in the form such as spherical in Korean eel goby Odontamhlvopus lacepedh (Park et al 2003b), pear or flask- shaped in air breathing tleost M. arroatus (Zaccone 1981 b) or both forts as found in Korean bullhead P. fidvidraco (Park et aL 2002). The nucleus is basal and spherical and cytoplasm either strongly basophilic as in C. calla and C. carpio or mildly in L. rohita and C. mrigala (Singh and Mittal 1990). In the present study on two catfishes, we have not been able to observe any distinct nuclei but a situation as described in the above carps where the secretary contents supposedly push cytoplasm along with flat basal crescentric nucleus to form a thick or thin peripheral laver in MCs (Singh and Mittal 1990) have been observed in present study. Mucous cells do not get stained with H/F, with the exception of some dragonet species (Sadovy et al 2005) but stained with PAS and AB (pH 2.5) with varying degree depending upon the relative concentration of glycoproteiH moiety. The middle layer of epidermis in both catfishes under present study are largely occupied by multilayered club cells with 1-2 centrally placed nuclei in FL fossilis multiple nuclei in C. batrachus. The club cells are elongated and columnar shape in C. batraclras and spherical in II. fossilis which occupies relatively smaller area compared to C. butrachvs. 48 These club cells in both these fishes have a consistent cap-Like vacuolar area and darkly stained ecsinophilic area at the base which has been attributed to fixation artifact. These club cells stain positively with 111E but show negative response with PAS and AB (pH 2.5). However, they do take up blue slain when treated with the combination of Aldehyde fuchsin and AB (pH 2.5). Amongst the large size cells in the middle layer of epidermis, the occurrence of club cells is perhaps most predominant. Sadovy et al (2005)observed that the distribution of club cells is confined typically in Ostariophysi and Anguilliformcs but their distribution is spread in a much wider range such as catfishes IL fossilis, C. barrachus and Corydoras aeneus (Raiphs and Benjamin 1992), other air- breathing fishes e.g. Iksookimia koreensis (Park 2002), Solea senegalensis (Arellano et al 2004), P. fulvidraco (Park et al 2002), in many carps such as C. carpio, L. rohita, C. cat/a, C. mrigala (Singh and Mittal 1990), Acanthophthalmuc .cemicinctus and Botia home (Ralphs and Benjamin 1992) and many other teleosts like Rita rita (lblittal 1968), Barbus sophor (Mittal et at 1976a) and also in Lamprey (Downing and Novels 1971and Sato 1981). However, with such wide spread distribution, it does not seem possible to correlate the occurrence of these cells with any specific ecological and functional adaptation of these teleost fishes. The club cells in most of these teleostei have the same structural features as observed in the present study on the catfish i.e. oval and spherical, usually uni- or multinuelcated, homogenous cytoplasm with peripheral shrinkage and staining positively with WE and Massons Irichrome. Ralphs and Benjamin (1992) have differentiated them with MCs by lack of opening into epidermal surface, the position of nuclei and negative reaction to PAS staining. It is possible that the club cells release theft contents into intercellular spaces to diffuse upward to act at the surface (Downing & Novels 1971). As to the function of these cells, their secretion may contain pheromone to induce fright reaction or alarm signal (Smith 1966), eel skin club cells have been shown to secrete mucus hemagglutinin (Suzuki and Kaneko 1986), contain sulphated glycosamino glycans (GAGS) in catfish and loaches (Ralpha and Benjamin 1992) that could help create jelly bandage which many seal the damaged tissue (Whitear and Mittal 1983). Some evidence has also been provided to show that fish skin secretion possibly from club cells stimulate the rate of wound healing in animals and diabetic foot ulcer in human (Estehane 2012). Hence, in general, the club cells are likely to play a protective role in different ways. Park et al (2002) have mentioned that the thickness of the epidermis depedds on the size and layers of the club cells in the middle region which, however, is not the case in the present study where club cells length in C. hatrachus is almost twice as much as FL fossilis but epidermal thickness is more or less same. Further, Singh and Mittal (1990) have suggested an inverse relationship between the density of MCs vis-a-vis the density of club cells per unit area in few Indian major carp. A second distinctive cell type, the sacciform cells have also been reported to be present in the noddle layer of epidermis and notably occur in Acanthopterygii and Paracanthuplerygii, Cottifunaes and Chaunifurmes (Takashima and Hibiya 1995). Among the reported species with the presence of sacciform cells are dmgonet species S. splendidus, S. picturatus (Sadovy or al 2D05), pufferfish Takifugu niphohles (Takashitna 49 and Hibiya 1995), Brown trout Salmo trutta, Arctic chary Salvetinus alpinus (Pickering and Fletcher 1987), M armatus (Zaccone 1981b), Cling fish Diademichthys lineanes (Hori et al 1979), Monopterur cuchia (Mittal and Agarwal 1977), C. striata (Mittal and Banerjee 1975). The sacciform cells are large, flask or ovoid shaped occupying the entire thickness of epidermis from end to and as in S. splendidus in a single layer or covering the major epidermal area as found in other dragonet fishes. the secretion of these cells is granular and contains a mixture of many chemical substances. In dragonet fishes, the secretion is positive with PAS and AB at both pI-I (1.0) and (2.5) and pale grey coloration with Sudan black B indicating the presence of glycoproteins and also lipid (Sadovy of al 2005). In M armatus, the sacciform contents show the presence of protein and lipid material rich in cysteine, tryptophan, tyrosine, cholesterol, SHT and also a possible source of catecholamine (Zaccone 19816). The function of these cells is largely uncertain but it is generally believed that the secretion may exercise protective influence. Since their prevalence is found to increase following infection with shin parasite (Pickering and Fletcher 1987). It has also been suggested that sacciform cells produce substances with repellent and toxic activity which may have haemagglutinic and hemolytic activity (Whitear 1986). Interestingly, these saccifomm cells resemble MCs in terms of secreting mucus and giving vacuolated appearance but are different due to its large size which has never been found even in the largest MCs. Similarly, these cells resemble club cells with reference to size but are vastly different because sacciform cells unlike club cells, show vacuolated appearance. Another very interesting cell type named swollen cells occupying the middle layer of epidermis, found in some but not all cutaneously respiring fishes are extensively described by Park et al (2002), (2003 a,b), (2004). Curiously enough, there is no other mention, to the best of my knowledge, of this cell type by any other investigator. Another peculiar feature of these cells is that, unlike mucous, club or saccifonn cells, these cell types are non-secretory and hence are considered a simple modified former of the epithelial cells and are negative for any routine histological or histochemical stains such as H(E, PAS, AB (2.5), toluidine blue. These characteristic features establish the independent entity of these cells. Straciurally, they are oval, multiiayered with an occasionally visible central nucleus and homogenous cytoplasm with a large vacuole. Their presence has so far been reported only in the cutaneously respiring fishes such as amphibious P. lwhlrenteri (Whitear 1986), Periophthalmus modeetus (Park of at 2002), P. maguuspinaatus (Park 2002), mudskipper Boleuphthalmus pectinirostris (Park et al 2003a), Korean eel goby Ddontamblyopus lacepedi (Park et at 20036). However, there are overwhelming number of other cutaneously respiring fishes such as Monopterus, Mastacembehus, Amphipnous, Micgurnus, Liohagrus, Iksookemia and Pseudobagras where the swollen eel Is do not occur. There is no suggested functional role of these cells but their presence exclusively in cutaneously respiring fishes indicate that it may have something to do with respiratory feature of these fishes but their total absence in some other air breathing fishes make this assumption less tenable. Ilene, it is clear that there are three major types of secretary structure in the epidermis i.e. the mucous or goblet cells present primarily in the uppermost but also in the middle epidermis, club and so sacciform cells which are much larger compared to MCs but are confined to the middle region of epidermis and account for its major thickness. The other type i.e. swollen cells, though occur in the same domain but are reportedly non-secretory and restricted exclusively in some cutaneously respiring teleost fishes. In addition, mention has also been made of cuticle-secreting cells in nine-spired stickleback Pungitius pungitius (Solanki and Beugamin 1982) and Lep(ocottus armalus (Marshal 1979) and others (Whitear 1970). However, these cells are very small, confined only in the upper epidermal part and are reported to contain sulpbomucin (Whitear 1970). A reference of a few other cell type in epidermis such as ionocyte in Senegal sole (Arellano et at 2004) and Blennius pholls (Nonnotle et at 1979) and Merkel cells (Whitear 1989, Zaccone et at 1994) have also been made. But these cells do not occur in significant numbers and do not occupy a sizeable surface area to contribute to the thickness of the epidermis. The last layer of epidermis, the stratum germinativum observed in both the catfishes in the present study is universally occurring layer made up of cuboidal to polygonal cells. This laver with the same nomenclature has also been reported in higher vertebrates groups including mammals where it is supposed to regularly multiply hence also called malpighian layer and the cells are pushed upward and become progressively flattened to form an intermediate segment of transitional layer. The cells of the outer most layer in these group of vertebrates may lose their nuclei become dead and cornified hence called stratum corneum. Hence, it is clear that there is a vast difference between the fish and other vertebrate epidermis since in fishes all the epidermal epithelial cells including the last stratum gerrninativum are metabolically active and transitional layer and outermost stratum cornenm are absent. One may wonder if it is at all justified to call stratum germinativum since all the epithelial cells lying above are metabolically active and dividing. The presence of a horny scaly layer forming the outermost covering over the skin which is made up of cysteine rich scleroprotcin called keratin is normally found in amphibians, reptiles, birds and mammals. 1rterestingly, in certain teleosts such as Bagarius bagarius (Mittal and Munshi 1970), Notopterus notopterus, Barb us sophor (Mittal and Munshi 1974 and Mittal et at 1976a), the outer most layer has been reported to be keratinized. Basically, fish integument is mucogenic and a layer of mucus with varying thickness covers its integument and there is a well established inverse relationship between keratinization and mucus secretion by unicellular and multicellular glands. Mittal and Whitear (1979) have stated that some teleost are capable of producing keratin usunlly in specialized structures such as homey lips and breeding tubercles as found in some male cyprinids. Hence, in such fishes the entire skin may not be keratinized parts of the epidermis such as in Indian catfish B. bagarius where unicellular mucous glands, ampu:lary neuromast organs and taste buds are found. The keratinized cells react positively to keratin specific histochemical stain and is further complemented with electron microscopy studies in if begarius (Mittat and Banergee 1974a). A similar but somewhat weaker reaction to histochemical stain was found in superficial epidermal cells of Notopterus notopterus (Mittal and Banerj ee 19746). The process of keratinization involves cells death which make up the layer which is regularly sloughed off as found in R. bagarius (Mittal and Banergee 1974a). to some other teleosts such as s1 Leptocottus armatus (Marshall 1979), Al. cuchia (Mittal et at 1980) and P. pungitius (Solanki and Bengamin 1982), cuticular mucus in the form of covering over the external epidermis has been described. This mucus has the same histochearical properties and functions as described to fish mucus in the general sense and may be the specific secretion of cuticle-secreting cells described in P. pungitius (Solanki and Bengamin 1982).We have also observed the presence of lymphatic spaces in the stratum germinativum which have been extensively described in most of the teleost fishes including air-breathing fishes (Park 2002, Park et at 2002, 2003). The lymphatic spaces contain lymphocytes wluch have deeply stained nucleus and narrow rim of cytoplasm. These lymphatic spaces are said to supply nutrition to stratum gerininativum and to provide protection against microorganisms since these spaces get enlarged under pathological conditions. The other features observed in the epidermis of both fishes under present study are taste buds and apical pits which have been reported in variety of teleosts (Singh and Mittal 199D, Park 2002, Park et a] 2002,2003b). A thin non-cellular but well-demarcated basement membrane distinctly stained with ABlPAS which separates the epidemds from the dermis and reported as almost universal feature of the integumentary system has also been observed in the present study on the catfishes. Just beneath the basement membrane lies an almost continuous string of the chromatophores lodged in the upper part of dermis which is the characteristic feature of poikilotherms including teleosts. We also observed a few sporadically scattered chromalophores in the last layer of skin i.e. the sub cutis which has also been reported in Anguilla australis (Elliott 2000). Interestingly, chromatophores are absent in C. mrigala and C. carpio while they are reportedly present in other Indian major carps such as C. calla and L. rohita (Singh and Mittal 1990). No plausible reason is forthcoming for such anomalous distribution. In the present study, the dermis of both the catfishes correspond to the typical teleostean plan of being formed by an upper loosely arranged stratum spongiosum and lower compactly packed stratum compactum collagen fibers. The demarcation of two layers is well-marked in C. batrachus while in H fossilis it is not so clearly visible. In H. fossilis stratum spongiesum occupies much wider space as compared to C. batrachus where the width of both these strata is almost equal. With regard to staining affinity, we have observed A/E +ve response for both catfishes where stratum compactum takes up darker stain possibly due to compact nature of the fibers. Both layers of dermis show —ve staining response with PAS, AB (2.5), AB (1.0) whereas Aldehyde fuchsin-AB (2.5) shows positive staining. The most successful stain in most of the teleost (Park et at 2002, Park 2002) is Masson trichrome specifically stains the collagen fibers which constitute the major bulk of dermis. Generally, these collagen bundles are oriented horizontally in stratum spongiosum but in the form of vertical strands in stratum compacrum. This network of connective tissues of dermis contains abundance of blood vessels, nerve endings, sense organs and assortment of epidermal and dermal derivations. Since epidermis is without any vascular supply, the vasculrization of dermis is essenfial to meet the nutritional requirement of epidermis through stratum germinativum. A well- 52 defined hypodernus or subcutis which is the innermost layer of the integument anchors the with the underlying muscles. It is characterized by the large number of adipose cells with in-between empty spaces in these catfishes as also other teleosts and is also said to have blood supply and nerve endings in other teleosts which, however, was not visible in the present study under low magnification. In this study, the sporadic presence of chromatophores was also observed in this layer. In the present study, we observed the significant variations in ratio of the epidermis versus dermis which in Ii fossflts and C. batrachus was two and four times respectively implying thereby that dermis of C. balrachus is twice that of H. fussilis. No reason seems apparent to justify such a difference except that it could simply be a species variation. The thickness of the epidermis in both these catfishes is approximately same and the mean thickness 155µm (range L31-176 pm) and 157 pin (rang 113-177 µm) in H. foss/Us and C. batrachus respectively. Park et al (2003a) and others have stated that amphibious fishes which are capable of cutaneous respiration are characterized by the presence of th ek epidermis since it contains several kinds of epithelial glands for partial Oz uptake_ However, critical evaluation of the available data on the epidermal thickness of these air breathing fishrs such as Misgm'rrus fossilis (338.7 µm) Mastacembelus panvalus (44 km), Amphipnous cuchia (119 µm), Monolpierus albvs (75 pm) with those of non-air breathing fishes such as C. carpio (126 pm), C. catla (142 µm), L. rohita (132 µm) and C. mrtgala (148 pm) (Singh and Mittel 1990) does not support such an assumption. Moreover, it has been clearly established in amphibious mudskipper B, pectinirostris by Park et at (2003a) that there is a significant variations in the epidermis thickness in the various regions of the body and none of the studies quoted above have ensured the uniformity of the skin sampling site makes such comparisons untenable. Hence, Elliott (2000) remarked that thickness of epidermis verses with fish species, age, size, different regions of body, degree of sexual maturation, sex and environmental conditions. It has also been argued that thickness of epidermis varies on the size and number of (he cells such as club cells, saccifomt cells etc. which constitute the predominate middle Layer of the epidermis. However, this does not appear to be the case in our study where club cells of C. batrachus are much taller than those of H. fossilis but the epidermis thickness is more or less same in both these catfishes. Such lack of relationship is also evident in many Indian major carps and common carp (Singh and Mittal 1990). This anomalous situation can be explained in the catfish where the area of club cells and MCs follow the inverse relationship leaving the effective thickness of the epidermis not being altered due to this compensation. Based on the aforesaid discussion, it can be concluded that the histological features of both these catfishes i.e. C'. batrachus and H. fossilis under the present study broadly conforms to the basic histological architecture of a typical teleostean species but few interesting deviations from broad generalization do exist which make this study significant. 53 B. Distribution of Glycoproteins (GPs): In the present study, the skin MCs of if fossilis exhibit the predominance of neutral GP whereas that in the C. batrachus it is the acidic. Furthermore, two different tissues i.e. skin and gill in the same species i.e. H. fossilis or C. batrachus show tissue specific variations in the type of predominant GPs in the MCs of both these organs. While in H. fossilis it is neutral GP in the skin and a mixture of all GPs in the gills, in other catfish C. batrachus it is the predominance of acidic and neutral OP in the skin and gill tissues respectively (see also chapter III for gill). The literature survey of this aspect reveals that such species-specific and tissue-specific variations within the same species do exist in many other species such as plaice P. platessa, rainbow trout Salmo gairdnert, flounder P. flesus (Fletcher et al 1976) and many other species. It is also evident from the available data that the acidic UP is found to be a predominant both in the skin and gill MCs (chapter III for gill). In skin, acidic GPs predominance is reported in eel Anguilla japonica (Asakawer 1970), brown trout S. trutta (Harris et al 1973), plaice P. platessa and rainbow trout Sc/no gairdneri (Fletcher et at 1976), spiny eel M armatus (7accone 1981b), senegal sole S. senegalensis (Arellano et at 2004) and cutaneously respiring fishes such as Korean spined ]each I koreensis (Park 2002), amphibious mudskipper B. pectiniroslris (Park ci a] 2003a) and Korean eel goby O. lacepedii (Park et al 2003b). Among the acidic dominant GPs in skin, the relative proportion of dominance of sulpho- or sialomucin seems almost equitable in different species, for instance, sulphomucin is predominant acidic moiety in plaice and nine-spired stickleback (Fletcher et al 1976 and Solanki and Benjamin 1982) but in rainbow trout and brown trout the sialomucin predominant as acidic GPs (Harris et al 1973). In gill MCs, however, the aulpbomucin distribution predominates over sialomucin in total acidic GPs distribution (see chapter III gills). It is interesting to note that the dominance of neutral GP in the skin MCs is very limited and reported to occur in flounder P. flesiu (Fletcher et al 1976) and green puffer fish Teh•uodon iuviatilis (Mittal and Bareijee 1976) and in the latter species, neutral GP is reported to be the only type of GPs. In the present study, while in C. hatrachus we observed the conventional trend of acidic GPs predominance in skin MCs but H. fossilis falls into that other category of few species where neutral UP exists predominantly. There are two more important observations which are documented in literature one related to the fact that in C. calla and C. mrigala the MCs in initial stage of development located in deeper layers appear to synthesize only neutral GP and the acidic moiety both sulphated and non-sulphated gradually gets incorporated upon further differentiation of these cells which are progressively pushed towards the surface (Singh and Mittal 1990). In another instance, there is an ontogenic change in the skin GPs moiety as observed in plaice where larval plaice integument contains only neutral OP but sulphates? GP makes its appearance by the 30'" day after hatching (Roberts el al 1973). There are large numbers of studies where only type and qualitative presence of GP in skin MCs have been described but no quantification to enable the determination of relative abundance of the various kinds of GPs has been given (Park et al 2002). It is now established that the distribution of different types of GPs in the skin does not conform to any specific pattern of either phylogenetic kinship, salinity based habitat i.e. fresh/sealbrackish water or any 54 other morphological or structural demand of a particular species?One_heeds'f probe deeper to ascertain if this distribution is simply random or is based an functional requirement of the animal. interestingly, there may be differences in the qualitative and quantitative distribution pattern of the GPs in the same species even in two closely located organs such as skin and the gills as is evident in H fossilis and C. bafrachus in the present study and also in flounder and others (FIetcher at al 1976). This necessitates the elaboration of the functional role of specific OP type such as neutral and acidic and in the latter, the sulpho- and sialomucin which together constitute acidic GP. One clear cut difference in the functional role of neutral and acidic GPs is that the former produces less viscous mucus whereas the latter produces more viscous. What has this property of mucus viscosity got to do with its functional significance has been elaborated by different authors in the different ways. Moron at al (2009) have mentioned that low mucus viscosity produced by neutral OP may protect the gill epithelium against physical injury. However, Pinky et al (2007), wile highlighting the function of GPs with 0- suiphated ester (acidic GPs) in hill stream Garra lamb stated that this OP produces high viscosity mucus which helps to lubricate heavily the rostra) cap and adhesive pad to provide protection against mechanical damage of these structures. Similar observations have also been made by Zaccone (1983), Whitear and Mittal (1984), Park et al (1987) and Park et al (2003). It is thus, obvious from this comparison that both high and low viscosity of mucus is essentially doing the same function of lubrication and protection. Similarly, it has been extensively documented that sulphated OP protects proliferation and inhibits invasion and colonization of pathogenic microorganisms on the epithelial surface but the mechanism of action of such a response has not been elaborated (Pinky eta! 2007, Mittal at al 1994 a, b, 2002, Supraset at al 1986, 87, Gone 1979). However, the similar functional role of prevention of fish from the pathogenic microorganisms has also been ascribed to carhoxylated OP. But in the latter case, it has been elaborated that the sialic acid coat covering the host eel I surface may prevent the pathogenic agents such as toxins, bacteria, viruses, prowzoans uud other exogenous macroruolccules by acting as the receptor site for binding the pathogenic agents (Ramphal and Pyle 1983, Schulte and Spicer 1985, Schauer and Kamerling 1997). Similarly, many other workers such as Culling et al (1974). Rosenberg and Schengrund (1976), Herder at al (1995) and Wharton et of (1989) have described the protective role of sialomucin against any bacterial or viral invasion. In the present instance, too, the overall function of both type of acidic GPs i.e. sulpho- and sialomucin is the sane but the mechanism of action may be different. In our study on catfish, while H fossilis has neutral predominance in skin MCs but it is acidic in C. batrachus. Both these fishes have striking similarities in term of habit, habitat, phylogenic proximity and apparently similar morphological and physiological requirements but the difference in the slain mucus (:Ps profile suggests that some more and, as yet unknown factors, are governing these variations in the skin MCs OP distribution. It is, therefore, not prudent to associate the occurrence of any specific GP moiety to only one specific function since each such function is governed by multiple types of GPs as has been amply illustrated above. Another role of GPs is in osmoregulatory homeostasis of teleosts but the precise details are mostly lacking (Diaz et al 2005, Moron et al 2009, Roberts and Powell 2005). However, Solanki and Benjamin ss (1982) elaborated that mucus layer over the skin surface may create a polyanion rich microenvimnment which may act as inn trap. Any cation effluxing from a freshwater fish would be attracted to polyanioaic glycoprotein coat which will also be extracted from the surrounding water and could then be absorbed by the fish by active transport. This will considerably retard the loss of ions from to body fluid in a freshwater fish which is of great osmoregulatory value for a FW teleost. The freshwater sticklebacks have large amount of sulphated GP than those of SW counterparts. Clamp et al (1978) have stated that sulphated glveoproteins are highly charged and can attract cations strongly. However, both the catfishes under present investigation which are the inhabitants in PW do not have the predominance of sulphornucin. Mckim and Lien (2001) have observed that skin mucus layer controls the water movement across the skin of the fish by establishing an osmotic gradient which is set up in the unstirred mucus layer. In case of freshwater, it is highest next to skin surface which shifts the osmotic gradient in favor of less water movement of fish across the skin from outside (hydration) which gets reversed in seawater fish where ion gradient is higher on water side of mucus layer and lower next to skin which curtails the water loss (dehydration) in SW fish (Shepherd 1981). C. Salinity Induced Changes: Our results on the increased ambient salinity on skin MCs number and size (area) in H fossilis show a biphasic response. A significant hyperplasia and hypertrophy in the MC is observed up to 30% SW but these values decrease significantly in 35% SW. Such a response of skin MCs in higher salinities is quite in contrast with that observed in the gill MCs which do not show any substantial changes in number and size in 25% and 30% SW but register a significant increase in both these parameters in 35% SW (chapter 111, gill). Our results on the salinity- induced changes in MCs in skin correspond with those in nine-spined stickleback P. pungitius where there is a dramatic increase in the epidermis goblet cells after transferring the fish to seawater followed by a gradual decline by 9`h day in seawater (Solanki and Benjamin 1982). However, the salinity increase did not causes any change in the gill MCs in the above fish up until 21th day when a distinct decline was recorded. But these two instances are not strictly comparable since the present study protocol is based on progressive salinity increase (TW, 25, 30 and 35% SW) whereas in P. pungitius, it is the variability of the duration of stay in of full strength SW. There are other studies on skin of brown buihead Ierahurus nebulasus (Zucholkowski et al 1986) and C. batrachus (Singh and Baaerjee 2008) where the authors observed hypertrophy and hyperplasia in skin MCs within 2 d and 12 hr of acid and arsenic exposure respectively. However, there are overwhelming numbers of studies which report the decline in MCs number and size following transfer to higher salinity as found in Sarorherodon mossambicus (Wendelaar Bonga and Meis 1981) and Cichlasoma biacellatum (Mattheij and Siroband 1971). Other stimuli resulting in the decreases of MCs number and size include the ectoparasitic infection with monogenea (Sterud et at '.998) and the sea louse in the Atlantic salmon (Nolan et a] 1999), beginning and end of smoltification in Atlantic salmon (0' Byrne-Ring et al 2003), spawning season in male brown trout S. trutta (Pickering [977), almuniun toxicity in Atlantic salmon S. salar with initial decrease followed by the subsequent increase (Bemtssenl et al 1997). These apparently conflicting observations in the literature on increase of MCs number and size in some and decrease in others have been convincingly explained by these authors by the fact that the reduction in the number of MCs is probably the result of exhaustion of MCs caused by extensive mucus secretion (Iger et al 1988, 1994, Roy 1988). The strong decrease in skin MCs is associated with general mucification of gill and skin. This phase is followed by the generation of new skin MCs which show a varying time span which ranges from 5-6 d in carps C. carpio (Iger et al 1988), Artie char Salvelinus alpines (Pickering and Macey 1977) and brown trout Salmo lruna (Pickering et al ]982). In rainbow trout Oncorynckus myldss gill and brown bullhead Ictalurus nebulosus skin exposed to acid toxicity a much shorter period of 2 d is required for generation of new MCs (Zuchelkowski at al 1986 and Audetand Wood 1993). A period of 72 h has been reported in freshwater C carpie for the new populations of MCs to appear following transfer to sublethal saline concentration (Abraham et al 2001). It is thus apparent that a clear cut biphasic response occurs following transfer of fish to altered salinity or due to parasite infection or multi source toxicity. The initial response is that of the sudden exudation of mucus over the surface forming a conspicuous protective mucus covering resulting in the exhaustion of MCs reflected in their substantially reduced number. This is followed by a more sedate phase where after a certain lapse of lime, the generation of new MCs where generation time frame varies from fish to fish and this proliferative phase of MCs result in their substantial increase. This has been amply demonstrated in C. carpio where a thick mucus coat over the epidermis became conspicuous a few hours after introducing the fish to sublethal saline concentration and an instance holocrine activity resulted in the loss of MCs. This followed, after 72 h, the emergence of new population of MCs near the outer boundary and their number became overwhelming subsequently in higher salinity (Abraham or at 2001). A similar biphasic response of initial decrease and subsequent increase in the MCs population was reported in Atlantic salmon (.S. salar) smolts following almunium exposure (Berntssenl et at 1997). It is therefore, abundantly clear that the apparent anomaly in the different reports on MC number dynamics following salinity or other stimuli may possibly be due to random sampling at varying time whereas the instances quoted above clearly underlines the need for time-course study. In our observations on catfish H. fossilis, the sampling was done after 7 day of residence in each salinity and it is quite likely that we have sampled the fish at a time when the catfish would be well into the 'recovery phase" where the number of MCs enhanced due to proliferation of immature MCs. But the clear-cut graded increase in 25% and 30% SW and then a substantial decrease in 35% SW with the uniform duration of acclimation in each salinity clearly points to the role of MCs in osmoregulatory homeostasis of H. Jbssilis in higher salinities although the precise machanism is not evident as on now. 57 D. Seasonal Changes in MCs Size, Number and Sulphomucia Contents: Our observations on annual seasonal variations which were documented on monthly basis on the size, number and sulphomucin content of the skin MCs of H. fossilis reveal an almost parallel profile changes in number and size of MCs. The changes in the above two parameters are marked by two major peaks- one spring peak during April and the other winter peak during December-January. The sulphomucin contents also register high values during March and June —February with highest levels during January but a near total absence is recorded during April and May. The profile almost matches with the number and size variation from July-August onwards. There are hardly any studies on seasonal variations in MCs dynamics possibly due to its arduous execution and also due to difficulty in interpretations of observations. It is logical to believe that the fish in its natural habitat is governed by the wide multiplicity of the factors and it becomes virtually impossible to quantity all of them. In the instant case, it is difficult to predict whether the observed changes in mucous cell number, size and sulphomucin contents vary either due to changes in ambient salinity or temperature or reproductive state or some other unspecified factor or due to collective response of consortium of these factors. Also, it becomes difficult to assess whether any one of the above factors exerts an overriding influence. The MCs seasonal profile change in the present study show no apparent correlation with its reproductive stages where values are just moderate between Junc-October and spaurning period occurs between June-August. Gonadal recrudescence resulting in the enlargement of gonadal size particularly the ovary may well cause a physical stress to the fish and so also spawning phenomenon which should get reelected in MCs hypertrophy and hyperplasia. However, contrary to this and except for an initial spurt in April, the MCs values remained moderate throughout this period ruling out any visible involvement in the spawning process. It is further strengthened by the fact that, all MCs parameters show a dramatic increase during peak of winter months i.e. December-January when the catfish is reproductively quiescent and not subjected to any gonadal size stress. Wilkins and Janesar (1979) in Atlantic salmon S. salar and Burton and Fletcher (1983) in winter flounder P. americanus recorded contrary observations where low temperature showed decrease in mucous cell frequency compared to a highly significant inciease observed in the catfish. Interestingly, in male brown trout the concentration of epidermal MCs dropped during spawning season (Pickering 1977). Thus, based on the seasonal profile of skin MCs in the catfish, no clear-cat involvement of MCs is envisaged in different reproductive phases particularly spawning season. The other likely isolated factor could be fluctuation in ambient temperature where both high as well as low temperatures appear to be stressful resulting in enhanced mucous cell activity. This is borne out by the observations on catfish H. jbssilis MCs profile where an increase is registered during April which coincide with the sudden spurt of temperature followed by a dip which show a period of adaptation up to October/ November. This period is followed by a sudden drop of temperature precipitating another stressful condition for catfish which again manifests in enhanced MCs activities. This is further corroborated by the fact that during the very next month i.e. February when the temperature starts gradually rising that the MCs activity show a gradual decline. A 58 similar explanation also holds true for seasonal variations in gill MCs which has been elaborately discussed in chapter III of this thesis_ Thus, it is logical to conclude that the ambient temperature, and not so much the reproductive stale, appears more causally Linked with seasonal variations in skin MCs profile. The changes in sulphomucin seasonal prolife in catfish integumentary MCs are very distinct and interesting where this GP moiety is totally absent during April and May and then record a progressively moderate increase up until October/November and then show a sudden spurt in December, reaching the peak during January followed by a declining trend. These observations on sulpbomucm clearly suggest that this GP moiety is not at all required during April and May resulting in its ail concentration and is maximally required during the months when temperature is low. 'Cite documented role of sulphomucin include the imparting high viscosity to mucus to serve the lubrication function, protection against invasion of pathogens, prevention against desiccation in air- breathing teleosts during air emersion and to increase negative charge in polyanionic surface mucus to attract cations for maintaining ionic equilibrium in FW leleosts (Solanki and Benjamin 1982, Mittal et a: 1994 a, b, 2002 and Pinky or al 2007). it one considers all the above documented functions of sulphomucin critically, its total absence in April-May and highly elevated concentration during winter months as observed in present study on the catfish does not fit into any of its documented roles. While its role in FW osmoregulation is elaborately described but any of its role in low temperature adaption process has not been suggested in Literature. But this decrease in temperature corresponding with a dramatic simultaneous increase in sulphomucin GP in this catfish seems clearly established. This provides us an important lead to further pursue this line with more detailed experimental studies to ascertain in unequivocal terms the role of sulphomucin in low temperature adaptation in this catfish and so also in other teleosts. We are into an exciting prospect of adding one more function to sulphomucin in the existing list of its multifarious roles. E. Ultrastructural Studies: The SEM study of catfish H foss{lis integument shows the overwhelming presence of pavement cells which border the outermost epithelial layer. These cells are characterized by conspicuous, concentrically arranged microridges with a single layer border. At the junctions of these PVCs, there are occasional openings presumably of the MCs which lie buried under these PVCs. These openings are clear, unblocked and are the exit points of the MCs for secretion of mucus which extensively fills the space between the microridges. Since the skin of this catfish is scaleless and is also devoid of any other epidermal or dermal derivatives which may project out of the skin, the entire integument surface under SEM view shows the predourinance of only PVC& and the opening of the MCs. The SEM study of the catfish integument has not revealed any other cellular or other structural details. The perusal of literature related to the SEM study on the integumentary surface structures show relatively fewer studies as compared to TEM studies on the same structures which may be largely due to the fact that SEM studies have the drawback of revealing only the limited information. For instance, any study dealing with SEM of integumentary surface shows the extensive appearance of pavement 59 cells which are essentially same structurally as observed in the catfishes. These cells are characterized by ring like microridges which are concentrically arranged in the catfish and are much the same in Senegal sole S. senegalensis (Arellano et al 2004), Favonigobius reichei (Ghaffar et a! 2006), Pseudorashoro parva parva (Fukuda 2001) and C. carpio (Abraham et al 2001). However, a few minor structural variations are seen in Periophthalmus chrysospilos where microridges are haphazard without any distinct concentric arrangements and there is a doubled-ridged border between the pavement cells unlike single-ridged border observed in the previously described teleosts (Ghaffar et at 2006). Similarly, African lungfish Protopterus annectenc show complicated arrangement of microplicae over PVCs giving honeycomb or sponge-like appearance (Sturla et at 2001). the other structure which becomes visible under SEM study of skin are the openings of MCs which are mostly located at the junction of two PVCs as in catfish but may also occur as centrally located pits in P. annectens which are filled with mucus (Sturla et al 2001). Any environmentally altered condition such as ambient salinity, sudden change in pH or temperature leads to copious exudation of mucus which covers the pavement cells over skin. A similar effect has been observed in the present study when the catfish has been transferred to 25% SW which has resulted in the copious secretion of mucus which filled the microridges to the extent of blurring its contours. In C. carpio, patches of mucus were seen in saline-stressed fish (Abraham eta' 2001), huge mucus accumulation occurred in Channa argus with sudden change of pH (Fukuda 2001) and a thick mucus covering over the skin surface in the aestivating African lungfish P. annectens (Sturla et al 2001). Interestingly, it is only in P. chrysospilos that the MCs orifices have been found to have microridges, though fewer in number, whereas in all the other teleosts quoted above for integumentary, SEM studies have clear MC opening without any traces of microridges-like structure. The functional significance of microridges are PVCs as being responsible to trap, hold or distribute mucus and to increase the surface area of apical membrane has already been discussed in detail in chapter III on gills and need not be repeated here. Under TEM observations of the skin, almost all the different cellular components can be observed quite unlike the SEM study which largely captures the view of surface lined pavement cells and the orifices of the MCs. Understandably, this is because of the plane of visualization. However, even under TEM studies, the observed predominant cells are MCs and pavement cells, which are universally present in the skin of al] tcicost fishes. The other cell-types which have been described under TEM integument studies are filament cells reported in Oncorhynchus mykiss, Gillichthys mirabilis, C. carpio, cuticle-secreting cells and undifferentiated cells in G. mirahilis, merkel cells in 0. mykiss and club cells in C. carpio (Marshall & Nishioka 1980, Iger et al 1994 and Abraham et al 2001). Chloride cells, in addition to gill epithelium, have also been found to occur in large number of euryhalinc teleosts but are mostly absent in stenohaline fishes (Abraham et at 2001). Rodlet cells which are likely to form non-specific defense mechanism of the skin have also been reported in epidermis of the fish exposed to stressors (Iger and Abraham 1997). M In the present study on H. fossilis, we have clearly deciphered the presence of MCs, pavement cells and the club cells within the limited observations made by us. Mucous cells clearly show the intermingled mucosomes of electron lucent (pale) and electron dense vesicles in the tap water maintained catfish. Interestingly, there is a well-marked regional aggregation of these mucosomes into distinct patches of electron dense and electron light granules. A somewhat similar observations reported in 0. mykiss where thermal stress causes the aggregation of electron dense granules in patches (Iger et at 1994) and also an intermingled arrangement of electron dense and light granules in the MCs has been reported in C. carpio (Abraham et al 2001) and freshwater chum salmon larvae Oncorhynchus kela (Wilson & Laurent 2002). An entirely different situation exists in j W pancalus (Mittal et at 1995) when three distinct types of MCs designated as Type A, B & C have been described on the basis of granules electron density and the position and the shape of nuclei. While we have observed a large number of chloride cells in the gills of both FW & 25% SW maintained Ti fossilis but these cells are conspicuously absent in the skin of this fish. A similar absence of these cells has been reported in the skin of C. earpio (Abraham et at 2001) even though chloride cells have been reported in large number of euryhaline teleosts such as Aphanius dispar (Chelouche et aL 1992), Gillichlhys mirabilis (Marshal and Nishioka 1950), Anguilla rostrara (Leonard and Summers 1976). and in the opercular skin ofRivulus marrnoratus (King et al 1989). Based on the above observation, it is tempting to suggest that the stenohalinity of the teleost may perhaps be in some way related to the absence of the chloride cells in the skin which will limit the ability of these fishes to make forays into higher salinities. Cl I Chapter III: Gills Chapter-Ill (Gills) I. Introduction: The gills are the major respiratory and osmoregulatory organs of aquatic vertebrates including fishes and have functional comparison with the lungs which are the counterpart respiratory organ of terrestrial vertebrates. However, it is interesting that both these major respiratory structures of diverse environments do have an intimate connection with the pharynx which is a cavity just behind mouth opening. Additionally, pharynx also functions as a passageway between the mouth and oesophagus as part of the digestive system and it is in the walls of the pharynx that swallowing movements are initiated. However, the association and the ultimate development of respiratory organs bath gills as well as lungs are more intimately linked with pharynx. The gills are the primary site of net Nat& Cl- transport actively taking up salt in fresh water and secreting them in seawater (Marshall and Bryson 1998, Evans et a] 1999, Chang et al2001, 2002, Marshall 2002 and Chen et al 2004). Teleosts generally lose ions passively through the permeable body surface such as the skin and gill (Percy 1997 and Kamaky 1998). The mechanism for balancing the ion loss and osmotic water gain is through the active uptake of ions across the gill and opercular surface and production of hypotonic urine (Perry 1997 and Karnaky 1998). The gills are the major site for not only gas exchange but also for ion transport, acid-base regulation, and nitrogenous waste excretion. The multiple functions of gill are comprehensively documented by Evans (1987) and some other reviewers on this subject (Perry 1997, Claiborne 1998, Kamaky 1998, Marshall and Bryson 1998, Walsh 1998, Evans et al 1999 and Evans et al 2005). Origin of Gills: One of the three prime diagnostic characters of the chordates is the presence of pharyngeal pouches during some stage of life cycle (i.e. embryo, juvenile or adult) located on either side of the pharynx. The numbers of these pharyngeal pouches are greatest in lower vertebrates and least in higher classes. Interestingly, in fishes and larval amphibians, perforation occurs in these pharyngeal pouches connecting them with the outside and these pouches are tamed gill clefs which are separated from each other by the skeletal structure called gill arch or visceral arch. These gill pouches develop vascular and lamellar extensions from the epithelial surface and may become functional gills in aquatic vertebrates. in lampreys and elasmobranch fishes each gill pouch opens separately to the outside whereas in higher fishes like teleosts, an operculum covering over the entire gill chamber is present communicating to outside only through a single exit. Most fishes have the internal gills but some in addition may have external gills at least during larval life. In amphibians, external gills occur which normally disappear upon metamorphosis and gill slits get closed. In other terrestrial classes such as salientians, reptiles, birds and mammals, pharyngeal pouches never open outside and 62 they get modified into other structures. The first gill pouch gives rise to Eustachian tube and middle ear and remaining pouches get modified into tonsils, parathyroid, thymus and other epithelial bodies. The lungs develop posterior to last gill pouch as diverticulum from the floor of the pharynx and if lungs are distally located, they are connected with the pharynx by trachea. Gross Anatomy of Teleost Gill: Basic functional anatomy of teleost gills-. The basic functional anatomy of the teleostean gill has been elaborately described by many workers and notable among them are Laurent and Dunel-Erb (1980), Laurent (1984), Satchel) (1984), Evans (1987 ), Evans of all (1999), Wilson and Laurent (2002) and Evans eta! (2005). Fig. A- Schematic teleost fish gill [Source: www.samey.is] Fish gills are built on a fundamentally similar plan to suit primarily its respiratory network. interestingly, such an organization is almost identical in higher groups of fishes such as'1'eleostei, Holostei but differs considerably in Chondrichthyes and Cyclostomes and this pattern is greatly modified in living Dipnoi. Hughes (1984) and Laurent (1982, 1984) have described the generalized basic structure of the gill system in teleost. In teleost fishes, four pairs of gill are present A complete gill is holobranch and each holobranch is made up of two hemibranchs. Both hemibranchs are connected to each other by gill arches. Gill rackets are present in one or two rows on the inner margin of each gill arch (Fig. A). The gill rackets may be soft, thin thread like or hard, flat and triangular or even teeth like, depending upon the food and feeding habits of fish. They form a sieve to filler out the water and protect the delicate gill filaments from solid particles. Each tacker is lined externally by an epithelial layer containing taste buds and mucous secreting cells. The taste buds help the fish in detecting the chemical nature of the water flowing through the gill slit. The perfusion of gill epithelium (both in the filaments and lamellae) is under the control of a variety of endocrine and paracrine 63 factors (Olson et al 1997) which may play a role in controlling the permeability of ionic transport in the gill. The gill epithelium is composed of several distinct cell types (Lament 1984 and Wilson and Laurent 2002). The major among them are pavement cells (PVCs), mitachondrial rich chloride cells (Ccs), mucous cells (MCs) and pillar cells (Laurent and Perry 1990 and Evans at a] 1999, Chabrillon et al 2004 and Diaz et at 2005). More than 90% gill epithelium of lamellar surface consists of PVCs which play an important role in gas exchange. (a) Structure of Gill Arch: Teleosts have four or five pairs of gill arches and each gill arch encloses afferent bronchial vessel and nerves. It is covered externally by a thick or thin epithelium in which a large number of mucous glands, club cells and taste buds are present. Each gill arch has one set of abductor and a set of adductor muscles for the muvements of the gill filaments during respiration. The abductor muscles are present on the outer side of gill arch connecting it with the proximal ends of the gill. The abductor muscles are present in the interbranchial septum and across each other so as to become inserted on the opposite gill rays. (b) Structure of gill filaments (Primary lamellae): Each gill arch bears two rows of the gill filaments towards the outer side of buccopharygeal cavity. In most telcost, the interbranchial septum between two rows of filament is short so that they are free at their distal end. The filaments are supported by gill rays which are partly bony and party cartilaginous and are connected with the gill arch and with each other by fibrous ligaments. Each gill ray is bifurcated at its proximal end to provide a passage for the efferent branchial vessels. (c) Structure of Secondary lamellae: Each gill filament bears large number of secondary lamellae on both sides. These flat leaf-like structures are the main site of gaseous exchange and vary in their shape, dimension and density per unit length of the gill filaments in species living in different ecological habitats. The secondary lamellae are free from each other but may be fused at the distal end of the filament. Basement Fig.B- Structural features of single gill lamellae 64 They vary from 10-40 in numbers on each side of primary lamellae being more numerous in active species. The central vascular regions of the secondary lamellae are composed of pillar cells covered by a basement membrane and an outer epithelium. The pillar cells are characteristic structure of teleostean gill and separate the epithelial layer of opposite side of lamellae. Each cell consists of a central body with extension at each end. These extensions are called the pillar cell flanges, which are connected from the neighbouring cells and form the boundary of blood channels in the lamellae (Fig. B). Gill Vasculature: Vascular components of gill epithelium: Broadly, in teleosts and so also in other fishes, there are broad divisions of vascular system- one called as arterio-arterial vasculature also referred to as respiratory pathway because it is mainly responsible for exchange of gases between the blood and circulating water current. The other type named arterio-venous vasculature is often referred to nourespiratory pathway with the likely function to provide nutrients to filament epithelium and to enter venous circulation without traversing lamellae. A brief description of arterio-arterial blood supply in teleost gill is given below. Artcrio-arterial blood supply in Telcost Gills: artery carotid Sinn ar artery venos Fig.C• Artedo•arterial blood supply in branchial and head region The entire cardiac output received sequentially through sinus venosus, atrium, ventricle, bulbus artriosus and finally into branchial artery enters the gill arch into afferent arch artery (AAA). Each AAA feeds filaments on the hemibranchs via afferent filament artery (AFA) which travels along the length of filaments (Fig. C). Some teleosts such as channel catfish Ictalurus punctatus, European perch Perea fluviafilis and rainbow trout Onchorynchui mykiss contain blebs or dilations which are speculated to perform The role of dampening pulsatile blood flow. Each AFA feeds one or more lamellae breaking into afferent lamellar arteriole (ALA) fine capillaries to facilitate the gas exchange becoming more oxygenated. Blood is collected from the lamellae, efferent lamellar arteriole (ELA) which drains into efferent filament artery (EFA). The EFA travels the entire length of filatucnt >;fferent side and joins with effercat arch arteries (EAA) which finally drains 6S into efferent branchial artery (EBA) (Fig. D). The oxygenated blood thus collected by EBA is drained into dorsal aorta from where it is distributed for systemic circulation. In arterio-venous pathway, the blood in the EFA is distributed to arterio-venous circulation via interlamellar vessels through post-lamellar anastomoses or via nutrient artery which arises from EfA or EBA and drains into bronchial vein which returns the blood to heart. Afferent arch artery arch artery Nutritive uecsel tilam Ent artery lamellar artery fitment artery Fig.D- Schematic diagram showing blood flow In lamellar region Cellular Components of Teleost Gill: The epithelium that covers the gill filaments and lamellae provide a distinct boundary between external environment and extracellular fluid of fish, and also plays an important role in the physiological function of the fish gill. This epithelium consists of different type of ccLis, primarily PVC 6 (Squamous) 90%, Cc, and MCs (goblet cells) together constitute 10% of remaining cells. 1. Pavement Cells (PVCs): The majority of the gill, filament epithelium covered by PVCs (more than 90%), they are considered to play a passive role in gill physiology of most tiahes. PVCs are assumed to be important for gaseous exchange and acid base regulation because they are thin squamous or cuboidal cells with an extensive apical surface area and are usually the primary cell types which cover the site of the lamellae. The apical membrane of pavement is characterized by the presence of microvihi and fingerprints like circular nderoridgcs which have elaborate arrangements that vary among species. These apical projections likely increase the functional surface area of the epithelium and may also play a role in anchoring mucus to surface. These cells also cover the surface of gill arch and gill rackers and their surface ridges often appear in coiled forms. Typically, PVCs in teleost do not contain the mitochondria but are rich in cytoplasmic vesicles or have distinct golgi apparatus (Laurent and Dunel-Erb 1980 and Laurent 1984). However, in Gii agnathans and elasmobranchs, these cells possess subapical secretory granules or vesicles that contain mucus and fuse with the apical membrane (Maillett and Paulsen 1986, Elger 1987, Bartels 1998 and Wilson and Laurent 2002). According to Evans et al (2005), the PVCs in freshwater teleost gill play an active rolc in km uptake and acid- base regulation. Laurent et al (1994) demonstrated the presence of studded vesicles near the apical membrane in the freshwater bullhead Ictalurus nebulosus gill epithelium resemble vacuolar-proton-ATPase (V-ATPase)-rich vesicles. 2. Mucous Cells (MCs): The mucous cells (MCs) distribution on gills are variable among species to species (Laurent and Dunel-Erb 1980) The MCs are abundantly present in epithelium covering the head of the gill arches and occur in relatively smaller number on the gill filament (primary lamellae) and vary in the secondary lamellae. In a cross section of the epithelium of the gill arch, the shapes of MCs vary from oval or pear shaped to round and these cells posses basal nuclei. In different species, the number and location of gill MCs axe quite variable. The mucous cells are absent from the secondary lamellae of unstressed rainbow trout so that only the filamental surface is coated (Handy and Eddy 1991). A histological study of the gill rackets and branchial arches in 0. niloticus revealed that two morphologically district types of MCs (Northcott and Beveridge 1988). Saboia-Morace et al (1996) described four different types of MCs on the basis of shape and size in the gill epithelia of guppy Poxci(ia vivipara. From a histological study of the gills of plaice, flounder and trout, Fletcher et al. (1976) suggested that the type of mucus produced by MCs in the arches of fish could vary depending on the habitat of each species. MCs secrete mucus which serves as the protective barrier of an individual. The mucus secretion in gill plays an important role in disease resistance, respiration, osmoregulation and also acts as natural defense against parasites. The mucous cells are ion exchange material which rapidly absorb H', particularly, it has the ability to increase the concentration of Nay, K`, CC in mucus on the gill surface (I Tandy et al 1989). Thus mucus may buffer incoming water passing over the primary lamellae, contributing to buffering capacity of the bronchial microenvironment (Playic and Wood 1991). The structure and function of MCs in the gills have been described by different authors (Hoar and Randall, 1984, Saraquete et al 1998, 2001, Arellano et al 1999, 2004 and Chabrillon et al 2004, Calabro et al 2005, Vigliano et al 2006, Cinar et al 2008, filer and Cinar 2009, 2010 and Diaz 2010). The precise role of MCs in osmoregulation is still unclear (Laurent 1984, Zuchelkowski at al 1985 and Shepherd 1994). However, the fact that MCs are most abundant in fresh water suggests that they might control the loss of ions or water influx (ion regulation). Prolactin administration brings about increase in the mtmher and density of mucous cells in freshwater fishes. The mucus secretion also plays an important role in disease resistance and respiration (Laurent and Perry 1990, Shepherd 1994 and Diaz et al 2001) as well as in natural defense against parasites and pathogens (Fletcher 1978). The functions of mucus cells are closely related to the kind of glycoprotein (GPs) produced by mucus layer (Gona 1979) which is involved in 67 mechanical, toxicant, pathogen defense and in osmotic insulation, ionic regulation and respiratory gas exchange (Shephard 1994). The MCs secrete mucus which contains different types of GPs in different teleosts which is divided into two groups i.e, acidic and neutral GPs (Zaccone 1972, 1973, Ojha and Munshi 1974, Mrmshi, 1996, Saboia-Moraee et al 1996, Powell of al 2001, Roberts and Powell 2003, 2005, Calabro et al 2005, Cinar et al 2008,Kurna i et at 2009, Oiler and Qinar 2009, 2010, Diaz et al 2010, Srivastava et at 201 land Patil et al 2012). The acidic mucin is made up either carboxylated or sulphated matins which can be differentiated by alcian blue 80X at pH 1.0 & 2.5. This can be fur her confirmed by methylation and saponification and can also be differentiated with AB (pH 2.5)/Aldehyde fuchsin (AF). Both acidic and natural GPs have been demonstrated by using the AB-PAS technique in many studies such as Jones and Reid (1993 a,b), Fletcher et al (1976), Ojha and Mishra (1987), Roberts and Powell (2003 and2005), Cinar et al (2008) and Pafil et al (2012). In some teleost, MCs secreting acidic GPs are more predominant compared to those secreting neutral ON (Fletcher eta! 1976). Interestingly, a mixture of acidic and neutral GP is present in the same cell which undergoes transformation from one type to the another type of glycoprotein under different ecological conditions (Olha et al 1982). The gill filament epithelial MCs of the freshwater mullets, Rhinomugi( corsula and Sciamugil cascasia have a mixed population of mucus secreting MCs having both acidic and neutral GP (Qjha and Mishra 1987). Quantification of MCs population by histochemistry is a widely used method for pathological assessment (Jones and Reid 1978, Ferguson et ul 1992 and Rubens and Powell 2003). The above light microscopy approach will not detect MCs which have recently discharged their glyceprotein content. It has been suggested that mucus with charged acidic groups may have increased particle retention properties (Nerthcott and 11everidge, 1988). Smaller goblet cells lined the anterior face and side of the gill arches secrete neutral or neutrallacidic mucus. This mucus may be less viscous and could aid in transport of captured particles towards the oesophagus (Northcott and Beveridge, 1988). In Oreochromis mossambicus, the proportion of mucosubstances present in the oral mucosa varied seasonally. During mouth-breeding, the concentrations of glycogen, sialomucins and sulfomucins increased compared to non- breeding seasons (Varute and Jirge, 1971). Thus, the oropliaryngeal mucus of 0. avreus may differ in acidity and viscosity from that of 0 nitotleus, and consequently differ in function. Under transmission electron microscopy, there are marked differences in the appearance of mucous and chloride cells which are distinguishable only under TEM and not by SEM. MCs appear generally smaller than Ccs and have distinct intracellular contents which comprise large mucin-contaning vesicles. Gill and skin show concentrically arranged rings of PVCs covered with floculent mucus and only the openings of chloride and mucus cells may be visible which cannot easily deciphered. The mucous cells number and dimension are dependent upon pathological or inflammatory processes in response to environmental conditions The abundance of MCs may also be affected by salinity changes. Generally, the increase in ambient salinity 68 simultaneously decreases the number and increases the size of MCs. In sea water, MCs are less in number on the leading and the trailing edges is comparison to the freshwater eel (Laurent 1984). In the former, however, Ccs developed mainly on the trailing edge, where they were seen to replace the MCs. Yadav er al (1983) examined the effect of salinity on gill MCs density and blood serum ion concentration in the fresh water fish Clarias batrachus. Gill MCs show more active development by Prolactin (Bern 1993), which reduces the gill water permeability (Ogawu 1973, Parwez et al 1984). In hypoohysectontized Fundulus heteroclitus, MCs show degeneration (Burden, 1956), a situation that could be counteracted by injection of pituitary homogenate. in the teleost, CichIasoma biocellatun MCs of the gills are not affected by osmotic conditions (Mattheij and Stroband 1971). Sotauki and Benjamin (1982) described the changes in GPs of MCs in the gills of the nine-spined stickleback Pungitius pungitus L., after transferring the fish to sea water. According to Dunel-Erb and Laurent (1973) the population of gill MCs is quite variable in number and location and their population might also be affected by changes in salinity (Roberts and Powell 2003, 2005). In fresh water telcosts Gambusia afnis and Carla ca/a, MCs disappear completely in chloride- containing media even at low concentration (Ahuja 1970). In Anguilla japonica it has been shown that the numbers of MCs decrease after their adaptation to seawater for a month (Lament 1984). In Barbrus flamentasus MCs are found mostly in interlamellat epithelium and the same authors have also reported, on the basis of light microscopy, that MCs arc able to transform into (Des after transfer from fresh water into 8% salt water (Zaccone 1981a). Such observations which need further evidence from electron microscopy, have already been reported in fresh water teleost gill (Das and Srivastava 1978) or in the abdominal epidermis of guppy (Scherdtfeger 1979). A euryhaline fish Etroplus maculatus adapted gradually from fresh water to 100% salt water showed an increase in size but decrease in number of MCs. At 100% sea water, MCs are reduced to negligible number in E. maculatus (Virabhadrachari 1961). The changes in structure and size of MCs in higher salinities have been documented in freshwater stenobalfine fish Clar(as batrachus (Yadav et al 1993), euryhaline fish Etroplus maculatus (Virabhadraehari 1961), Soekey Salmon Oncorhynhus nerka (Franklin 1990), seabream Sparus aurata (Bordas 2003), Atlantic salmon Salmo salar (Roberts and Powell 2003) traira Hoplias malabaricus and jeju Iloplerythrinus unitaenialus (Moron et al 2009). Mucus producing cells are numerically and morphologically affected by different conditions as do other cells localized in gill epithelium. Increase of MCs number is reported with different conditions such as bacterial gill disease (Ferguson et al 1992), amoebic gill disease (Monday et al 2001, Powell et al 2001, Shane and Powell 2003), high concentration of ammonia (Ferguson et al 1992, Hilary et at 2003), salinity (Franklin 1990, Berths or al 2003, Roberts and Powell 2003, 2005, Moron et al 2009), acidity (Charles and Terry I997, Ledy et al 2003) and high pressure and low temperature (Dunel-Erb et al 1996). On the other hand, under the conditions of high concentration of ammonia (Hilary et al 2003), low pH (Wilson et al 1994) high concentration of aluminum (Playle and Wood 1991), heavy metals (Jezierska and Witeska 2004), substrate of diazinen (Dutar et at 1993) and acid plus aluminium (Charles and Terry 1997), the size of MCs showed an increase. Burton and Fletcher (1983) suggested that in winter flounder Pseudap1esranctes amer-icanur, MCs exhibit seasonal variation. Changes in the number and dimension of MCs may be indicators of pathological or inflammatory processes induced by adverse environmental conditions (Lemoine and Olivereau, 1971, Wendelaar Bonga and Lock 1992 and Ortiz et al 1999). However, the above studies do not provide sufficient data to describe different types of GPs in MCs and their density and size changes following transfer to higher salinities particularly in freshwater stenohaline teleosts_ 3. Chloride Cells (Ces): Keys and Wilmer in (1932) for the first time described the Ccs in gill filament. These cells, also called mitochondrial rich cells due to the presence of large number of mitochondria, are responsible for the Cl- secretion in sea water adapted teleosts. Copland (1948) in his study designated these cells as Cos. The specific involvement of the hranchial Ces in Cl' secretion in seawater fishes, although originally disputed by Bevelander (1935), the mechanism of both Nay and Cl excretion across the gill of marine teleost fish is now well established (Silva et al 1997) and reviewed by Fuskett (1983), Kamaky (1986), Shutticworth (1989), Avella and Bornancin (1990), Wood bt al (1994). Marshall et at (1995), Petry (1997) and Evans et at (2005). These cells constitute only a small proportion of the branchial epithelium on gill surface, whereas PVCs are found in all regions of gill filaments. Ccs are usually more numerous on afferent (trailing) edge of filament as well as the region that runs between individual lamellae termed the interlamellar region. Also, chloride cell are usually not found on the epithelium covering the lamellae, however, certain environmental conditions are associated with the presence of lamellar mitochondrial rich Ces (Laurent and Dunel- Erbl9S0, Pisam 1931, Karnakey 1986, Laurent et al 1994, Perry 1997, Strula et at 2001, Wilson et al 2000, Chen et al 2004, Evans et al 2005, Sasai et al 2007 and Azizi el a! 2010). The morphology of the Cos is unequivocal when observed at ultmstructural level or by using light microscopy with specific staining (Watrin and Mayer-Gnstan 1996, Arellano et at 1999 and Calabro et al 2005). They are sensitive to environmental changes such as salinity (Laurent et al 1985, Pisam and Rambour 1991, Guner et al 2005 and Rrkmen and Kolankaya 2009), calcium concentration (Perry and Wood 1985 and Avella or al 1987), acidity (Lein et al, 1987 and Wendelaar Bonga et al 1990) and toxicants (Erkauen and Kolmikaya 2000). Localization, size and number of thcs© cells can change in relation to adverse environmental conditions (Watrin and Mayer-Gostan 1996). The chloride cells are large, ovoid shaped and their cytoplasmic mitochondria is closely related to the site of appearance for the active transport enzyme, Na`/K'-ATPase, which indirectly energizes Na4 and Cr secretion by these cells in SW teleosts. In fresh water teleost, Cos are predominantly found on the trading edge and interlamellar region but in some species, these cells may also be found on the lamellae where they are responsible for ion uptake. In several species of teleosts fishes, distinct morphology based Ces 70 subtypes named a and @ Ces have been found in gill. The a Ccs have light cytoplasm, usually a smooth apical membrane associated with numerous subapical vesicles, a well developed tubular system elongated in shape and are found at The base of lamellae. In contrast, (i Ccs have dark cytoplasm, usually with complex apical membrane projections associated with an extensive subapical tubular vesicles, a less developed tubular system which is ovoid shaped and occur in the interlamellar region. The a and I3 types of Cos are thought to be associated with seawater and freshwater osmoregulation respectively (Perry 1997 and Evans et at 2005). In several species of teleost fish such as Atlantic salmon Salmo solar, Salmolratta, Poeci(ia reticulate, Cob ills (aenia, Gobio gobio and 0. nzlolicus Ccs subtypes have been detected in the gills (Pisani et al 1987, 1988, 1990, 1995, 2000, Evans et at 2005 and Lee et at 2008). Seawater Cos are large and generally more numerous than those of fresh water (Shirai and Utida I970, Pisam 1981, Hwang and Hirano 1985, Uchida et al 1996, Schreiber and Specker 1999, Strula et al 2001, Chang et at 2002, Parashar and Banetj ee 2002, Arellano et at 2004, Chen et al 2004, Sasai at at 2007 and Erkmen and Kolankaya 2009). Generally, the apical surface of seawater-acclimated fish opercular epithelium has pockmarked apical crypts of varying size, each surrounded by flattend PVCs and fresh water adapted fish has Ccs covered with pavement cell ( Marshall and Bryson 1998). In fresh water teleosts. Ces are found on the trailing edge and interlamellar region of gill filament but lamellar Cos arc also present in some species (Uchida or al 1996 and Shikano and Fujio 1998). The ultra structure characteristic features of Ces in fresh water teleost share some similarity to sea water, such as extensive basolateral membrane and tubular system associated with mitochondria] and subapical tubular vesicular system (Laurent et al 1994 and Perry 1997) and differences such as the accessory cell and multicellular complex associated with Ccs not found in fresh water. Significant structural changes in the Ccs occur when the fish is t artsferred from freshwater to sea water (Karnaky et al 1976 and Marshall and Bryson 1998) and vice- versa (Copeland 1948, Threadgold and Houston 1964, Doyle 1977, Laurent and Dunel-Erb 1980, Hossler 1980, Foskett or at 1981, Zaccone 1981n, Lee at at 2008, Etknren and Kolankaya 2009 and Mitrovic and Perry 2009). The above studies have clearly shcnvn that Ces increase in size, number and exhibit darkening of cytoplasm when the fish is transferred from the fresh water to sea water. Some techniyues arc employed fur the luealization and identification of Cos such as fluorescent mitochondrial marker (Karnaky 1986, Perry et at 1992) or histochemical (Ahuja 1970, McCormick 1990, Lin et al 2003, Chen et at 2004,Nebel et at 2005, Mitrovic and Perry 2009 and Azizi or at 2010) or immunohisiechemistry (Ura et at 1996, Wilson et a12000, Uchida et at 2000, Chen ct at 2004. Hiroi and McCormick 2007 and Allen et at 2009). Marshall and Bryson (1998) used DASPEI fluorescence and scanning and transmission electron microscopy technique to map the surface and subsurface structures of Ucs both in fresh water and sea water acclimated Killifish. 71 4. Accessory cells (ACs): The ACs are similar to Ces in terms of structural details except that these cells are much smaller with less developed tubular system and lower expression of Na`, Pt-ATPase as compared to Ccs. Earlier the ACs were found to be exclusively present in SW teleosts but some later studies reported their presence also in FW teleosts such as loath Cobitis taenia and gudgeons Gobio gobio. The transfer of euryhaline fishes such as vigil cephalus, Fundulus hereroclilus and Salmo gairdneri following transfer from SW to FW results in the disappearance of ACs. In SW teleosts these ACs share the apical crypt to give rise to complex interdigitation which forms a paracellular pathway for the ionic excretion. Hence, these cells are also called companion cells to Cos in SW teleosts. These cells have also been regarded as the developmental stage of the Cos which finally get fully transformed into matured Ces. 5. Pillar cells (PC): These cell types unique to the fish gill are basically modified endothelial cells located in close proximity with the blood spaces within the secondary lamellae. The cells have centrally located polymorphic muscles and each comer side of these cells flare out to form flanges which virtually enclose the blood channels and extend up to the next pillar cells. These flanges are thin, devoid of organelles except microfilaments and column of collagen bundles intertwined on the vascular side to prevent the collapse of blood space. These cells contain mitochondria, free ribosomes, Golgi apparatus and rough and smooth endoplasmic reticulum (RER & SER). 6. Neuroepithelial cells (NECs): Neuroepithehal cells (NECs) found in the gills of clasmobranchs and teleosts are generally located on the efferent border of the filament mostly restricted around filament tip region. These cells lie closer to the nutrient and interlamellar blood vessels. The NECs are believed to serve as oxygen sensors since they become degenerated lithe fish is exposed to hypoxia. Laterestingly, the dark-cored vessels contained in these cells are rich in serotonin and are involved in the regulation of blood flow. The identification of these cells is largely based on the detection of biogenic amine fluorescence. 7. Rodlet cells (RCs): The rodlet cells are rarely occurring cells reportedly present on gill arch, filament, inter lamellar and basal areas of the lamellae. These cells are characterized by numerous electron dense spindle or club-shaped rodlet sacs. Another distinguishing feature of RCs is the presence of unusually thick fibrous border immediately beneath the cell membrane. These cells have also been encountered in wide range of tissues such as skin, kidney tubules, intestine and also in mesothclia and endothelia of freshwater and marine teleosts (Manera and Dezfuli 2004). S. Undifferentiated cells (UCs): As the name implies, these cells are undifferentiated in nature and serve as progenitors for other cell types such as PVC or Cos. The defining feature of these cells is a high 72 nucleus to cytoplasm ratio and large number of free ribosemes relative to PVC. They are more abundant in filament than the lamellar epithelium and connected with desmosomes with PVC and hemidesmosomes with basal lamina anchoring the epithelium (Wilson & Laurent 2002). STATUS OF THE STUDIES ON THE GILLS OF THE TELEOSTS OF INDIAN SUBCONTINENT: The survey of literature reveals that considerable amount of work on the structure and function of the fish gills have been done on large number of fish species of Indian subcontinent. A major quantum of work is focused on the au-breathing fishes such as catfishes H fossils, C. batrachus, dnnbas tesrudineus, i61anpterus cuchia and four species of C) onna. The studies on the gills have been carried out along four major lines i.e. general morphology based on light and electron microscopy, toxicological effects on the surface of gills, salinity-induced changes on gill architecture and qualitative and quantitative study of glycoprotein moieties on the gill epithelium. Of the above, the morphological studies are perhaps most abundant and carried out on ten different species belonging to different genera. The major contribution on this aspect has been made by Munshi and cc-workers (Hughes and Munshi 1968, 1973. 1978, 1979 and 1986; Munshi et al 1978,1980, 1984, 1986 ; Munshi and Singh 1992). Some of the fishes which have been studied for branchial ultrastructural details are H. fa.ssilis, C. batrachus, Channa striata, Anabas testudineus, Cierhinus mrigala, Rita rito and Setipinna phasa (12ajbanshi t977, Hughes and Munshi 1978, 1986, Kumari eta! 2005, Kumari eta' 2009, Naskar et at 2009 and Moitra et a] 2009). The toxicologal studies on the gill epithelium are mostly confined to the air-breathing fishes such as H. fussifis, C. 6curuchus and two species of Channa in addition to Indian major carp Cirrhinus mrigala. The major toxicants used in these studies are cadmium chloride and lead nitrate (Parashar and Ranerjee 2002 and Sosithn et at 2007), ammonium sulphate (Paul and Banerjee 1996b), zinc chloride (Hemalatha and Banerjec 1997), mclathion (Roy and Munshi 1991 and Patil et al 2012) and acid and aluminium (Naxkar et al 2009). The salinity induced changes in the gill epithelium have been studied in Etroplus maculaluc (Virbbadrachari 1961), C. batrachus (Yadav et al 1983), Ganthusia q finis &Calla calla (Abuja 1970) and Setipinna phasa (Moitrn et al 2009). There are only limited studies on the localization of Ups on the gill epithelium notably on Mgcrogrethus aculeaium (Ojha and Munshi 1971), Cirrhinus mrigala (Srivastava et at 2011), Rita rha (Kumari el a] 2009) and Channa punctatus (Patil or al 2012). From the foregoing description, it seems clear that a large number of species of Indian subcontinent have been investigated on the morphology and structural alterations in gill epithelium under different stimuli and environmental conditions which could largely be possible because Indian subcontinent harbours a wide piscine biodiversity. 73 OBJECTIVESOF STUDY: In our laboratory, we have covered major osmoregulatory aspects of H fossilis (Parwez et al 1979, 1984, Goswaai et al 1983 and Sherwani and Parwez 2008) and to a lesser extent on C. batrachus (Patwez et al 2001). The structure and function of the air breathing organs of H. fossils has been described in detail by Munshi (1996), and toxic impact of lead salts on the respiratory organs of air breathing fish H. fossilis and C. hatrachus by Parashar and Banerjee (1999 a, b, c, 2002), Chandra and Banegee (2003) and Naskar et al (2009). Susisthra et al (2007) have demonstrated the toxicopathological effects of the heavy metal salt, cadmium on the respiratory epithelium of air breathing catfish Ii fossilis. However, no systematic investigation has been carried out to study the presence of different types of glycoprotein moieties in MCs, their seasonal alterations in population density and GPs contents and the effects of salinity variations on the MCs and Ccs in gill epithelium which play an important role in fresh and salt water osmoregulation. In the present study, an attempt has been made to study the aforesaid aspects on the gill MCs in H. fossilis and C. bab'achus. In the present study. therefore, the following objectives have been undertaken. i. Qualitative and quantitative assessment of the different types of glycoprotein moieties in MCs of the gills of H fossilis and C. batrachtes in fresh water. ii. To study the circannual profile (based on monthly sampling) of the changes in the glycoprotein moiety, number and size of the MCs in the gills of the catfish H fossilis. iii. To quantify the changes in glycoconjugate contents, number and size of MCs in the gill of the catfish H. Jossilis, following gradual transfer of the catfish from TW to higher salinities i.e. 25%, 30% and 35% SW. iv_ To localize the Ces by light microscopy in TW maintained catfish H. fossilis gill using variety of histochemical stains and to quantify the changes in their number and size following sequential transfer to 25% and 30% SW. On the basis of inununohistochenustry and electron microscopy (TEM & SEM), to further confirm the cellular entity of Ces in the gills of H. fossidis and to study their structural alterations following transfer to higher salinity i.e. 25% SW. 11. Experimental protocol: Like in the previous chapter II on integument, three major aspects were also studied on the gills of the catfishes i.e. H fossilis and C. batrachus. 1. The first aspect deals with the studies relating to general morphology and histology and also the qualitative and quantitative aspects of glycoeoniueate moieties of the gill mucous cells M H. fossifis and 74 C. batrachus. The details of the staining techniques, quantification of various glycoconjugate moieties and the statistical analysis have been described in Materials and Methods. 2. The experimental protocol on the second aspect on the salinity-induced changes in elycoconiue,ate moieties, number and size in pill mucous cells of IL fossrds is the same as described previously in chapter II on skin studies. In addition, the changes in the size and number of chloride cells in H. fessilis following transfer to higber salinities have also been studied. In this study, gill chloride cells are also localized histochemically using acid hematein, sedan black B, toluidine Blue and Champy Maillet's fixative/OZI and inununohistochemically (only in 25% SW maintained catfish) as per details described in Materials and Methods. 3. The third aspect is focused on the qualitative and quantitative seasonal chaoaes in the elvcoeoniaeates of eih mucous cells of H. fossi is. The details of the experimental protocol on this aspect are the same as described in chapter II on skin. 4. For ultrastructural studies of scanhine and transmission electron microscopy of H. fossils gills, five specitnens each from TW and 25%SW groups are sampled as per details described in Materials and Methods. III. Observations: A. External Features and Gill Morphokogy* of H. fossils; 1Se body structure of H. foss//is is elongated with compressed abdomen and narrow and terminal mouth. Head is greatly depressed, moderate in size and the external body is covered with scaleless skin. There are four pairs of barbels, one pair each of maxillary: easel and two mandibular. Dorsal fin is short and inserted slightly away from the tip of pectoral fin and is provided with strong serrated spine along the inner edge. Anal fin is long, just reaching the round caudal fm and separated from it by a notch (Fig-3.1). Like other teleosts, the gills of catfish consist of five pairs of gill arches where four pairs are complete with two holobranchs but the fifth pair of gill arch located towards the posterior side and consists of a single hemibranch (Fig- 3.2 B). The gill arches from anterior to posterior side get progressively reduced in size. In a complete gill architecture of H. fossils, each gill arch has two rows of hundreds of filaments which are also called primary lamellae and each filament bears the series of secondary lamellae arranged in two rows on both sides. At the opposite side of the filaments, there are short and spiny or stumpy structures called gill rackers which are also present in two rows on the each gill arch (Fig- 3.3 A, B). The two hemibranchs are connected with each other with an interbranchial septum (Fig- 3.3 B). 75 The longitudinal sections of a complete gill stained with H/E show that each filament which appears a leaf like structure is made up of cartilaginous support, a vascular system and single or multilayered epithelium (Fig- 3.4.1 A & B). The cellular components arc primarily made up of epithelial cell layers which may vary according to their location i.e. multilayered epithelium on filament and gill arch and a single layer on the lamellae which are supported internally by cartilaginous gill rays and pillar cells (Fig- 3.4.1C & F). The adductor muscles considered to be responsible for controlling the filament movement are situated at the head of gill rays (Fig. 3.4.1 B). The blood supply in gill arches is in the form of afferent and efferent arteries which enter and come out from each primary gill filament, run parallel with the gill rays and are finally connect with blood channels located in the secondary lamellae (Fig- 3.4.1 A & B). The secondary lamellae are made up of alternately arranged pillar cells with blood channels which contain one or two blood cells in it and all these structures are enclosed within a basement membrane (Fig- 3.4.1 F & G). The filament epithelium is multilayered whereas the epithelia] lining of secondary lamellae is only two layered thick. These epithelial cells have a predominant population of pavement cells which constitute about 90% of the total cells tbllowed by the mucous cells (MCs), chloride cells (Ces) and uudifferenciated cells (UC) making up the remaining 10% cells (Fig- 3.4.1C, D &E). The gill arch is covered by thick epithelium which is highly secretory due to the presence of large number of MCs and a large number of taste buds are also present at the margin of gill arch (Fig-3.4.2 H-K). MCs which stain positive with PAS-AB (pH 2.5) are found to occur on gill arch, gill filament and secondary lamellae and show a decreasing abundance respectively (Fig- 3.4. ID & 3.4.2 K). At the base of lamellae, the MCs may occur in a cluster of 2-3 cells or singly and are occasionally present on the secondary lamellae (Fig. 3.4.1 D). Mostly MCs are goblet or globular in shape throughout the filament but on gill arch these are globular in shape but change to flask-shaped with a distinct neck when the mucus oozes out on the surface (Fig-3.4.2 J). The chloride cells which stain positive with Acid hematein are mostly located on the interlamellar region of filament or at the base of secondary lamellae, rarely found on secondary lamellae and are totally absent on the gill arch (Fig-3.4.1 E). B. External Features and Gill Morphology of C. batracbas: The body structure of C. batrachus is largely similar to that of the H. fossilis in terms of body shape, size and contours except that the head which is dorsally covered with osseous plates and laterally form a cask. Like in H fossilis, the anterior portion of head is provided with four pairs of barbels, one pair each of maxillary, nasal and two mandibular. Dorsal fin, unlike H, fossitis, is long and without spine. Pectoral fm is below the origin of dorsal fm & its spine is finely serrated Loth externally and internally. Caudal fin is free and almost round whereas anal fin is long. The colour of this catfish is dingy green or dark brownish (Fig-3.5, 3.6 A) with five pairs of gill arches, four pairs are holobranch and last pair is hemibranch which is similar to that found in N fossilis (Fig-3.6 B). Some of the 76 unique9 and interesting features observed in C. batrachus are~h'etYiel~turdy a-" and thick gill arch, relatively short gill filament and fused gill rackets located opposite to the gill filament (c.f. Fig- 3.7 with 33). Further, The length of the secondary lamellae is significantly reduced and has a fleshy appearance due to the multiple epithelial cell layers which are absent in K fossiIis. The rest of the structural features of cellular, vascular and skeletal components of the gill in C. batrachus are the same as described for H fossilis (Fig-3.8.1 & 3.8.2). C. Glycoconjugates (GPs) in Gills Mucous Cells: H. fossllis: The relative abundance of glyeoprotein (GP) moiety in the branchial MCs of H. fossilis has been depicted in Fig 3.9 and 3.11. The number of MCs with different OF moiety counted per unit area with the use of different stains such as AB (pH-I 2.5) (Acidic), Methylation/AB (pH 2.5) (Sulphated), Saponification/AB (pH 2.5) (carboxylated), PAS/AB (pH 2.5) (Mixture of neutral and acidic) and PAS (neutral) have given the mean values of 88 t 6.23, 73 ± 5.8,75±6.8, 83 t 9.1 and 81 f 6.1 respectively. The perusal of Fig. 3.11 clearly shows that there is no statistical difference in the absolute numbers of different kinds of GP moieties such ass acidic, sulphated, carhoxylated, neutral and mixture of acidic and neutral. In order to doubly check the presence of sulphated glycoconjugate moiety, the gills have been stained with AR (pH 1.0) (Fig. 3.9 B) which shows almost the same number of MCs as observed after methylalion (Fig. 3.9 C) and total acidic MCs stained with AB (pH 2.5) (Fig. 3.9 A). Further, branchial sections nave also been stained with PAS/AB (pH 1.0) to differentially localize the number of neutral and sulphated glyoomoiety (Fig. 3.9 F). A close look of the sections reveals that there are neither pure blue nor pure magenta colour MCs indicating thereby the absence of pure, sulphated and pure neutral glycoconjugate containing MCs. The gill sections stained with AFIAB (pH 2.5) to differentiate the pure sulphated from the pure carboxylated GP also reveal that no such difference is visible (Fig 3.9 1-1). C batracbus: The qualitative abundance of glycoconjugate moieties in the gill MCs of C. balrachus shows a profile which is quite different from that of the H. fossilis (c.f. Fig 3.9 vs 3.10 and 3.11 vs 3.12). The major change in the qualitative and quantitative abundance of these GP moieties is largely because of the presence of pure sulphated and pure carboxylated out of the total acidic OP moiety but these pure forms arc, however, absent in FL fossilis. This is borne by the fact the mean number of pure sulphated moiety per unit area (12.5 ± 5.7) and carboxylated moiety (20.23 f 6.6) almost equals to that of the total acidic moiety (32.75 t 3.7) which is quite unlike of K fossilis where each of the above mentioned moiety has almost the same mean number per unit area. Another interesting observation is that the sulphated moiety number is comparatively much less than that of carboxylated moiety which is also corroborated by the perusal of Fig. 3.10 F & H where the gill 77 epithelium are stained with PAS/AB (pH 1.0) and AF/AB(pH 2.5) respectively. In C. batrachus neutral moiety shows the highest abundance which is different from H. jbssilis when acidic moiety seems more predominant. I-Iowever, none of these . values of predominant CPs are statistically significant Further, a close look of Fig 3.10 E clearly shows two distinct population of the cells where one type is stained pure magenta and other with the different shades of purple showing file presence of pure neutral and the mixture of acidic and neutral moiety. Such a situation, however, does not exist in H. fossilis where all the MCs stained with the combination of PAS/AB (p1I 2.5) show the purple colour cells which is a mixture of acidic and neutral glycoconjugates (c.f. Fig. 3.9 E vs 3.10 E). The number of MCs containing the mixture of acidic and neutral glycoconjugates are more or less similar in both the calfishes (c.f. 3.11 vs 3.12). The fig 3.13 summarizes the qualitative and quantitative changes in the GPs of MCs of H Jossilis and C. batrachus where the above described differences are apparent at a glance. D. Salinity Induced Changes in Gills Mucous Cells: The catfish H fossilis were sequentially transferred to 25%, 30%, 35% SW after 7 days residence in each salinity. There is no statistical difference in the relative abundance of different kinds of GPs moiety i.e. acidic, carboxvlated, sulphated, neutral and mixture of acidic and neutral in the gill MCs compared to those of the TW maintained (control) fishes (compare Fig. 3.9 with Fig 3.14, 3.15 and 116). Interestingly, the gill lamellae of the fishes maintained in 25% SW show profound swelling which, however, disappears completely in 30% and 35% SW maintained fishes (Fig. 3.14, 3.15 and 3.16). The number of the MCs also shows interesting variations in different salinities in as much as they show a significant increase (P<0.0S) over tap water control in 25% SW, followed by a decrease in 30% SW becoming almost equivalent to TW and then register a significant increase (P<0.001) in 35% SW. However, Sc size of MCs shows a consistently increasing pattern in different salinities when the increase in 25% SW, though distinct, is statistically insignificant, but in 30 and 35% SW the increases are more pronounced and statistically significant (P<0.05 and P<0.01 in 30% and 35% SW) (Fig 3.17). However, the profile of the size and the number of skin MCs following transfer to higher salinities is found to be quite different with that of gill profile. In skin, there is a significant increase in size and the number of MCs in 25% and 30% SW which, however, shows a sudden decline in both these parameters in 35% SW where these values become almost equivalent to those of the tap water control (Fig 3.17 and 3.18). F. Seasonal Variations in Gill Mucous Cells: In the month of March (Fig. 3.19.1 A, a), the MCs of catfish H. foss//is are distributed on the interlamellar, base of lamellae and occasionally on the secondary lamellae. Their average number and size per unit area during this month is 59 ± 2.5 and 46.72 ± 5.6 (µm2) respectively (Fig. 3.20, 3.21). They are goblet shaped and do not exhibit oozed out secretion and the granules are arranged in a condensed 78 fashion. The histochemical staining with AB (pH 2.5)/PAS shows purple stained MCs which indicate the mixture of neutral and acidic t'IPs. The month of March when weather conditions were moderate was taken as a reference month for the comparison of number and the size of MCs with the rest of the months of the year. Based on this comparison, the MCs number showed an increase over the month of March (59 t 2.5) during the months of April (1(13 t 1.7), September (105 f 7.8), October/November (85.75 f 4.7), December (160 ± 15) and January (135±8.3) (Fig. 3.19.1,2,3 B, b; F, f; G, g; H, hand I, i & 3.20). Interestingly, sudden surges in the number of MCs were observed from March to April, August to September and November to December where the last surge was most significant (P<0.001). Only the month of May (29.5f2.6) registered a significant decline (Fig. 3.19.1 C:c). Whereas the number of MCs was more or less similar during in June (48.25 ± 6.3), July/August (57 f 5.5) and February (68.75 f 19.2) when compared with the month of March (Fig. 3.20)_ The perusal of the Figs. 3.19.1,2,3 and 3.20show the seasonal (monthly) variations in the size of the gill MCs which shows a sustained increase from May to January compared with March and a spurt in the values from April to May and November to December recording the highest value during December. The values of the MCs area were almost similar during April and February when compared with the month of March (Fig. 3.20). Interestingly, the correlation between the numbers of MCs with those of its size shows an inverse pattern during April and May and an almost parallel pattern during the remaining months (Fig. 3.20, 3.21 upper panel). The shape of MCs are irregular in April, June and July/ August months and these cells stain purple with AB (pH 2.5)/ PAS showing the presence of mixed glycomoicties i.e. neutral and acidic (Fig. 3.19.1,2 B, b; D, d and E, e). The data reveal a significant vacuolaliion in the gill MCs which becomes visible during the months of October/November with the highest vacuolation degree in December which continues up to January (Fig.3.19.2,3 G, g; H, h; 1, i). Similarly, a predominant acidic glycomoicty is evident by blue colour staining of the MCs with AB (pH 2.5)/ PAS during the months of April, May, September and October/November (Fig.3.19.1,2 B, b; C, c; F, f; (3, g and H, h) whereas the rest of the months show the predominance of mixed GPs i.e. neutral and acidic which is evident by purple stained MCs by AB (pH 2.5)1 PAS. F. Gill Chloride Cells (Ccs): in the present study, out of the four used stains i.e. toluidine blue, Sudan black B, acid hematein (All) and Champy Maillet's fixative/ osmium zinc iodide (OZI), only acid hematein and OZI have successfully stained the chloride cells (Ces) whereas lulaidine Blue and ssulau Black B do nog give us pusilive results with reference to localization of Ccs. The gill lamellae show the predominance of PVCs which stain brownish followed by unstained MCs, a few accessory cells (ACs) and darkly stained Cos (Fig. 3.22). The Ccs are mostly confined in the interlamellar and tip region of filament either as a single or in cluster of two cells and occasionally 79 the number increased to four cells in the same cluster with varying size of cells (Fig. 3.22 A, B, C and E). In the secondary lamellae, the cells are mostly single (Fig. 3.22 A,B). The number of Cos per unit area is 66.25 t 6.3 and its average area is 24 f 0.4 is TW maintained fish. The Ccs stained blue with acid hematein and show clear-cut granulation of varying degree at higher magnification (400x, [000x) showing large granules (Fig. 3.22 C & E). Interestingly, the Ccs stain black with OZI and do not show any degree of granulation (Fig. 3.25 A,B&C). The nucleus which is brownish is displaced from the centre and located towards periphery (Fig. 3.22 E and F). In the catfish, the Ces are usually oval or round in shape with no location] variation in shape (Fig. 3.22 A, B). MCs remain totally unstained both with All and OZI and appear in the form of large vacuoles. The ACs which are fewer in number, confined mostly in the interlamellar region with hardly any in the secondary lamellae, stain light brown with moderate degree of granulation (Fig. 3.22 E &F and 3.23 E). When the catfish are transferred to 25% SW for 7days, the number of Cos (21.75 t 3.3) are significantly decreased (P<0.001) compared to TW maintained catfish (66.25 f 6.3) whereas the area of Cos show slight but insignificant decrease in this salinity when stained with acid hematein and OZI (Fig 3.23 A,B,C,D & E, 3.25, D, E & F 3.26 and 3.27). Likewise, the ACs number registers an increase in 25% SW but their size remains more or less same (Fig 23 D). There is also a significant aggregation of the ACs in the secondary lamellae and tips of filaments and they are hardly present in the interlamellar region (of Fig. 3.23C& D and E 3.22 D, E & F). Gradual transfer of the catfish FL fossilis from 25% to 30% SW for 7 days results in a dramatic change in the status of Cc which register a highly significant increase in number (168.5±7.2) which is three times higher in 30% SW (Fig. 3.24) compared to TW and 25%SW. On the contrary, the size of Ccs significantly decreased (P<0.001) compared to TW and 25%SW (Fig. 3.24, 3.26 and 3.27). Interestingly, the Ccs are present in extensive numbers almost all over the gill epithelia with their maximum number on the secondary lamellae (Fig. 3.24 B,C &D). The ACs show further increasing numbers in 30% SW maintained fish and are present largely in secondary lamellae (cf pig. 3.24E, 3.23D and 3.22C). G: Imm unobistochemistry: The immtmoreactive Ces were clearly visible in the interlamellar region which are varying in shape from ovoid to polygonal. Some Cos are also present in the lamellar region of FW maintained fish even though their numbers are relatively very few. In 25% SW maintained catfish, the Cos are distributed in more or less same fashion as those found in FW maintained catfish. However, in some 25% SW maintained catfish, there is greater abundance of Ccs both at the lamellar and interlanrellar region and huge cluster at the apical] trailing region (Fig-2c). In some sections, usually two chloride cells occur within one interlamellar region. In limited sections observed by us, we have not observed any appreciable difference 80 in the size or the abundance of Cris between TW & 25% SW maintained catfish. It is likely that if sufficient of individual fishes arc observed, a more authentic conclusion in terms of Ces population in higher salinities may be made. The blackish stained RBC.s are also abundantly present. However, they are clearly distinguishable from Cos by being relatively smaller and being present abundantly in the lamellar region. EI: Electron Microscopy: i. Scanning electron microscopy (SEM): Under scanning electron microscopy each holobranch gill of K foss!Its, shows two rows of well-organised primary filaments with leaf-like secondary lamellae lined up along both sides (Fig. 330.1 A-C & 3.31.1 A-C). The secondary ]amellae are wrinkled and backwardly curved a little before the tip region which is free at the distal end (Fig. 3.30.1 C & 3.31.1 B. At higher magnification, the filament and gill arch epithelium show the presence of polygonal, whorled, fingerprint-like pattern on the pavement cells with wel: organised concentrically arranged surface microridges (Fig. 3.30.1 E, F & 3.30.2 L). These pavement cells have the intermittent openings of Ccs and MCs over their surface (Fig. 3.30.1 E & F). On the contrary, the PVCs surface on the secondary lamellae appear different from the filament pavement cell in as much as the surface microridges are irregular, rings are less conspicuous and the mucus covering is also considerably reduced (Fig. 3.30.2 J). There is a clear whitish coating over the PVC concentric rings presumably the mucus film making these concentric rings thicker in appearance (Fig. 3.30.1 E &3.30.2 G). A floccclent mucus seems to be oozing out through the MCs which is supposedly buried under the PVCs (Fig. 3.30.1 F). This surface mucus in the form of spherical globules may be either due to the sudden overwhelming oozing or balling up of surface sheet (Fig. 3.30.1 F &3.30.2 1, J). In H. fossilis the appearance of Cos under SEM appears to be in the form of swelled structures which, interestingly, have a ridged appearance like that of PVCs (Fig. 3.30.1 C &3.30.2 M. These Ccs have a very small opening at the side which may correspond with the apical pit opening observed under TEM (Fig. 3.32.1A). There is no difference in the topographical appearance of the Ccs which under SEM appear to be all the same type. The surface view of catfish gill has also revealed, particularly at the gill arch, the existence of prominent protuberances and each of which has distinct We bud located at its apical end (Fig. 3.302 K). Following transfer the H. fossilis to 25%SW, the number of MCs openings have increased considerably as compared to the TW maintained fish so much so that there seems to be the MCs openings are seen almost at each junction of PVCs (Fig. 3.31.1 E & 3.31.2 0,11). Further, unlike the TW fishes, the mucus layer seems to be more dispersed and the big spherical globules observed in the TW maintained fishes appear fragmented and mere widely scattered (Fig. 3.31.1 F). There are, 81 however, no significant differences in the abundance of Cos in 25% SW maintained fish (Fig. 3.31 .2 I,J) . ii. Transmission electron microscopy (TEM) Ultrastructurally, the gill epithelium of the catfish H. foss/Its clearly shows the occurrence of the Cos, PVCs, ACs and pillar cells (PC) which are largely confined to the secondary lamellae. The Ccs are quite abundant in number, mostly ovoid in shape and show all the ultrastructural characteristic features which are associated with a typical Co. The Cos are characterized by high mitochondrsal density, extensively amplified basolateral membrane made up through an invagination and a narrow apically located vesiculotubular system. The shape of the apical membrane varies from being mostly flat to sometimes convex with a virtually smooth topography and without the presence of microvilli (Fig. 3.32.1 A). A close look at the Cos show an appearance of two different types characterized by the electron dense and electron lucent (pale) designated as CcI and Ccli in the present study (Fig. 3.32.1 B & C). These two different types of Cc are further differentiated on the basis of the shape of the mitochondria which in the case of Delis round and in Cell both round as well elongated (Fig. 3.32.1 B C & D). The other cell organelles such as golgi apparatus, a distinct round nucleus and large number of vesicles scattered all over the cytoplasm are seen (Fig. 3.32.1 D). At higher magnification, the rod shaped cristae inside the mitochondria and tubular system in continuity with the cell membrane darkly stained with osmium in the form of banded structures are clearly visible (Fig.3.32.2 G & I). Interestingly, in certain instances the extensive tubular network seem to form a ring like structure around mitochondria (Fig. 3.32.2 1). In mbulovesiculo system, light and dark veicles are apparent (Fig. 3.32.2 G). Along with the Cos, a large number of PVCs characterized by the presence of microridges at the apical surface, low mitochondrial density, unelaborated basolateral membrane have also been observed (Fig. 332.1 C). Further, the presence of typical intracellular organelles such as golgi apparatus, eadoplasmic reticulum, lysosomes and dark vesicles of different electron densities with an apical plasma membrane covered by a thin dark layer of glycocalyx are also seen (Fig. 3.32.1 A). Interestingly, such PVCs are in close association with the Ccs and joined together by numerous tight junction (Fig. 332.1 A & 3.32.2 H). These intracellular junction between PVC and adjoining cells are extensive and multistraaded which makes these junctions tight and impermeable to ions (Fig. 3.32.2 J & 3.33.2 K). The apical surface of the Cos and PVCs seems to be in continuation and is demarcated by the presence of microridges in the latter. The PVCs are normally peripherally located in the form of elongated cells sharing a common pit/junction with Ccs near the apical surface. This overlaying of the Cos by the PVCs leaves only very small surface of Cos directly in touch with the external environment (Fig. 3.32.1 A). In the secondary lamellae of branchial epithelium, the typical pillar cells characterized by large nucleus and extended flanges on either side in the vicinity of blood spaces are visible (Fig. 3.33.1 A & B). The end of the flanges of PC are 82 formed by flaring out at the point of contact with the adjacent basal lamina of the lamellar epithelium and are extended up to the neighboring pillar cells (Fig. 3.33.1 C). The details of the cells organelles in the pillar cells are not distinctly visible at the present magnification. Another type of cells, though fewer in number but characterized by a high nucleus to cytoplasm ratio generally termed undifferentiated cells (DC) in literature, have also been observed both at the imerlamellar and secondary lamellae (Fig. 3.32.1 A & 3.33.1 A & B ). The 25% SW maintained catfish shows interesting ultrastructtnal changes in its various epithelial cells. as show the presence of deep apical crypt (Fig. 3.33.2 ]) relatively reduced number of mitochondria and less extensive basolateral tubular system (Fig. 3.33.1 F & Fig. 3.332 1). However, both Ccl and Celi types of cells are clearly observed in SW maintained catfish (Fig. 3.33.1 E). The striking feature is the sudden abundance of large number of Cos in the secondary lamellae (Fig. 3.33.1 A, B, C & D) which are, however, not observed in FW catfish FFig not shown). Another interesting feature is the sudden predominance of typical accessory cells which are characterized by less number of mitochondria and less developed tubular system and being relatively smaller as compared to Cc (Fig. 3.33.1 A B &3.33.2 G). A ruptured MCs showing the presence of typical election light and electron dense granules has also been observed in SW maintained catfish (Fig. 3.33.2 G). III. Discussion: The significance of gills as an organ with multifunctional entities for successful adaptation of fish in diverse environment cannot be overemphasized. In order to have a comprehensive understanding of the functional role of this organ, it is essential to study its general morphoanatomy, and more importantly, the role of various epithelial cell types which constitute this organ. Of these, the important cell types i.e. pavement cells (PVCs), Chloride cell (Ces) and mucous cells (MCs) need to be studied in detail and particularly the Latter two which have been associated with osmoionic regulation in addition to other physiological functions. In pisces, the gills playa very important role in ion regulation, in addition to its functions such as mapiratu y gas exchauge, excretion of nitrogenous waste and acid base regulation. The gills are highly sensitive to many factors such as stress, environmental pollution and changes in the ambient salinity (Pisam et al 1988, Franklin 1990, Cioni et at 1991, Kultz or at 1995 and Uchida et al 2000). Such multifaceted role of the gills is largely due to the fact that the gills are the primary corridor for exchange between the internal milieu of fish and their external environment and provide the largest surface area for such exchanges (Olson, 1996). Due to this multifunctional role of the gill, literature abounds with large number of studies which deal with gill apparatus and also accessory respiratory organs of the several air breathing fishes such as Lepidocephalichfhys guinea, Anahas testudineus, H. fossils, Hypostomus plecastomus, Channa striate, Periophthalmuc chrymspilos and C. hairachus (Singh 1966, Munshi et at 1986, Olson et 33 Fig-3.1 External features of Heteropneustes fossilis. Pectoral fin 1 Fig-3.2 (A) Head of Heteropneustes fossilis showing the arrangement barbels, eyes and pectoral fin with spine (B) In situ position of gill in H. fossilis. Fig-3.3 (A) A complete (holobranch) gill of H. fossilis. (B) Magnified view of a complete gill showing filaments with lamellae and gill rackers attached with gill arch and gill septum of H. fossilis 83(i) Epithelia cells of .,-- i rch Mucous cells; - _ : : : '•. : • • •~ ~~ • ~¶ « • • • •. i q) , 4: 'S; .\ y •Ehloridef t!s '~ , Bement mEi~1 e •8lo~d ri. channels` bra :t'Y' i' I-ig.i.4.I IIistolog of gill of f/. fi».si/is (. ) & (13) Longitudinal sections of gill filaments through the cartilaginous supporting rods stained %%ith Harris hematox\lin/ eosin, 5Ox. (C) Magnified view of single filament and epithelium cells, 1'a\ ement cells laser on filament and lamellae stained ith Harris hematocs len / eosin, 4(111k. (I)) The mucous cells present in abundance at interlameller and .econdan lamellac, stained %-with .AB (2.5)/ l S, 11111. (F) The chloride cells present on interlamellar and on secondary lamellac, stained %%ith Acid hematein, 4011x. (F) & (() sho~~ in(; pillar cells, blood channels and basement membrane in secondary lamellae, stained sith B (2.*)/ PAS and hi/F, 400x. 83 (ii) 4 4 Taste bud . 1 1 ~• Mucous cells 0; 1 400x Gill rackers Mucous cells ~ • ..ate ~ y r~t~• ~ , '~ " r • J:;IIIlsJ ''nj. ;o :' • '~ K Fig 14.2 (H) Epithelial cells of gill arch.ho,.%ing mucous cells and taste buds stained Hith Harris hematoxylin/ eosin, 11111x (I) & (J) Magnified view of taste bud Nith Po different stains AB (2.5)/ PAS and H/F:, -Alts (K) gill rackers and mucous cells arrangement on gill arch stained with AB 0 (2.5)/ PAS, I9th. 83 NO Fig-3.5 External features of Cbrias batrachus. Fig-3.6 (A) Head of C. batrachus showing the arrangement barbels, eyes and pectoral fin with spine and (B) In situ position of gill. G II r;i( kern Fig-3.7 A complete (holobranch) gill of C. batrachus. a 83 (iv) of gill WO /: cMuusco cells • ' ." D' 1 1.1,1 1''- 'q~r D Fir. 3.lt.1 'I he general hi%toIoq of gill of C. horrurhrts ( %) S (K) I.HWgifiittinal sections of gill filament% through the cartilaginous supporting rude stained %sith llarris hcmatox% (ini eosin, 5ti . U) Magnilicd -,iew of singkk filament anal epithelium ctil%. Paycment cells Ia'crun filatncut and lamellae stainer! II ith 11-F, 41111x. (I)) The nnucous cells present in abundance at intcrlameller and secodar% lamellae of filament, stained with AK (25)/ t .. , IMIx. (F) the prc%umptine chloride cells present on interlameller and on secondary lamellae of filament, stained with Harris hemato%%lin/ eosin, 31111. (F) & ((:) Shm%ing pillar cells and blond channels in seeundar% eel with AI] (2.5)/ Pt and H/E, x1111%. jJ 4 IGIU s ce ' • '::: .'; ' = Gill rackers 1l .a \\ ' J 4 Taste Ards tA lI 3.8.2 (11I.',Ii ino gill rack r•. and nnucuu, cell% arraWgrnient on gill arch stained v itlr .1 It / !'AS, 100% (1) Epithelial cells of gill arch showing taste bud stained with Harris atox,lint eosin, 11)Ox (.1) & (K) Magnified iew of taste bud with two different stain AR I/ I'.-S and H/E, 400x. 83 (vi) • 1 ` . ` ` _ Fig-3.9 Different types of glycoprotein moieties in mucous cells of branchial epithelium of H. fossilis maintained in TW, 100x. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moiety. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moiety. (C) Mucous cell following active methylation (AM/AB (pH 2.5) showing sulphated glycoprotein moiety. (D) Active methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylated s glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour, a mixture of glycoprotein moieties (F) Mucous cells stained with AB (pH 1.0) - PAS showing few purple colour cells which are a mixture of sulphated and neutral and the magenta colour cells indicating neutral glycoprotein. (G) Neutral glycoprotein in mucous cells stained magenta with PAS. (H) Acidic (Carboxylated and sulphated) glycoprotein moiety in mucous cells stained AF • AB (pH 2.5). (Neu. Gly. , Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly. , Mixed glycoprotein). 83 (VII) Sul. Gly. ti AP -~ • Carbo. Gly. - • 'r Sul. Gly. box • C ioox D Fig-3.1O Different types of glycoprotein moieties in mucous cells of branchial epithelium of C. batrc(hus maintained in 1W, 100x. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moiety. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moiety. (C) Mucous cells following active methylation (AM/AB (pH 2.5) showing sulphated glycoprotein moiety. (D) Active methylation followed by saponification (KOH) and then stained with AB (pH 2.5) showing the carboxylated glycoprotein. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour, a mixture of glycoprotein moieties where magenta colour indicating neutral glycoprotein. (F) Mucous cells stained with AB (pH 1.0) - PAS showing few purple colour cells which are a mixture of sulphated and neutral and the magenta colour cells indicating neutral glycoprotein. (G) Neutral glycoprotein in mucous cells stained magenta with PAS. (HI Acidic (Sulphated and carboxylated) glycoprotein in mucous cells stained with AF - AS (pH 2.5). (Neu. Gly., Neutral gtycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly. , Mixec glycoprotein). 83 (viii) Gill 35 30 C 0 25 uZ NS NS NS 4-20 NS N_ 4"15 4) U X10 0 5 Li Acidic Sulphated Carboxylated Neutral Mixture Fig.3.11 Per cent distribution of glycoproteins in mucous cells of the branchial epithelium of catfish H. fossilis. Each bar represents percentage of different type of glycoproteins. Significance calculated with reference to Acidic glycoprotein. NS = Not Significant. 35 30 C 25 20 C 15 a U 10 5 J Acidic Sulphated Carboxylated Neutral Mixture Fig-3.12 Per cent distribution of glycoproteins in branchial mucous cells of catfish C. batrachus. Each column represents mean t SEM for five fish, '(P<0.05), (P< 0.001) denotes significant difference calculated with reference of neutral versus sulphated, mixture, acidic and carboxylated glycoproteins. 83 (ix) 35 H. fossilis * * C. batrochus 30 * C 25 0 -20 .. 15 C U a 10 5 0 Acidic Sulphated Carboxylated Neutral Mixture Fig.3.13 Relative per cent distribution of various type of glycoproteins - containing mucous cells in H. fossilis & C. batrochus. The line diagram depicts the mucous cells of the gills epithelium in both the catfishes. Each point represents mean t SEM for five fish, `s (P<0.01), "s (P< 0.001) denotes significant differences from glycoproteins of H. fossilis versus C. batrachus glycoproteins. 83(x) '(uia3oJdo3Ag8 paxuw' -Ajg •xiw fuialoado3Ajg pale4dinS "A •!nS •uialoido,Aj3 pale(AxogJe '-AMg •ogie~ !u!ajojdo3AiB'!pPPV ' •A1 9 PV 'u!aloido)A!8 !ejlnaN '•A19 •naN) -(S-Z Hd) gd - Iy pau!els sIIa3 sno3nw u! Ala!ow u!a3oJdo3AIB (pa3e!Axogie~ put palegdinS) "!P!3v (H) •SVd 41!M ewua3ew pau!eis s!!a3 sno)nw u! u!aloido3AI9 !ei1naN (g) •u!a3oJdo3A!8 ie~lnau put palegdins;o amlx!w e aie 4314M s~ia3 ano~o3 aldind Mal Su,Moys Syd - (0.1 Hd) 9V yl!M pau!els sa3 sno3nyy (1) sa!la!ow u!aloJdo)AjB;o aJnlx! w e'ino~o3 aldind Su!moys SYd - (S-Z Hd) 9d 43!M pau!e3s sila, sno)ny4 (3) •Alaiow u!aloido3A!8 paleµxogie3 a43 8u!M04s (S-Z Hd) 9V/(HO1 ) uo!ie~l;!uodes/uo!le!A4law aAlpe Su!Mo!!oj spa3 snomyy (Q) -Ala!ow u!aloado,AIS paIe4d1ns Su!Moys (S-Z Hd) 9V/ AJV) uo!le~Aglaw aA!he Su!MO~~oj s~~a~ sno,nyy (D) •Ala!ow ap!Jey»eSAJodo~nw palegd~ns 8u!MOys (0'T Hd) an~q ue!)Iy 41!m anlq daap pauweis s~~a) sno3nW (g) •Aia!ow apue4»esAIodo)nw 3!p!Pe 8u!Mo4s (S'Z Hd) aniq ue!3!y 4un~ an!q daap pau!els s~!a, sno'nyy (y) xooT 's!~a3 sno~nw pue 3uawaned u! A4douuiadA4 aloN 'MS %SZ ui paueelu!ew sysssoJ •H jo wn11a43!da !e!43ueJq Jo s!la) sno~nw w sa!ia!ow u!aloado)AIB ;o sadAl luaJaj!Q bj•£ -31 j • x00T F OOT 0- •1 . -'' ,- • •tiD -X!w' . • ' v y • •r • h huII •~ • g Y • bf.:: . S .'. IA \• 8 • 1i v x00T • w~ ✓ • T / •• .• ~•MIS '.• ', re • " Au. Gly. , • • L•• ~ , • , : • Sul. Gly: • • •• .. B ION •,~ • •• A 100x • 2 • • -p ' •~ • • Sul. Gly. ,~ • Carbo. Glyn.. • C 100 :A . ,T. Mix. Gly. , ': !:: • lJ,, .( i r\.' . • l..M 1 • i .. t ~ '! ~ Er ti Fig- 3.15 Different types of glycoprotein moieties in mucous cells of branchial epithelium of H. fossilis maintained in 30% SW. Note hypertrophy only in mucous cells, 100x. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moiety. (8) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moiety. (C) Mucous cells following active methylation (AM/AB (pH 2.5) showing sulphated glycoprotein moiety. (D) Mucous cells following active methylation/saponification (KOH)/AB (pH 2.5) showing the carboxylated glycoprotein moiety. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour, a mixture of glycoprotein moieties (F) Mucous cells stained with AB (pH 1.0) - PAS showing few purple colour cells which are a mixture of sulphated and neutral glycoprotein. (G) Neutral gtycoprotein in mucous cells stained magenta with PAS. (H) Acidic (sulphated and carboxylated) glycoprotein moiety in mucous cells stained AF - AB (pH 2.5). (Neu. Gly. , Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly., Mixed glycoprotein). 83 (xii) Sul.Uly. •- • ?~ ~. -~ z Wit_- _ • ••• ♦ • •• • • • • Sul. ly.b • • _ • •~ a Q c ` ` • e• Car. 1 „♦ • • ,V, , • lb :.!,,;A ".• :•: U' : ' tUOx %, cY •: $ • Mix. Gly '• o • ' • I. • • • 4•. • Mix. G'..• . . ♦ ~ ' • •• 1 •. •. • • ioAx • • E F ~ . .. I Neu. GP•i~ s '\ .G Fig-3.16 Different types of glycoprotein moieties in mucous cells of branchial epithelium of H. fossilis maintained in 35% SW. Note hypertrophy only in mucous cells 100x. (A) Mucous cells stained deep blue with Alcian blue (pH 2.5) showing acidic mucopolysaccharide moiety. (B) Mucous cells stained deep blue with Alcian blue (1.0 pH) showing sulphated mucopolysaccharide moiety. (C) Mucous cells following active methylation (AM/AB (pH 2.5) showing sulphated glycoprotein moiety. (D) Mucous cells following active methylation/saponification (KOH)/AB (pH 2.5) showing the carboxylated glycoprotein moiety. (E) Mucous cells stained with AB (pH 2.5) - PAS showing purple colour, a mixture of glycoprotein moieties (F) Mucous cells stained with AB (pH 1.0) - PAS showing few purple colour cells which are a mixture of sulphated and neutral glycoprotein. (G) Neutral glycoprotein in mucous cells stained magenta with PAS. (H) Acidic glycoprotein moiety in mucous cells stained AF - AB (pH 2.5). (Neu. Gly. , Neutral glycoprotein; Aci. Gly., Acidic glycoprotein; Carbo. Gly., Carboxylated glycoprotein; Sul. Gly., Sulphated Glycoprotein; Mix. Gly. , Mixed glycoprotein). 83 (xiii) NUMBER & SIZE OF MUCOUS CELLS 140 140 • SIZE Gill • NUMBER 120 120 C N E 100 ti ff# 1o0.` d H U in U 80 80 0 V 0 60 60 O 40 40 E Q z 20 20 0 0 TW 25% SW 30% SW 35% SW 140 140 • SIZE ■ NUMBER Skin 120 120 C N X100 100. dU N 80 80 0 U O U 60 60 O] O 40 40 E Q 3 Z 20 20 0 0 TW 25% SW 30% SW 35% SW Fig-3.17 Abundance of mucous cells of the gills (upper panel) and integument (lower panel) of catfish H. fossi/is following transfer to 25, 30 and 35% sea water. Each bar represents mean ± SEM, * * (P< 0.05), * * "(P< 0.01), "* "" (P< 0.001) denote significant difference calculated with reference to TW mucous cells. 83 (xiv) 120 140 MC number in skin ** ** MC MC 120 100 —MC 100 E y 80 U 80 60 U 0 C 60 c w 0 ao C a, n a E 20 a Z 20 0 0 TW 25% SW 30% SW 35% SW Fig-3.18 Number and size of branchial and integumentary mucous cells (MC) of catfish H. fossilis following transfer to 25, 30 and 35% sea water. The line diagram represents the mean ± SEMI * " (P < 0.05), " " (P< 0.01), "' (P< 0.001) significant difference calculated with reference to TW mucous cells. 83 (xv) • tçolh cell .., •Mtkous cells' • :% <;_, :' .. * . . • • • .~box~ , •. Mck~ • a .r ► M &Cgu3 Is , j So • ~ ► ;{ Muc ~S • ioox • ArjI 1 - b ♦ Mucouscells V Mucous cells \~ 4 . lOOx May C •; R'~ ,~ ! tMu Is : Mucous cells t „ ' '•~ Q III ~.Ox• June dx s,, Fig-3.19.1 Seasonal variations in the abundance and size of mucous cells in gills of H. fossi/is during March to June represented by panels A-D respectively. Mucous cells stained with AB-PAS(pH 2.5), 100x. Magnified view of mucous cells of the above panels represented by panel a-d respectively, 400x. Note the significance decrease in number and migration of mucous cells towards the filament tip during May and June. 83 (xvi) [. Mu(' ells " • u 1 1 . •~ t. ~ ~ , .1, 1&b 4°°x .,ti ; !uty/August I' t ' " •« • tli1 ue0t 'cep t 4 • • ..:. 4 . "._.cam . ` •' + Mucoi ceH -- _ -r7_ .,— _ _,• . .• * • - Mucouscelt~;r -_—_; ; s. t qo "' s • .,+ - . Mucous c&R t ` , • I. • - ' I s . - - --- ioox ' Ott&w/ November G ;'4OOx—$ • ~. ~, ro, Luu~ .eii5 e x~ate• Mucous c~tls • ~.rsti• oQ/CembPw '•' H 400x -. 1] Fig-3.19.2 Seasonal variations in the abundance and size of mucous cells in gills of H. fossi/is during July to December represented by panels E-H respectively. Mucous cells stained with AB-PAS (pH 2.5), 1 00x. Magnified view of mucous cells of the above panels represented by panel e-h respectively, 400x. 83 (xvii) MuCc1Sseus •,"i ;a • •r . "i - 4O9x _ l~ ' ~M cous cells UCOUS Ceel . V. •' sla ' t - f .~ Fig-3.19.3 Seasonal variations in the abundance and size of mucous cells in gills of H. foss//is durir January and February represented by panels I & J respectively. Mucous cells stained with AB-P~ (pH 2.5), 100x. Magnified view of mucous cells of the above panels represented by panels i 8 respectively, 400x. 0 83 (xviii) Number 180 M a 160 140 120 a E F a 40 3 20 Z 0 a`r PQ~. ~,a.~ 1JCc J~Jy~. ~~~~ ~oJ¢. ~¢~ ~' Qe o~ eye c e oC Size 180 160 r4 E 140 `LA 120 d U N 100 0 80 60 0 40 20 0 w >JCe 4- des oJe Jam Ja~~ \Po `ec~` ec~ lac eoc ~J~a " Oho oet' ~c Fig.3.20 Seasonal variations in number (upper panel) and area (lower panel) of branchial mucous cells of catfish H. fossilis. Each bar represents the mean ± SEM, * (P< 0.05), (P < 0.01), "•' (P< 0.001) denote significant differences calculated with reference to the March value. 83 (xix) SIZE & NUMBER OF MUCOUS CELLS Number — Size GILLS 181J .80 16C .60 !~ 14C .40 E N 12C L20 u47 L00 0 10 u O 10 Q 10 ~eo ` Oho• pe~~ ~a Skin Sc 00 45 0 - 4C4C C 35 dJ 3( U 0h 2[ U 2( O 1! a £ 1( !0 Q O 2 LO < Je• 4' ac\ act e~ ~~ e~ acJ ocJ 1J\~\ ' `'~Q~ O~o' ec. ~e Fig.3.21 Seasonal variations in number and area of branchial (upper panel) and integumentary (lower panel) mucous cells of catfish H. fossilis. The line diagram represents the mean ± SEM, * (P < 0.05), ** (P< 0.01), "' (P< 0.001) denote significant difference with reference to March value. Comparisons have been made amongst the values of the same parameter. 83 (xx) M ` ~ •~ ,rat• ~~ !~ v1 . - 1 .. ~ •a'! leox /~ 400x • • -• ,r ti Goox C 400x • • 1 1000x Fig-3.22 Histolocalization of chloride cells in the sagittal sections of gill epithelium of TW maintained catfish H. fossilis stained with Acid hematein. Chloride cells are present in the interlamellar and lamellar regions (indicated by ► arrows), mucous cells (indicated by ► arrows ) and accessory cells (indicated by .. arrows ), at different magnification, 100x, 400x and 1000x. Note the abundance in the number of chloride cells for comparison with 25 and 30% SW maintained catfish shown in the next plates. 83 (xxi) 10ox A ' lie ~a • ,~i 1' t~J ` 400x 4* c 400x Fig-3.23 Histolocalization of chloride cells in the sagittal section of gill epithelium of catfish H. fossilis maintained in 25% SW stained with Acid hematein. Chloride cells are present in the interlamellar and lamellar region (indicated by ► arrows), mucous cells (indicated by ► arrows) and accessory cells (indicate by ► arrows), at different magnification, 100x, 400x and 1000x. Note a significant reduction in the number of chloride cells in 25% SW maintained catfish compared with TW control, see Fig. 3.22 . 83 (xxii) N ~ - ; '" t 400x ~► • `•.-•~ 400x - ?T0 K.y St. Fig-3.24 Histolocalization of chloride cells in the sagittal section of gill epithelium of catfish H. fossilis maintained in 30% SW stained with Acid hematein. Chloride cells are present in the interlamellar and lamellar region (indicated by ► arrows), Mucous cells (indicated by ► arrows) and Accessory cells (indicate by ► arrows), at different magnification, 100x, 400x and 1000x. Note a significant increase in the number of chloride cells in 30% SW maintained catfish compared with TW control, see Fig. 3.22. x3 (xxiii) Fig-3.25 Histolocalization of chloride cells in the sagittal sections of gill epithelium of catfish H. fossilis localized with Champy Maillet's fixative/OZI, maintained in TW (panel A-C) and 25% SW (panel D-F). Chloride cells are present in the interlamellar and lamellar region (indicated by ► arrows) and Mucous cells (indicated by .arrows ), at different magnification, 100x and 400x. Note a significant reduction in the number of chloride cells in 25% SW maintained catfish. 83 (xxiv) 200 Number *** 180 •`= 160 140 120 a 100 0 = 80 V 60 40 E 20 Z 0 TVV 25% SW 30% SW 30 SIZE ,. 25 E• 20 d 15 L V 0 10 d a 5 0 TW 25% SW 30% SW Fig-3.26 Abundance (upper panel) and size (lower panel) of chloride cells of the gills of catfish H. fossilis following transfer to 25, 30 and 35% seawater. Each bar represents mean ± SEM, *** (P < 0.001) denotes significant difference calculated with reference to TW chloride cells. 83 (xxv) NUMBER & SIZE OF CHLORIDE CELLS 30 NUMBER —SIZE 180 25 160 140 20 n 120 U 4) 100 15 0 80 L V 10 0 60 4J 40 E 5 Z 20 0 0 TW 25% SW 30% SW Fig-3.27 Number and size of branchial Chloride cells of catfish H. fossilis. The line diagram represents the mean ± SEM, "'' (P< 0.001) significant difference calculated with reference to TW chloride cells. 83 (xxvi) T !I 44 FI F , Pus r ~aa Fig.3.28 Immunolocalization of Na', K'-ATPase rich cells (chloride cells) in the sagittal sections of gill epithelium of TW maintained catfish H. fossilis. Immunoreactive cells are present in the interlamellar and lamellar region (indicated by arrows ►) F= Filament, L= Lamellae, Magnification is given in each panel. 83 (xxvii) 4 Fig-3.29 Immunolocalization of Na',K'-ATPase rich cells (chloride cells) in the sagittal sections of gill epithelium of 25% SW maintained catfish H. fossilis. Immunoreactive cells are present in the interlamellar and lamellar region (indicated by arrows r) F = Filament, L= Lamellae, Magnification is given in each panel. 83 (xxviii) 104x Gill arch ,, `{fir Gill arch r 4- .. ...100 t~ ~ k~.~.~ ~ '~- - -'~►~ ~~µrn t .t ,.~ 88 if.:ms 4610K Y PVC t; 1 ~... M '~ pvC 1 M1 M 2 prn ..-~I ' 1µm Fig-3.30.1 Scanning electron microscopy of gill of catfish H. fossilis maintained in TW. (A) Gill filaments from two hemibranch of a gill arch. (B) Magnified view of gill filaments with secondary lamellae. (C) Single filament showing chloride and mucous cells opening on surface and secreted mucous on secondary lamellae. (D) Arrangement of secondary lamellae on the filament. (E) Magnified view of gill epithelium showing concentric rings of pavement cells, chloride cells and mucous cells openings. (F) Showing concentric ring of pavement cells and mucous released on the surface. [Filament (F), Secondary lamellae (L), Pavement cells (PVC), Chloride cells (a), Mucous cells opening (4) and mucus (M)]. 83 (xxix) 22460x ' 81OOx PVC ~, G /' ~•1 ¼•14 - 4 cj Fig-3.30.2 Scanning electron microscopy of gill of catfish H. fossilis maintained in TW. (G) Magnified view of mucous cells opening and arrangement of concentric ring of pavement cells. (H) Showing the single chloride cell and opening of mucous cell. (I) Cluster of mucus on the surface. (J) Opening of mucous cells in interlamellar region. (K) Gill arch epithelium with taste bud. (L) Magnified view of pavement cells and opening of mucous cells. [Pavement cells (PVC), Chloride cells (a-), Mucous cells opening (4) and mucus (M►]. 83 (xxx) gr 395x • 111 w, ,► IIL .1 Gill ar ':!cr'p.w I .. Lt\JG% 1y5x ` 1 2 Fig-3.31.1 Scanning electron microscopy of gill of catfish H. fossilis maintained in 25% seawater. (A) Gill filaments attached with gill arch. (B) Magnified view of gill filament with lamellae. (C) Some filaments showing hypertrophy of branchial epithelium. (D) Magnified view of branchial epithelium and interlamellar region with chloride and mucous cells. (E) Opening of mucous cells and secreted mucus all over the filament and concentric rings of pavement cells. (F) Magnified view of the mucous cell opening. [Filament (F), Secondary lamellae (L), Pavement cells (PVC), Chloride cells (*), Mucous cells opening (4) and mucus (M)] K3 (xxxi) %4 178SOx ~ 178dx-`- / f ~9'i• . PVC "Y , - _ . PVC 4i1 . H.. Fig-3.31.2 Scanning electron microscopy of gill of catfish H. fossilis maintained in 25% seawater. (G) Showing the magnified view of secondary lamellae epithelium. (H) Magnified view of interlamellar region. (I) Magnified view of chloride cell opening. (j) Magnified view of branchial epithelium with chloride cells, mucous cells and pavement cells with concentric rings. (K) Opening of mucous cells in filament and pavement cells with concentric rings. (L) Magnified view of the mucous cell opening with mucus. [Pavement cells (PVC), Chloride cells (q-), Mucous cells opening (4) and mucus (M) 1. 83 (xxxii) :,.. Mr S. r • • PI.VC !'j A • y V.4 Z4I 4 portion •+► I N. C 1• 500 rim T •~ t PVC i Microridges Apical portion i M:! Fig- 3.32.1 Transmission electron micrograph of catfish H. fossilis gill epithelium maintained in TW. (A) Chloride cells (Cc) with adjacent pavement cells (PVC ) and undifferentiated cells (Uc). Cc shows organelles such as mitochondria (Mt), tubular network (Tu), vesiculotubular system (V), microridges (Mr) at apical portion and tight junctions between Cc and PVC, 14500x. (B) Cc coverd with PVC. Note two different types of Ccs differentiated by dense cytoplasm due to extensive tubular network (Cc I) and enlarged Cc with less dense tubular network (Cc II), 14500x. (C) Two different chloride cells Cc I and Cc II clearly visible, 14500x. (D) Cc covered with PVC, V and Golgi apparatus are clearly visible, 29100x. (E) Magnified view of Cc showing Mt and Tu, 46500x (F) Apical portion of Cc with light vesicles(Lv) and dark vesicles(Dv), 69800x. 83 (xxxiii) Fig- 3.32.2 High magnification transmission electron micrograph view of Chloride cells (Cc) of catfish gill epithelium maintained in TW (G) Cc with elongated and spherical mitochondria (Mt) and tubular granules, 116000x. (H) Tight junction between (Cc) and pavement cells (PVC). (1) Tubular network (Tu) between Mt and light vesicles (Lv) in Cc, 116000x. (J) Desmosomes (D) between Cc and PVC, 69800x. 83 (xxxiv) Micro ridges, Flange Pc ' ... ` ~' V t• :QY. Pc Cc Uc , • -. Uc . , PVC , , Pc. • v PVC ~ a 'r Ac r C r 1kc • J 2 micro • W (. 2 microns Flanges ` ~ ~~ ~Y•~ .Y r' `~— Cc / __ -' , PVC ' i5p0 nm — S00 n 4ft __ L ~~ ~`; ,~.,I=+'t {: '.-- • ~. yr t • . ,• Lay °i .41 ~r►' s,►... mss: '-___ _ . s - .. _ Fig- 3.33.1 Transmission electron micrograph of gill epithelium of catfish maintained in 25% SW. (A Secondary lamellae showing chloride cell (Cc), undifferentiated cells (Uc) and flattened pavement cells (PV( with microridges, pillar cells(Pc) with flanges around blood channel (Bc) 5820x. (B) Base of secondar lamellae showing Cc, Uc, PVC and Pc between the Bc and accessory cells (Ac), 5820x. (C) Magnified view c Pc, flanges and Bc, 14500x. (D) Magnified view of fully matured Cc with mitochondria (Mt) and nucleus (N 14500x. (E) Two different type Ccs with mt, Tu in interlamellar region. Cc I shows the dense cytoplasm due t extensive tubular network and Cc II with sparse tubular network (Cc II) 11600x. (F) Magnified view of singl Cc showing Mt, Tu and vesiculotubular system (V), 12000x. 83 (xxxv) .+ a 500 0% • } - . • '~ ApicalApicalcrypt dc •rt~ .~ Pvc 'e ; f ,,~ g ~, ~" ~• yam . V 4 .. lr yS t ~ ~ i✓ ~,= I et: 500 nrna • • Ti rte ' q-: ! ~''r .`''`~ , ter PVC .. ,~ ~~x. •. d r s C . ~. . Fig- 3.33.2 Transmission electron micrograph of gill epithelium of catfish maintained in 25% Sea water. (G) Magnified view of secondary lamellae showing flattened pavement cells (PVC) and accessory cells (Ac), 11600x. (H) Magnified view of mucous cells (Mc) with light and dark granules, 6980x. (I) Magnified view of Chloride cell (Cc) with mitochondria (Mt) and vesiculotubular system (V), glycocalax (g) at the PVC margin, 34900x. (J) Magnified view of apical crypt of Cc. Note glycocalax (g) at the apical crypt of CC, 69800x. (K) Magnified view of junction of Cc, PVC, Uc and Ac with desmosomes (d). Note the presence of most of the constituent cells of gill epithelia at one site, 23200x. 83 (xxxvi) at 1986, Fernandes and Perna-Martins 2001, Parashar and Baneijee 2002, Chandra and Banetjee 2003, 22004, Ghaffar or at 2006 and Naskar et at 2(109). A. Gross Gill Morphology: It is generally agreed that the majority of teleosts have five pairs gill arches although in some, the fifth pair of gill arch is without any lamellae and has been provided with the pharyngeal teeth for trituration of food (Stever and Hume 2004). In the present study on catfish C bairachu.s and IL fossils, we have described five pairs of gill arches where four pairs are complete gills (holobranch) and the fifth gill arch is provided only with the gill rackets. Similar observations are described in Solea senegalensis and Clarias gariepinus (Arellano et at 2004, Zayed and Mohamed 2004 and Atoned el al 2008). However, in some teleosts fishes such as Lepidocephallchthya guntea, Gastero.rteus aculeatus, Coelorhynchus coelorhynchus and Steindochnerina brevipina, only four pairs of gill arches have been reported (Singh 1966, Leatherland and Lam 1969, Calabro or al 2005 and Diaz et al 2010). It is indeed surprising that Parashar and Banetjce 2002 and Chandra and Banerjee 2003 have reported the existence of only four gill arches in C. batrachus and IL fassilis which is contrary to our observations on the above two fishes. In order to be doubly sure, we examined large number of individuals and found our observations of the existence of five gill arches consistent. Tt is likely that the last gill arch which was without lamellae must have been disregarded by these authors. In the present study, there is a distinct difference in the cartilaginous component of the gill apparatus in both the catfishes. In K fossitis, gill arch is rod like cartilaginous structure with two rows of relatively longer and spiny gill rackets whereas in C. bairachus, the gill arch is extremely sturdy and thick and gill rackers are short and fused with each other. Generally, in fishes, the gill rackers act as trappers of the rood particles which correlate with the feeding habit. According to Evans et at (2005), the gill septum supports several rows of fleshy gill filaments that run parallel to the gill rays on both the cranial and caudal side gill arch which is also clearly seen in A. fossilis and C. batrachus. The occurrence and degree of development of gill septum varies in different teleosts e.g. it is found well-developed in Garra lamta and greatly reduced in Colisa Jbsciatus and Wa/lago atta (Ojha et at 1982, Singh 1986 and Singh 1990). Hughes (1984) suggested that different degrees of development of interbranchial septa among teleosts would influence the path of the water current. The blood supply in the gills arch of the teleosts is generally provided through the afferent and efferent arteries which are clearly observed in the longitudinal sections of both catfishes gill arcbs. These arteries ran parallel to gill filament cartilaginous support and finally enter the lamellae. In the present study, we have observed the adductor muscles on the head of gill rays which arise from the basal extremities of the two successive gill rays of hnmibranch and are inserted into the gill rays of the opposite hernibranch. Singh (1966) has also described these muscles in Zepidocephalichthys guntea which are known to perform the function of the movement of filament. In both the catfishes H. fossils &C. batrachus, a complete gill called holobranch is made up of hundreds of filaments or primary lamellae which is also called the unit of gills. The 84 tilamenis of IL josiiis are spiny or conical towards the tip side but is C. batrachus these are blunt (c.f. Fig 3.4.1 A, 3.8.1 A). The size, shape and number of gill filaments may vary in different fishes. The Carla calla and Cirrhinus mrigala have large number of gill filaments whereas Opsanus tau, Anabas testudine s, H. fossiiis, C. batrachus, Lepidocephalichthys ganlea, Colisa fasciatus, Boleophthalmus boddaerf, Pseudapocryptes lanceolarus and Pertophthalmodon schlorseri have short filaments with smaller numbers (Hughes and Gray 1972, Hughes et al 1973, Hughes et at 1974, Munshi et al 1980. Niva et al 1981, Singh et at 1981, Hughes and 0jha 1985, Roy and Munshi 1986, Singh 1986, Yadav and Singh 1989 and Yadav et al 1990). The survey of the literature shows that most of the workers have failed to demonstrate a clear anatomic arrangement of the pillar cells and also the blood clnnnels which are the main stmemral features of secondary lamellae. Most of the details about these structures are largely based on ultrastrucnue based studies. However, in the present study, we have clearly demonstrated the presence of pillar cells along with its vascular components in both the catfishes. The filaments of both these catfishes bear a series of two parallely arranged rows of secondary lamellae with numerous blood capillaries which originate from the branchial filamentous arteries. These blood capillaries form central blood channels which contain different type of blood cells. Such blood channels are alternated by the presence of pillar cells which extend their flanges sideways and cover the blood channel from either side. These pillar cells along with their flanges provide the cartilaginous support and also ensure uninterntpted blood supply to the lamellae which is covered by the basement merhbrane (c.f Fig 3.4.1 F & G, 3.8.1 F & (I). It has been speculated that the blood pressure at the secondary lamellae is fairly high and the overall coverage by the basement membrane which is reportedly invested with collagen fibers prevent the outward ballooning of the secondary lamellae (Olson 2000). Gill cell hypes: Generally, gill arch, filaments and secondary lamellae are covered with the branchial epithelium which is multilayered on gill arch and filaments and bilayered on secondary lamellae. In the present study, both the catfishes i.e. H fossilis and C. batrachus display more or less similar pattern which has also been described in many other teleosts (Morgan and Toyed 1973, Laurent 1984, Evans 1987 and Evans at al 2005). However, secondary lamellae do exhibit different features e.g. the epithelium in C. barrachus is thick and its size is reduced whereas these are long and pointed in H. fossilis. Hughes and Munshi (1979) suggested that the thickness of lamellae in air-breathers serves an oxygen-conserving device or to prevent oxygen loss when the surrounding water has low oxygen tension. It is a well accepted that functionally, the filament epithelium is non respiratory whereas the secondary lamclLae epithelium is intimately involved with the respiratory function. The cellular features of the teleost gill epithelium clearly show that it is made up of largely three ditlerent cells types i.e. pavement cells (1'VCs), chloride cells (Ccs) and mucous cells (MCs) and the occurrence of other cell types such as rodlet cells and undifferentiated cells (UCs) is very rare and reported in very limited fish species. According to Evans et al (2005) the gill epithelium primarily consists of nearly 85 90% PVCs, and 10% of Cos and MCs at the epithelial surface area. Our study on both catfishes i.e. H fossilis and C. batrachvs shows the occurrence of only three above- mentioned cell types and no rarely occurring cells such as rodlet cells, undifferentiated cells etc. have been observed under Iight microscopy and the same features have been described in other teleosts (Fernandes and Perna-Martins 2001, Cannon et at 2004, Calabro et at 2005, Diaz 2005 and Diaz Cal 2010). In the present observation, there is an overwhelming number of PVCs followed by MCs and then Ces. However, we have not been able to calculate the exact quantitative proportions of each of these cell types. Typically. PVCs are functionally involved in the respiration but some studies have suggested that the lamellar PVCs could also be involved in acid base regulation (Nam uptake) H" excretion) in freshwater teleost (Perry et aI 1992, Perry and Laurent 1993 and Goss et at 1994). In the present study, we have clearly localized the Ccs in H. fossilis gills with acid hemalcin and they are present in the interlamellar and Lamellar region. But in C. batrachus gill, the above stain has not been tried even though the presumptive Ccs based location by TILE staining do seem to be present though in reduced numbers. Abuja (1970) has localized the Ces with Sudan black B, Acid hematein and Mercury- Bromphenol blue in Gambusia afnis and Catla catla and Fernandes and Perna-Martins (2001) localized the Ces with Toluidiuc blue in armoured catfish. The critical evaluation of the localization of Ccs in the gill epithelium reveals that it is often not vety easy to localize the Cos with routine histological stains and only few histochemical stains seem to work. The occurrence and distribution of MCs in the gill filaments of H fossilis and C. batrachrrs are largely similar to those found in the gill epithelium of other teleosts (Roberts et at 1973, Takashima and Hibia 1995 and Fernandes and Perna-Martins 2001). However, their distribution and number in gill epithelia may vary from species to species (Laurent 1984). In both catfishes, MCs are mostly present in interlamellar and occasionally on secondary lamellae which is similar to those reported in Catla catla (Ahuja 1970), Poecilia vivipara (Saboia-Morace et a1 1996), Acipenser naccarh (Carmona et at 2004), Channa striata (Chandra and Banerjee 2004) and Cyprinas carpio (Cinar et at 2008) and Steindachnerina brevipina (Diaz et at 2010). The predominant presence of MCs in the interlamellar region and their relatively reduced number in the secondary lamellae is intriguing. It is likely that the abundance of MCs in the interlamellar region may serve as an immediate source to provide mucus for lubrication function whereas their reduced number in the secondary lamellae causing less mucus production may facilitate more efficient respiratory function performed at the level of secondary lamellae. This assumption gets credence from the observations of Handy and Eddy (1991) who reported that the MCs are completely absent on the secondary lamellae of unstressed rainbow trout. Similarly, studies of Ahuja (1970), Fernandes and Pema- Martins (2001) and Dilcr and Cinar (2009) on teleost fishes like Gambusia al/This, Hypostomus plecostomus and Dicentrarchus labrax have reported that MCs are confined in the interlamellar regions of filaments. 86 B. Glycoprotein Distribution in Mucous Cells: Glycoconjugates, are the main moiety of the MCs present in the different organ system including gills. Broadly, two major type i.e. neutral and acidic types of GPs are found and the latter type is made up of either carboxylated or sulphated or both types. Each component of the glycoconjugates has been associated with multifunctional roles and their occurrence and abundance has been correlated with the different type of ecological niche which the animal occupies. Such information becomes more critical in case of aquatic fauna particularly fishes which occupies diverse environment of divergent salinities. Hence, there has been considerable interest amongst fishery scientists to study the distribution and abundance of various glyeonjugate moieties in the gill MCs- A variety of histochemical approaches and most notably Periodic Acid Schiff (PAS) and Alcian blue (AB) at pH 2.5, t.0 and 0.5 followed by active methylation and soponification and AB (pH 2.5)1 Aldehyde fuchsin have been used either alone or in combination with each other to localize the presence of various type of glycomoieties in the gill MCs of fishes. The details of these histochemical methods along with other alternate approaches such as lectin have been elaborated in the introductory preface of this dissertation. Mucous cells, next to PVCs are most abundantly occurring cell types in gill epithelium. Though mucopolysaccharides have been quite extensively investigated in higher animals, comparatively much less is known about the mucus secretion in fishes. Hence, the data generated in this study on the different components of mucins present inside the branchial MCs and their correlation with various functions have contributed significantly to unravel the role of branchial mucin in teleosts in general and H. fossilis in particular. GIvconrotcina (CPs) in FW maintained H. farsil'Lc and C. batruc%ors all MCs: In catfish H fossilis, it seems evident that each MC contains the small moiety of each different type of GPs i.e. neutral, sulphated and carboxylated and that no MC has been found to exist with only one type of glycomoiety. this is clearly evident from the fact that the total number of MCs stained with AB (pII 1.0) and also followed by methylation which selectively suppresses the carboxylated type equals with those of carboxylated types which are selectively stained following saponification which suppresses the sulphated moiety. The number of these sulphated and carboxylated types of MCs almost equals with those of total acidic type. Interestingly, there may be distinct variations in the staining intensity when the sections are stained with AB (pH 2.5) which stains both sulphated as well as carboxylated moietes in the same MC. The perusal of Fig 3.9 (A) shows that AB (pH 2.5) stained sections are more darkly stained compared with those with AB (pH 1.0) or methylation or saponification. Obviously in AB (pH 2.5) stained sections both sulphated and carboxylated moiety take up stain in the same MC and hence, these sections appeared more darkly stained. Similarly, the fact that the number of neutral as well as acidic MCs equal with those of the other sections stained with both PAS and AB (pH 2.5) together fu ther suggest that no pure neutral or acidic glycomoiety exist and that each MCs contains a small proportion of each of these moiety. 87 An extensive survey of all the available information on GPs of gill MCs reveals a very diversified picture of the occurrence and distribution of such a glycomoiety in fish gill MCs. Some workers have merely recorded the qualitative presence of some of the glycomoiety with no effort to quantify the relative abundance of each type of glycoconjugate. In literature, some contradictory interpretations have been described due to the lack of proper execution or comprehension of histochemical procedures. Our observations on the catfish H. fossilis clearly suggest that each Of MCs contain all the different types of GPs in relatively different proportions and that no individual MC contains any specific type of exclusive moiety. Such an observation has also been reported in Hoplias malabaricus and Hoplerthrinus unitaeniatus (Moron at al 2009)whcre a single MC has been shown to contain three types of GPs (neutral, carboxylated and sulphated) which were altered by the exposure to different environments. However, in the present investigation on another catfish C. batrachur, glycoconiugate profile is quite different from the FL fossilis. in C. batrachus, unlike, H. fossilis sulphated and carboxylated acidic GPs existed as pure moieties and latter was relatively more abundant than the former and the two together almost equal with that of the total acidic GPs quantified by AB (pH 2.5) staining. The relative proportion of the acidic, neutral and mixed glycoconj ugate was found to be almost equal to each other. Any instance of glycoconjugate qualitative and quantitative abundance similar to that of C. bairachus in the present study is not documented in literature. Interestingly, both these catfishes are phy]ogenetically quite akin and occupy similar kind of habitat but their gLycoconjugate profiles seem vastly different from each other. However, such an occurrence does not seem absolutely unique since Gona (1979) working on the oomparttive profile of GPs of gill MCs of four different species of the same family Belontidae found vastly different profile in each species. These findings led the Gana (1979) to conclude that similarities in ancestry, in social habits and habitats of different species may not necessarily generate the same mucus GP profile. Differences in mucus profile of even closely related fish species have been reported (Fletcher et al 1976, Shephard 1994, Sarasquete et al 2001 and Fast eta! 2002 a,b). The perusal of literature reveals that there has been considerable variation in the glycoconjugate distribution profile in gill MCs of teleosts. No clear-cut correlation with regard to either phylogenetic kinship or habitat similarity with that of GP distribationat pattern seems to exist. However, a few interesting generalizations are apparent on the basis of documented information on GPs composition in different teleosts. Of the available fishes surveyed, it is interesting to note that the acidic glycomoiety is quite widely distributed as the major GP component in the gill MCs of the teleost fishes. The teleosts where acidic GPs are an almost exclusive GP component include Channa striata (Chandra and Banerjcc 2004) where no neutral moiety is detected. This is followed by lrichogester (99% acidic UP) M opercularis (95% acidic) and Colisa lalia (55% acidic) (Gona 1979). Other teleosts where acidic predominance has been reported are Odontesthes bonariensis (Diaz et at 2010), Solea senegalensis (Arellano et at 2004), Sparus aurata and Acipenser baeri (Sarasquete et al 2001) even though the exact relative 88 proportion has not been described in these studies. In contrast, there are only very few teleosts where neutral GP has been reported to be predominant which include Berm splendens reportedly with the entire GPs moiety as neutral (Gona 1979) and Atlantic salmon Salmo salar with predominant neutral moiety (Roberts and Powell 2003). Another interesting feature is the predominance of sulphomucin over sialomucin out of the total acidic glycoconjugates which is a more common phenomenon and has been reported in most of the teleosts such as So/ea senegalensis, Sparus auralus, Ac(enser baeri and Odontesthes bonuriensis (Sarasquele et al 2001, Arellano or at 20C4 and Vigliano et at 2006). Curiously, we have not come across any study where carboxylated acidic predominantes over sulphated moiety except our own observation in the present study on C. barrachus where carboxylated moiety outnumbers sulphated moiety even though statistical significance may not exist. The extreme case has been reported in Aphanius anatoliae sureyanus (Diler and Cinar 2010) where sulphated GP is reported to be totally absent suggesting the exclusive presence of carboxylated moiety in the fish_ There are good number of studies where the relative proportion of all the constituents GPs of gill MCs have not been described such as in Channa puclulws where neutral and sulphomucin designated as M- land M-2 types have been mentioned with no reference to any other GPs (Patil et al 2012). Similar partial quantification of various GPs has been recorded in Salmo salar, Cyprinus carpio, Micropogonias )urnieri and Odontesthes bonariensis (Roberts and Powell 2003, Cinaret at 2008, Diaz et al 2001. and 2010). This partial information fails to give a comprehensive profile of relative abundance of various GP constituents in the aforesaid teleosts which is mainly due to incomplete usage of the available quantification techniques. It is well documented that different GP moieties in the mucus play different roles such as lubrication, preventing dehydration and antimicrobial and ion regulation. It may be both difficult as well as simple to ascribe and justify the specific functional role where only one type of glycomoiety is present in the mucus such as in B. splendens (Gong et aI 1979) or Channa striala (Chandra and Banerjee 2004) where only neutral and acidic moiety is present. Are the functions associated with other GP moieties which are absent, not needed or compromised due to their absence is a question which needs to be answered. The relative predominance of one GP moiety of the teleost can be explained on the basis of greater predominance of one function over the other. However, the most interesting situation is observed in the present study with regard H. fossilis gill MCs where each MC shows heterogeneity of moiety in each cell same as also reported in H. malebnricus and H. unitaeniastus (Moran et at 2009) and Cynoscion guatucupa (Diaz et at 2005). This has prompted Diaz or at (2005) to suggest that gill mucus in these fishes is involved in multifarious functions of gas exchange, osmoregulation, pathogenic protection as well as binding and uptake of xenobiotics (Randall at at 1996). It is likely that the heterogeneity occurring in the glycoconjugates in H fossilis branchial MCs would contribute to various functional roles quite unlike C. batrachus where such functional diversity may be limited to only restricted functions. E9 The variability in the staining response to MCs has been attributed by Harrison et al (1987) to the temporal sequence of granules formation. The biosynthesis of mucin gtycoconjugatcs involve two post-transcriptional modifications to the protein moiety i.e. glycosylation of protein and subsequently modification of sugar moiety. Hence PAS negative staining indicates the stage up to protein and PAS positive indicates glycosylated proteins. Alcian blue (pH 2.5) stains positive when glycoproteins have been carboxylated and also possibly sulphated but positive staining with AB (pI-I 0.5) would coincide when sulphated groups have been conjugated with glycoproteins. This is an interesting hypothesis but needs to be confirmed on the basis of rigid experimental validation in different teleost species. C. Salinity Induced Changes in Glycoconjugate Moiety in MCs in H. fossilis: Variations in the occurrence of various types of GPs in the same species under divergent environmental salinities have been reported in literature (Solanki and Benjamin 19g2). The available information suggests that, by and large, neutral OP is more predominant in freshwater teleost whereas acidic GP particularly sulphated type is abundant in seawater (Fergusan et at 1992). This has been most comprehensively illustrated in three species of salmon where a general trend of shift from neutral mucin in FW to more acidic mucin in SW is recorded (Robert and Powell 2005). Such acidic mucin containing MCs contribute to significant increase in branchial MCs after acclimation to SW. This pattern of branchial MC glycoconju1,nte changes in response to salinity also correlates well with the studies of Powell et al (2001) and Clark (2002) where marine Atlantic salmon recorded notable decrease in acidic mucins following FW exposure. The predominance of neutral moiety in FW has also been reported in H. malabaricus following 2-day of exposure in distilled water (Moron et a] 2009) and in fresh water rainbow trout Oncorhynchus mykiss (Fergusan et al 1992). Conversely, a reduction in neutral OP in high ion concentration seawater in II unitereniatus has been observed by Moron of at (2009). Similarly, the predominance of acidic/ sulphatedtcarboxylated moiety in seawater is reported in Atlantic salmon S. salar and reduction of this GP moiety in DW/FW is documented in H, uflitaeniatus (Moron et al 2009) and in marine Atlantic salmon exposed to FW for 2 hr (Powell et al 2001 and Clark 2002). [-[owever, there are also contrary reports which do not conform the above convention pattern. These include predominance of neutral mucin in SW-acclimated Acipenser 9acearu (Carmona et at 2004), decrease in acidic and sulphated moiety in high ion concentrated water in H malabaricus and H. unitaeniatus (Moron et at 2009). Based on the above generalized pattern and the contrary instances, it is logical to suggest that while the salinity may be a strong determinant to the glycoconjugate distribution, it may not be justified to link GP distributional pattern in branchial MCs to a mere single factor such as salinity. It is quite likely that other physiological conditions such as reproductive state etc. which is known to affect MCs density may also play a role. The present study on two catfishes shows that each MC contains all types of glycomnieties in H. fossilis where acidic moiety almost equals with that of neutral type of GPs in TW fishes. However, in another catfish C. batrachus, the profile of mucous cells GP is quite different and we do encounter MCs with exclusively sulphated and carboxylated gill MCs where carboxylated are predominant over the sulphated containing MCs. The total neutral GPs are slightly more than the total ncidic counterpart. If one considers the specific functional role of various type GPs, there is a considerable overlapping in the reported functions of each OP. For example, protection against bacterial and viral invasion has been ascribed to both carboxylated (Herder et al 1985, Suprasert et al 1987) and sulphated moiety (Ojha and Munshi 1974, Mittal et al 1995). This protection against pathogen may be achieved by masking the receptor sites for viruses and mycoplasma species (Pajah and Danguy 1993 and Zimmer or at 1993). However, one unequivocally established function of acidic GP (both sialo- and sulphomucin) is to increase the viscosity of mucus which may be related to deionization of ingested SW in marine teleast (Suprasert et al 1986, 1987 and Loetz 1995),may help in adhesion of particles in suspension in water (Sibbing and Uribe I985)and provide protection against dessication during inunersion in air- breathing fishes (Mittal ct al 1994a, Parasker and Banerjee 1999 a, b, Chandra and Banerjee 2003 and 2004). On the contrary, neutral GP imparts low viscosity to mucus which may protect and lubricate gill epithelium against physical injuries (Moron et a] 2009). The occurrence of heterogeneity in the gill mucus OPs in H. fossilis suggests that the protection against pathogenic microorganisms accorded generally by acidic GP and the lubrication of gill epithelium provided by neutral GP may be of crucial significance to this catfish. However, the relative abundance of neutral GP and exclusive presence of carboxylated and sulphated moieties in gill MCs of C. batrachus may conform to conventional pattern observed in other FW teleosts. Admittedly, while the available data in the present study on these two catfishes is strongly suggestive of the role of various GPs, more experimental evidence needs to be adduced to establish more definite functional roles of the GPs in the catfishes. Effect of salinity on GP moiety and the number and size of mucous cells: There is clearly no change in the GP moiety profile in H fossilfs following its transfer to 25%, 30% and 35% SW. This is in contrast to several reports on variety of teleosts particularly those on euryhaline fishes where a distinct shift from neutral mucin in FW to acidic particularly sulphomucin in SW has been reported (Roberts and Powell 2(03, Arellano et at 2004 and Roberts and Powell 2005). This lack of response is understandable due to the fact that H. fossili.r is a stenohaline fish and fails to osmotegulate beyond 10% SW and the survival up to 35% SW is though passive tissue tolerance. Since the sulphomucins which is said to increase the mucus viscosity which, in turn, facilitates SW-adaptation is not present in predominant proportion, the catfish presumably fails to osmoregulale or conversely since this catfish is stcnohaline due to the lack of other eruyhalinity features, the additional production of sulphomucin may simply not be required. 91 However, there are significant changes in the size and number of MCs profile of the 11. fossilis upon transfer to higher salinities. In 25% SW, there is distinct swelling of MCs which disappears in 30% and 35% SW. Similarly, the density of MCs counted as the numbers per unit area registers significant increase in 25% SW, followed by a significant dip in 30 % SW and then the MC density becomes normal i.e. equivalent to 'I'W control in 35% SW. The size of MCs in this catfish in higher salinities shows a progressive increase i.e. noticeable increase in 25% which becomes statistically significant in 30% and 35% SW. A similar profile of increase in MCs size and decrease in number in FW teleost Etroplus maculates following gradual transfer to full strength sea water has been observed by Virabhadrachari (1961). However, in alewives Alosa pseudohanangus, the number of MCs increase in marine environment but size remained statistically unchanged (Cook et at 1979). The available information on other teleosts on this aspect reveals three patterns of responses. In first category, the MCs number increases after transfer to FW as in Fundz+lus heferoclitus (Burden 1956), stenohaline Anoptichthys jordoni (Mattheij and Sprangeis 1969), second category shows no change as in Cichlasoma biocellatum (Matthoij and Stroband 1971) and Mugil cephalas (B1ane-Livui and Abraham 1970) and third category shows a decrease in MCs as observed in threespine stickleback Gasterosteus aculeatus (Leatherland and Lam 1969). It would be interesting to probe if this change in the MC profile in this catfish has any osmoregulatory role. It is well established that a distinct mucous film covers catfish gill epithelium in FW which becomes more abundant, at least temporarily, in high salinities. In term of osmoregulatory role, it is worthwhile to consider the suggestion of Kirscher (1978) who postulated that pnlyanioric mucus layer may serve to concentrate cations in freshwater and aid in the uptake of these ions. However, in higher salinities, such an action will be physiologically disadvantageous to the fish. What could then be the reason of enhanced mucus secretion, even though temporarily, following transfer to higher salinities both in stenohaline as well as euryhaline teleosts. It is also interesting to note that even the transfer from SW to FW evokes increase in MCs density. It is tempting to suggest that it may simply be a stress-induced response to allow the fish to adapt to the changed environment and as soon as adaptation process is completed, the mucus secretion is returned to normal levels. The above suggestion is based an the observations that in most of the fishes, the same response of increased mucus secretion is reported to both from FW to SW and vice-versa transfer. The variation in response pattern in different teleosts could be attributed to species-specific variations where environmental factors may not play a very overriding role. The multifunctional entity of mucus with specific focus on its osmoregulatory participation is a fascinating area which needs to be probed further to elicit more precise answers. D. Seasonal Variations in Mucous Cells Population of Gill in H. fossilis: The catfish H. fossifrs inhabits various freshwater ecosystem mostly confined to muddy and derelict ponds with low levels of water and dissolved oxygen. The survival in such oxygen-deficient water bodies is made possible due to its aerial mode of respiration 92 accomplished through the presence of accessory respiratory organs. Since this part of North India witnesses widely varying climatic conditions with large circannual temperature fluctuations ranging from as low as O-VC in winter months (December - January) to 45-4R°C in summer months (June-July). It is logically conceivable that such drastic temperature change coupled with other environmental factors would have concomitant changes in the physico-chemical characteristics of the water bodies inhabited by H. fossilis. These water bodies wilt be subjected to a cycGctrans- evaporation during elevated temperature leading to increase in salt concentration followed by its dilution during July-August due to influx of rainwater into these water bodies. It is, therefore, likely that the population of gill MCs and its glycomoiety may also exhibit some kind of seasonality in its population pattern and chemical moiety of its glycoconjugates. The present study on H. fossilis on this aspect has shown interesting results. We used month of March as a control when all environmental parameters particularly temperature is very moderate (20-25°C) the MCs present a normal appearance in term of their shape which is ovoid and regular, granulation status, col l number and size which, on average, is 59 ± 2.5 and 46.7 f 5.6 µm2lunit area respectively. However, their numbers record a sudden spurt from March to April nearly doubling during this phase and similar surges in number are also recorded from August to September and November to December. L ierestingly, these three surges correspond with three different events i.e. a sudden rise in environmental temperature from March to April, the onset of monsoon rain from Augi st to September and a precipitous decline in environmental temperature from November to December. The month of May registered the least number of MCs population whereas duruig June, July and February, the MCs population is the same as recorded during March. the reference month of this study. There are no changes in the major environmental parameters during the months when the MCs population was similar to its reference month i.e. March. The seasonal profile in the MCs area is quite different with that of its seasonal population density profile. There is a consistent increase of varying degree in MC area noticed from May up to January registering highest average size during December. The months of March, April and February showed, more or less, similar MCs area. It is evident from seasonal profiles that the number and size of MCs do not have a clear cut correlation. However, few interesting facts are worth noting. The number and the size of the MCs are maximum during December which is followed by a sudden decline in the next month i.e. January. Further, one of the common spurts both in the number and the size of the MCs, occurs from November to December. Also, there is an interesting inverse pattern of response in the size and the number of MCs during May when the number decreases whereas the size registers a significant increase. In a multifunctional environment where several environmental factors influence the physiology of as animal, it becomes often difficult to correlate all these changes on the basis of few variables. For instance, in the present study, the focus is mainly on the changes related to osrnoionic balance vis-a-vis MCs seasonal profile. However, other concurrent factors such as reproductive seasonality linked with the homeostatic balance of an animal may also influence MCs seasonal population. In winter flounder Pseudopleuronectes americanus an increase in 93 the number of MCs has been recorded in pre-spawning season (Burton and Fletcher 1983) and in brown trout Salmo trufta, MCs show decrease in spawning season (Pickering 1977). In Sockeye salmon Oncorhyncus nerka, more number of MCs are observed during January which was attributed to warm temperature during this month (Franklin 1990). In the present study on catfish H fossilis, it seems clear that major change in the MCs profile occurs during two temperature extremes i.e. high temperature period during April-June and low temperature period between Novembers to January. The first phase of high temperature i.e. April-June also coincides with the stage of reproductive recrudescence, when there is a sudden increase in the gonadal weight particularly the ovary which virtually fills the entire body cavity. Hence the catfish during this phase is subjected to both temperature and reproductive stress and hence MC population shows significant variation. We have also experienced greater mortality of this catfish during this particular period. The winter months spanning over November - January exert only low temperature stress and virtually no reproductive stress since the gonads are in the quiescent stage. Hence, the mortality during these months is comparatively much less and in fact the best survival time is during March —April when both temperature and reproductive stress is least and the survival conditions are most conducive. the data on the seasonal variation in difBrent GPs in the H. Jossibs show the biphasic predominance of acidic moiety during April - May and September - November and similar biphasic elevation of mixed moiety first during June - August and then during December — February. In another study on mouth breeding Tilapia rnossambicus, concentration of sialomucins and glycogen in oral mucosa increases considerably with the onset of breeding, reaching the peak during oral gestation and registering a drop to a minimum concentration during non-breading season (Vamte and Jirge 1971). However, in the same fish sulphomucins which make its appearance, though with low concentration during onset of breeding, disappears completely during non-breading season. The high acidic moiety is known to induce greater mucus viscosity and to prevent prolifetaliou of potentially pathogenic organisms in epithelial surfaces (Mittel et al 1994b). The mixed glycoproteins would also have sizeable proportion of neutral moiety to provide an overall balanced function associated with both acidic and neutral GPs. However, in a multifactorial ecosystem in which H fossils lives, it may be difficult to pin point the role of a single factor as being responsible for the observed seasonal variations in the GP contents of gill MCs of catfish H. fosailis. The foregoing discussion of the seasonal variations in the densLy, area and GP content of the gill MCs of H fossili.s suggest that these variations are governed by more than one environmental factors and the mechanism by which these environmental factor evoke seasonality in MCs profile it Hfossilis is still not cleaf.y known. E. Chloride Cells (Ces): Ccs are one of the important cellular components of the branchial epithelium and have been extensively implicated in the processes of ionoregulatory adjustment in fishes. Hence, one of the important pre-requisites to study the role of Cc involves its definite 94 identification amongst the different cellular components of gill epithelium. Such a task becomes even more challenging due to the fact that the Ccs, unlike PVCs, are not present in overwhelrning number. The general characteristic features of Ccs are great abundance of mitochondria and extensive tubular system which, however, are not visible at the magnification of light microscopy. It has, therefore, been somewhat difficult to localize the Ccs through light microscopy. The best identification at this magnification is largely based on the localization of these cells at interlamellar and lamellar region and its relatively bigger size compared to PVCs and MCs. The survey of literature reveals that large number of workers have used the conventional histological stains i.e. hematozylin and eosin followed by, in certain cases, the use of toluidine blue to localize their cellular identity. Obviously, such an identification would, at best, be tentative and would require other means of confirmation of this specific cell Lype. The workers who have used hematoxylin and eosin for the atbresaid purpose includes Ahuja (1970), Ura et al (1996), Katoh et al (2000), Strula at al (2001). Parahar and Banerjee (2002), Arellano at al (2004), Carmona at at (2004), Chen at al (2004), Sasai at al (2007) and Azizi at al (2010). Those who have used toluidine blue are Ahuja (1970), Schwerdtfeger and Bereiter-Hahn (1978), Foskett eta! (1981), Zaccone and Licata (1981) and Franchini et at (1994). In our own study on IL fossilis, we have used hematoxylin and eosin but with limited success since the presumptive Ccs stained with hematoxylin and eosin were much fewer in number than those observed with more definitive histochemical techniques. Hence, a large number of workers have used lipid/ phospholipid based specific histocheinical techniques such as sudan black B, acid hematein and Chaenpy Maillet's fluid (OZL). All these are based on the principle that the Ccs have a predominant presence of lipid/ phospholipid which are selectively stained with the above techniques. Ahuja (1970) has used a wide array of histochemical methods to localize the Ces in gill of Garnbusia a7nis affinis which has been subjected to high chloride/ high sulphate medium. The author has reported the positive response of sudan Black B, acid hematein and mercury-bromophenol blue to specifically localize the Ces in the gill epithelium of these fishes. The other stains which gave moderately positive response are propylene glycol—sudan method, rile blue method and methyl green-pymnin (Ahuja 1970). However, in our study on H. fossi&s, we did not get positive response with sudan black B and ioludine blue in this catfish even though other workers have reported positive resuhs in Poecilia reeiculgla, Xyrichlys novacula, Gambusia affinis affinis, Sarotherodon mossambicus and Carasalus carassius (Ahuja 1970, Schwerdlleger and Bereiter-Hahn 1978, Foskett et at 1981, Zaccone and Licata 1981 and Franchini et at 1994). It is interesting to note that some workers even tried alcian blue stain which is specific to glycoprotein to identify Ccs. While most of them i.e. Ahuja (1970), Watrin and Mayer-Gostan (1996) and Arellann at a] (2004) reported negative results but Guner or at (2005) observed that Ccs are specifically stained in gill of O. niloticus with the alcian blue. Such a response is difficult to understand since alcian blue is normally designed for specific staining of MCs. The other stains occasionally tried in selective fishes include mcthyLcac blue end Azuree II in F heteroclilus by Karnaky (1992), 95 Mallory's triple in Etroplus rnaculatus (Virbhadrachari 1961) and ALtmans acid fuchsin in Anguilla anguilla (Utida or al 1971) with varying success. Amongst the successfully used histochemical stains to localize the Ces is Baker acid hematein technique which selectively stains phospholipids known to be abundantly present in Ces. The cytoplasmic granules of Ccs stain blue/black with acid hematein. The fact that Ces granules contain phospholipid is proved by the use of hot-pyridin which extracts the phospholipid from the granules except the nuclei of gill epithelial cells. This method is also useful iii differentiating the types of granulation between the actively, non adapting and dying fish in different salinities. We have got excellent results with the use of acid :hematein to localize the Ccs in catfish Hfossilis. However, it is surprising that despite such great versatility of this method, there are very few reports where this method has been used (Ahuja 1970). It is strongly believed that this method may show very promising result in terms of the specific localization of Ccs in different salinities. One of the most extensively used techniques to selectively localize Cos under the light microscopy is Champy-Maillct technique, also e,a[Lcd Osmium Zinc Iodide (OZI). The Ccs o` large nulnber of leleosts such as tilapia (Oreochromis mossambicus), dwarf gourami (Colisa lalia), milkfish (Chanos chanos), sea-bass (Dicentrarchus labrax), goldfish (Carassrus auratus) and many others have been selectively stained with the help of OZI technique (McCormick 1990, Lin et al 2003, Chen et at 2004, Nebel et al 2005, Mitrovic and Petry 2009). This technique was modified by Maillet, (1963) from the original technique derived from Champy (1913). It was first used in the nefsous tissue study and other type of tissue such as epidermal Langerhans, thymus, submandibular and pancreas (Dagdeviren et a] 1994). This technique was first time used by Garcia-Romen and Masoni (1970) to stain the Ces in the gill epithelium of Anguilla anguilla and Platichthys Jlesus adapted to seawater (Garcia-Romeu and Masoni 1970, Masoni and Isaia 1980). According to Maillet (1963), the OZI technique acts to uncouple lipid moieties from lipoprotein complex and newly exposed groups of the lipid would then be available for increased deposition of metal. This technique allows recognition of most membrane components, including the extensive tubulovesicles and mitochondria thus preferentially stain Ces as compared to PVC which are not so abundant in these cells organelles (Wilson et al 2002). Osntidm tetra-oxide, the active component of OZI is known to react with the double bonds of unsaturated fatty acid and lipoproteins (IIayat 1970). One of the reasons why this technique is used extensively is that it omits the subsequent staining. The paraffin sections obtained following the fixation of tissue solution are deparaffinized and the sections are ready for viewing. This makes the technique extraordinary simple and extremely easy to use. However, one of the disadvantages of this technique is the appearance of uniformly black stained Ces with no visible granulation thus severely limiting interpretation based on the physiological status of Ccs. This is quite in contrast to the Ccs stained with acid hematein in the present study where the degree of granulation in the Ccs are quite elaborately visible. Hence, if one has to base observations on the change in size and number of Ces following experimental treatment, OZI is the most suited technique. M Some of the earlier workers (Munshi 1964, Jozuka 1967 and Abuja 1970) regarded the Cos as either being absent or, if present in the gill epithelium of freshwater stenohaline teleosts, they are of no functional significance. However; the presence of Cos were later reported in large number of freshwater stenohaline telcosts by Kikuchi (1977), Hwang (1988 a, b), Pisam or al (1990) and Lee et at (1996). While the role of Cos as the specific site of ion excretion in seawater is well-established but their role in freshwater as the site of ion uptake in salt deficient environment has been the subject of considerable debate. However, the fact remains that in the gill epithelium of teleost, Ces are the likely sites of ion transport. It has also been suggested that there are two suh-population of Ccs which can be differentiated ultrastructurally. These freshwater and seawater-type Ccs have been suggested to perform ion absorption in freshwater and ion secretion in seawater (Sardet et al 1979, Foskett et at 1981 and Pisam and Rambourg 1991). Hence, the subpopulation of this Ccs in branchial epithelium of teleost, may exist to deal with different ionic situations in external envirormtent. Shape: In H. Jossilis, there seems to be consistency in shape of Ccs which are round or oval both at interlamellar and also lamellar location. However, in goldfish the Cos show variations in shape according their location in gill epithelium: they are columnar in afferent and efferent location of gill filament, cuboida] in the interlamellar and flatten in the respiratory lamellae (Kikuchi 1977). In Hawaiian goby Stenogobius hawafensis which is euryhaline fish the Ces on primary filament are large columnar whereas those on secondary lamellae are somewhat smaller (McCormick et a] 2007). In Australian snapper Pagrus auratus, the Ccs located at the base of lamellae and interlamellar region were round or columnar (filament Cc) and those located at lamellae (lamellar Cc) were flat (Fielder el al 2007). In another teleost such as rainbow trout Salmo gairdneri round and flat Ces are present in the secondary lamellae which become progressively flattened in filament and in Poecilia reticulata, a Ccs present on the secondary lamellae are elongated and (i Ces located at the interlamellar region are ovoid (Avella et at 1987 and Slrikano and Fujio 1998). It seems from above discussion that the shape of Ces do show variations in different location and uniformity of size observed in the present study on catfish is not avery common feature. Distributional pattern of Ces in FW and in higher salinities: The distribution pattern of Cos in the branchial epithelium of teleosts varied from species to species. In our study on H fossilis, the Cos were largely confined to interlamellar or the tip region either singly or in cluster of 2-4 cells with varying size. Very few cells were observed in the lamellar region. A similar pattern of distribution has been recorded in most of the stenohaline freshwater teleost such as Cyprinus carpfo, Carasyius auratus (Lee et al 1996b) and Carassivs carassius (Kikuchi 1977). However, in some freshwater stenohaline teleosts such as Misgurnus anguillicaudalus, they are present more on lamellae (Kikuchi 1977). In seawater stenohaline teleosts such as turbot Senphothalmur maximus the Ccs have been found only on the interlamellar region (Watrin and Mayer- 97 Gostan 1996). The study on the distribution of Ces in Carassius auratus by Kikuchi (1977) contradicts the observation of Lee eta! (1996) on the same fish where Cos have been reported to be more concentrated in the lamellae than the interlamellar region. The distributional pattern of Ccs in euryhaline fishes maintained in fresh water has been, like stenohaline, quite varying. The survey of literature reveals that in majority of freshwater euryhaline teleosts, the Cos are localized both at the interlamellarand lamellar region with greater predominance in interlamellar region. This situation is exemplified in Onchorhynchus keta, 0. masau, 0. mykiss, Salvelinus namaycush, Salveiinus foutinalis, Salmo solar and many others ((Jra or at 1996, Witters et at 1996, Uchida et al 1997, Wilson or al 2000, Zydlcwski and McCormick 2001, Quinn et al 2003, Fielder or al 2007, 1- iroi and McCormick 2007 and Lee at a! 2008). Contrary to this, there is another group of freshwater euryhaline teleosts where Cos have been reported to be totally absent on the lamellae and are exclusively confined to the interlamellar region as found in Oreochromis mloticus, Paraiichthy denlatus, EpinepfieIus coioldes, Oreochronns mossambicus, and Lebistes reticulates (Avella or al 1992, Schreiber and Specker 1999, Cateroy and Quinitio 2000, Uchida et al 2000. Chang et al 2001, 2003 and Erkmen and Kolankaya 2009),I3owever, an interesting fact emerges that the Cos in seawater endemic eurvhaline fishes such as Japanese seabass Lateolabrax japonicas, seabass Dicentrachus labrax and Milkfish Chanos chanos are generally concentrated on the interlamellar region with very few, if any, found on the proximal region of the lamellae (Hirai at al 1999, Varsamos or at 2002, Chen et al 2004 and Nebel et & 2005). When the teleost fishes are transferred to divergent media i.e. freshwater to sea water and vice—versa and in some cases to deionized water, there is a change in the locational pattern of Cos following such transfers. In freshwater stenohaline fish such as Cyprinus carpio and Carassnrs auratus transfer from fresh water to higher salinities there is a significant reduction in the number of the filament Cos whereas the number of lamellar Ces which initially were very few do not show any change (Lee et al 1996b). However, when these fishes are transferred to deionized water, the number of lamellar Cos shows an increase whereas the interlamellar / frlamental Ces remain unchanged compared to fresh water. A similar phenomena of the appearance of lamellar Cos also occurs in some euryhaline fishes such as DW-adapted eel Anguilla angvilla, rainbow trout Salmo gairdneri (Laurent and Dunel-Erb 1990) and medaka O. Iatipes (Lec et a1 1996a). In our study on catfish H. Jossilis, we observed a biphasic response where the number of Cos registered a sudden decline in 25% SW and shows a dramatic increase in 30% SW particularly in lamellar region which till date, to the best of our knowledge, has not been reported in any other fresh water stenohaline teleost. In the euryhaline fishes, the locational changes in the Ccs following transfer from freshwater to sea water do not show a single consistent pattern. However, a few broad generalizations are evident in the form of three distinct patterns of response. In first category, there is absolutely no change in the number of Ccs either at the lamellar or interlamellar region which is exemplified in Onchorhynchus keta fry. Salvelinus namaycush, S fontinalis and Pagrus auratus (Uchida et al 1996, Fielder et al 2007 and Hiroi and McCormick 2007). In the second category, the enhanced salinity causes a decrease in the lamellar Ces whereas the number 98 :H EE of interlamellar Ces remain unchanged. Such a condition has been reported in Oncorhynchus mason (Ura et at 1996), Salmo solar (2liroi and McCormick 2007) and Alosa sapidissima (Zydlewski and McCormick 2001). In the third category, the salinity increase causes the decrease of both the lamellar and interlamellar Ces such as in Onthorhynchus keta (Uchida et at 1997). Interestingly, a consistent pattern of response has been observed in seawater endemic euryhaline telcosts when they are transferred from sea water to fresh water, The number of chloride cells in these fishes increased significantly is lamellar region which, otherwise are totally absent in sea water maintained fish. The number of interlamellar Ces, however, remain unchanged following such a transfer from fresh water to sea water (I-Thai et at 1999, Varsamos et at 2002, Lin eta! 2003, Chen et al 2004, Nebel et al 2005). As Described earlier the number of Ccs in IL fossUis showed the decrease in 25% SW which was followed by a sudden increase in 30% SW. The size of Ccs, however, was slightly reduced in 25% SW but considerably decreased in 30% SW. In most of the stenohaline teleost fishes studied so far such as Cyprinus carpio, Carassius auratus and Cichlosoma biocellalum the transfer to higher salinities invariably results in decrease in the number and size of Ccs (Mattheij and Stroband 1971 and Lee et at 1996b). These findings on different stenohaline teleosts compare quite well with our observation on H. fossilis transferred to 25% SW. It has been generally suggested that such a decrease in size and number may be due to the inability of the stenohaline fish to osmoregulate in higher salinities. This is further corroborated by the fact that in most of the euryhaline fishes such as Anguilla anguilla, C}prinrxdon variegetus, Cillichthyv mirnhi1i.v, Salmo solar, Salmo gairdneri, Oreochromis niloricus, Stenogobius hawaiiensis and Lebistes rr(ieula!us the transfer to higher salinities is followed by significant increase in the number and size of Ccs (Shirai and Utida 1970, Utida et at 1971, Karnaky et a] 1976, Marshall and Nishioka 1980, Pisam eta! 1987, 1988, Avella et at 1992, , McCormick et at 2003. tuner et al 2005 and Erkmen and Kolankaya 2009) which is necessitated due to accelerated requirement of salt excretion in seawater environment. However, there are certain exceptions such as trout Salmo Irulta (Brown 1992) and American shad Alosa sapidirsima (Zydlewski and McCormick 2001) to this generalized pattern where Ces number in euryhaline fishes decrease following transfer to higher salinities. Further, there arc also instances of euryhaline fishes such as Cyprfrodon variegatus, Pagrus avratus and Gillichlhys mirabllis (Kamakey et at 1976, Yoshikawa et at 1993 and Fielder et at 2007) where the number of Ccs remain unchanged following transfer to higher salinities. It has been suggested by Zydlewski and McCormick (2001) that these fishes maintain high density of excretory Ccs regardless of external salinities and hence their transfer to higher salinities dos not evoke any changed in the number of Ccs. A unique feature in our observation on If fossilir is significant increase in the number and size of Ccs in 30% SW which is preceded by a substantial decrease in the number of these cells in 25% SW. It seems that IT fassilis up to 25%SW is behaving like a typical stenohaline fish registering a decline in the Ces number. But in 30% SW, it is presenting a pattern of euryhaline fish which, by and large, registers significant increase in the 99 number of Ces in higher salinities. This, however, is somewhat knotty situation since H. fossitis does not suggest any indication of active osmonegulation at 30% SW which is evident from the elevated plasma osmolarity and unchanged levels of Na' /K`-ATPase in gills (Goswami et at 19R3 and Sberwani and Parwcz 2008). In the present study on K fossdis, we also observed the occurrence of some specialized calls which are somewhat lightly stained with acid hematein and show distinct granulation. We have been quite tempted to designate these cells as `accessory cells' which have been mostly described in seawater adapted fishes (Colemen et al 1977, Laurent and Dunel-Erb 1980, Cioni et at 1991 and Fontaine et al 1995) and also in some freshwater fishes (Pisam et all989, Cioni et al 1991 and Fontaine ct al 1995). Curiously, there is no report in literature except of the present study where these cells have been identified on the basis of light microscopy even though their ultcaslructura1 features are quite extensively described by various authors (Pisam at al 1989; 1990, Zydlewski and McCormick 2001 and Wilson and Laurent 2002). It is generally believed that ACs are the developmental form of the Ccs which participate in the pre-adaptation of fish in seawater and hence accounts for increase in their number in higher salinities (Pisam ct al 1988). A similar appearance of ACs in higher salinity (30% SW) and overall increase in the total number of Ces has also been observed in the present study. F. Im mu nohistoch emistry: For immunolocalization of catfish Ccs in gill epithelium, we have used commercially available antibody from the Developmental Studios Hybridoma Bank. This monoclonal antibody (IgG a-5) developed by D. M. Fambrough against the catalytic a subunit of chicken Na'/K'-ATPase binds specifically to the cytosolic epitopes and recognizes all three isofotms (al, a2 & a3) of NatV-ATPase in vertebrates and several invertebrates epithelia. b'fajority of the investigators have used this antibody to localize Na`/K'- ATPase in gill and other tissues of fishes which seem to work quite successfully (Witters et at 1996, Sardella and Kultz 2008). On the other hand, another group of workers, mostly from Japanese laboratories, have used polygonal antibody raised against synthetic peptide based on sequences of high homology of a-subunit ofNa`/K`-ATPase in various group of animals such as white sucker (Catostomus commersoni), electric ray (Torpedo californica), Drosophila, Toad (lenopus Iaevis), chicken, rat, sheep and human (Ura et a] 1996). However, since the commercial availability of this antibody is not known, most of the workers have used the former antibody more because of its wider applicability in different fish species. Ideally, the perfused tissue would be most appropriate for inuun!oealization since it eliminates all the RBCs which are the source of endogenous peroxidase activity. Since perfused tissues could not be used in the present study, the RBCs also took up positive reaction despite the fact that the sections were treated with H202 which possibly did not neutralize all the endogenous peroxidase activity. However, this did not pose any identification problem vis-a-vis Ccs because of relatively smaller size and wider distribution of RBCs particularly in the blood sinuses of the secondary lamellae in proximity with pillar cells. 100 The immunoreactive cells obseryad in our study designated as Ces and localized by immunostaining against a subunit of Nav 1K'-ATPase were found to be varying in shape ranging from ovoid to polygonal which are typically of the same shape as reported in many other teleosts . This is exemplified in Cichlasoma biocellalum (Mattheij and Stroband 1971), Oreochromis mossambicus and Oncorkynchus mykiss (Uchida et al 20(10 and Wilson et at 2000) Chanos charms (Chen et at 2004) and Salmo .valor (Birdi and McCormick 2007). Shikano and Fujio (1999) on Poecilia reticulata observed Large spherical cells on the lamellae and same was also observed by Urn et at (1997) in Oncorhynchus masou. We observed the presence of filament & lamellar Ccs both in TlWW & 25 /oS W maintained fish. However, there is no difference in their shape and size except that the number of lamellar cells appeared to be fewer. Further, there does nut seem to be any remarkable change in the distribution pattern of these two differentially located cells in TW & 25% SW. The presence of Ces based on their location in lamellar and filamental region has been reported in chum salmon (Uchida or al 1996), Japanese eel (Sasai et al 1998), Japanese sea bass (Hirai et at 1999) and American shad (Zydlewski & McCormick 2001). However, Allen et at (2009) observed the highest number of Ccs at the base of lamellae, intermediate on the gill filament and lowest on the gill lamellae of green sturgeon rlcipenser medirostris. In FW adapted milkfish Chanos chanos, Na} (Ki ATPase immunoreaetive cells were also found in lamellae (Chen et at 2004). Witters et at (1996) observed the Ccs in Oncorynchus mykiss at the base of lamellae and between the lamellae on the filament which were localized with Na' /K'-ATPase a5 antibodies. It is evident from these observations on different teleost fishes that the transfer of curyhaline fishes from FW to SW, but not vice-versa, is mostly followed by an increase in the immunoreactive Ces (Shirai and Utida, 1970; Utida et al 1971, Kultz and Jurss 1993, Urn et at 1997 and Varsamos et at 2002). However, in some cases such as mudskipper Gillichthys mirabilis, SW transfer was riot accompanied by increase in immunoreactive cells but their size got enlarged (Yoshikawa et al 1993). In stenohaline fishes, in general, the transfer from marine to FW or reverse is followed by the decrease in number of Ccs which is also accompanied by the loss of osmorea latory ability (Varsamos et al 2002). G. Electron Microscopy Studies: I. Scanning electron microscopy (SEMI: Under the scanning cLectron microscopy study of gill of H fossilts, each holobranch gill arch, the typically arranged primary filament giving rise to leaf-like secondary lamellae on either side are visible. These lamellae are wrinkled and backwardly curved close to apical end. This arrangement of secondary lamellae in telcost is quite common and has also been observed in senegal sole, So/ca senegalensis (Arellano et al 2004),orange toadfish Opsanus beta (Evans 1987)and many other stenohuline and euryhaline teleosts. It has been documented by Zayed and Mohamed (2004) that the teleosts which have greater respiratory requirement such as tilapia have long gill filament and lamellae 101 whereas those with limited respiratory requirement through gill with additional accessory respiratory structures such as catfish, have short lamellae and filaments. Another interesting observation made on amphibious mudskipper Periophthalmus chrysospilos made by Ghaffar el at (2006) is that the gill lamellae of these fishes in addition to large surface area are also provided with large number of distinct apical pores to meet its respiratory requirement during its short sojourn to the terrestrial environment. In certain other category of fishes such as striped marlin Tetrapturus audax, the terminal ends of secondary lamellae show a distinct fusion which has not been observed in present study (Wegner et al 2006). At higher magnification in the gill surface ultrastrucrure (SEN), the most visible feature is the presence of large number of pavement cells (PVCs) characterized by the presence of concentrically arranged micoridges. A survey of literature reveals that there are four different patterns based on the appearance of the microridges found on the surface stricture of PVCs. The first type which is observed on the filament body of walking catfish C. batrachus and many other teleosts have whorled-Eke concentrically arranged microridges giving a finger print like pattern. The second type shows more complex microridges with much less defined and irregular rings as found the lamellar surface of rainbow trout O. mykiss. The third category is characterized by few microridges without any well-defined concentric rings as seen in the lamellar surface of walking catfish C. barrachus and the last category is totally devoid of miorcridges or microvilli and are observed in lamellar PVCs of bluefish Pomatomus salratrix (Olson 2000). It seems in walking catfish, two types of PVCs with different microridges patterns exist. There is a general consensus that the filament and lamellar PVCs are different from each other in terms of the presence of microridges which are more extensive in the case of former than the latter. In ouf study on catfish, this architectural difference based on the microridges on the filament and lamellar PVCs is also distinctly visible where filament PVCs have much more microridges than the lamella PVCs and the similar observations have also been made on many other teleosts as cited above. Such a difference between filament and lamellar PVCs also have strong physiological basis since lamellar PVCs, also being referred to as respiratory epithelium, do require an unobstructive gill surface for an efficient respiratory function which would otherwise be hindered if more mucus layer is trapped due to large number of microridges. The well documented role of microridges presence over the l'VCs is that they trap, hold and distribute mucus and to provides structural integrity to epithelium and increase the surface area of the apical membrane (Olson and Fromm 1973 and Arellano at al 2004). We also observed the presence of taste buds located at the apical surface protuberance epithelial cells in gill arch. These taste buds correspond to the type I of the taste buds observed by Kumari et al (2005) in Rita rita. An extensive mucus covering either in the form of sheet or the spherical flocculent droplets of varying size lying close to MCs opening have been observed in the catfish gill surface structure. A similar kind of prolific exudation of mucus and its covering over the PVCs located between the microridges and also close to the MCs openings have been observed in large number of other teleosts such as rainbow trout O. mikiss (Powell et al 1112 1992), catfish C hatrachus (Naskar et al 2009), catfish Rita rita and mrigil carp Cirrhinus mrigala (Kumari et al 2005). The multifunctional role of mucus over gills and other organs have already been extensively described in earlier chapters and cannot be overemphasized again. The Ccs observed in the present study on H. fosrilic are somewhat unique and of single type characterized by the elevated swelled structure with the presence of faint rings resembling with those of the inicroridges of PVCs and with a very small inconspicuous side opening. To the best of our knowledge, such architecture pattern of Ccs have not been documented in literature for any other teleost fish. There are descriptions of only one type of Ces observed in Mugil cephalus by Hossler et al (1979) which shows a distinct apical pore of Ccs which are broad and shallow. In African lungfish Protopterus annectens, two type of Ccs comparable to that a and (i types of freshwater teleosts have been described by Sturla et al (2001). However, in large number of other tcleosts such as scalelesscarp, Gymnocypris przewalskii (Matey et al 2008), juvenile yellowfm seabream Acanihopagrus talus (Movahedinia ei al 2009), tilapia Oreochromis mossambicus (Lee et al 2000, Cheng et al 2003), siluriform Lophiosilurus alexandri (Cardoso et at 1996) and sea trout Sa/mo trutta (Brown 1992), three types of Ccc based on surface ultrastructure have been described. A close comparison of our results on the type of Ces observed under SEM and TEM in H. fossilis may appear anomalous since we have identified two types of Ccs by TEM whereas only one types is described under SEM. This apparent paradox is largely based on the fact that the criteria of differentiating two types of Ccs under TEM studies is based on the mitochondriul differentiation and the intensity of electron density in the cytoplasm which cannot be visualised by the surface study under SEM where only one type is visible. The only significant change in the salinity transfer of catfish to 25% SW is that the number of MCs openings show a remarkable increase. This observation is further corroborated by our light microscopy studies where the transfer to higher salinities have registered a considerable increase in the aumber of the MCs while under SEM study only the opening of these MCs can be visualised. II. Transmission Electron Microscopy (TEMI: Under transmission electron microscopy, ultrastructural details of Ces also referred to as mitochondrial rich cells or ionocytes have been extensively documented in teleost branchial epithelium. These cells are typically characterized by great abundance of mitochondria, extensively anastomosed basolateral tubular system, a site for ionoregulatory enzyme Nat /K'- ATPase and an apical tubulovesicular system (Philpott and Copeland 1963, Karnaky et at 1976, Doyle 1977, Schwerdtfeger and Bereiter-Hahn 1978, Wendelaar Bonga et at 199), Perry 1997, Wilson and Laurent 2002, Evans et a] 2005 Laurent et al 2006 and Saadatfar and Shahsavani 2009). Interestingly, the Ces show striking structural differences in freshwater and marine leleosts which are attributed to the differences in their functional attributes in divergent salinities. Briefly, SW adapted teleosts have much greater abundance of mitoohondria, more elaborate basolateral tubular system and a recessed apical pit unlike freshwater teleost Ces where it is normally convex. The survey of literature reveals that a large body information on the Ccs is based on most of the euryhaline fishes in divergent salinities. Studies on the 103 stenohaline fishes endemic to either fresh water such as Carassius auratus, Carassius carassius, Cyprinus carpio, Misgurnus anguillicaudatus, Gobic gobio, Cobifis taenia and Gymnocyprisprzewalski or sea water such as turbots Scophthalmus inaximus are very limited (Kikuchi 1977, Pisam 1990, Franchini et at 1994 and Matey et al 200R). The role of Cos in freshwater fish following its first description by Keys and Wilmer (1932) as the cells involved in the excretion of chloride ions in seawater, has been the matter of extensive debate. Some workers even described that the Ccs are totally absent in FW teleost such as Calla calla Laheo rohita, Rohtee colic, Wallago attu, Mystus aor, Anabas tesmdineuc (Munshi 1964, Ahnja 1970). However, the huge quantum of ultrastructural information on the cells which are typically characterized as Ccs occurring oven in large number of FW both stenohaline as well euryhaline fishes, established the ubicuitous presence of the cells both in FW and SW adapted telcosts. The role being ascribed to these cells in FW-adapted fish was that of active ion uptake from hypoosmotic media. The well marked ultrastructtaal differences in the morphology and cell organelles and other associated adjoining cells with these cells in FW and SW teleosts are in tandem with their functional requirement in direrse ionic environment of FW and SW. This seems to have settled the on-going debate considerably. However, the observation of the existence of two types of Ccs— one involved in SW adaptation and other in FW added another interesting dimension to this aspect. The current status, therefore, is that Ccs do exist both in FW and SW teleost with uLtrastructurally different features. In the present study on the ultrastructure of gill epithelium of catfish H. fossi/is by TEM has clearly established the presence of typical Ces along with the PVCs, accessory cells (ACs) in higher salinities and the pillar cells (PCs) which are confined to secondary lamellae in close association with blood channels. A few nondifferentiated cells (UC) lined along the basement membrane of secondary lamellae have also been observed. The Ccs are quite abundant in number mostly ovoid in shape and display all characteristic features of Cc such as high mitochondria] density, extensively amplified basolateral tubular system and vesiculoluliular system confined mostly at the apical surface. Like a typical FW stenohaline teleost, the apical membrane of the Cc of H . fossils is somewhat flat or at places slightly convex, mostly devoid of any microvilli projections. Such characteristic feature of the apical membrane have also been observed in few other FW stenohaline fishes such as scaleless carp (Gymno ipris przewalskii) and seawater stenohaline turbots (Scophthalmus maxim us) transferred to 5%o saltwater (Pisam et at 1990 and Matey et al 2008). However, there are good number of freshwater of stenohaline fishes such as goldfish Carassius auratus, common carp Carasstus carassius, leaches !Lfisgurnus angnilli caudatus and gudgeons Gobio gobio (Kikuchi 1977 and Pisam 199D) which do not correspond to this typical character of freshwater teleost and display a shallow or deep apical pit which is the character of of Cps of SW- adapted teleosts. We also observed two types of Ccs differentiated on the basis of elc0.ron dense (usmuplulic) or dark Ccs and electron lucent (pale) light Cos designated as Cut and Cell respectively. These two types are also differentiated on the basis of the shape of mitochondria which in the case of the former have only round shape mitochondria whereas in the latter type both round as well as elongated mitochondria were seen. There are large number of stenohaline fishes such as Carassius earassius, 104 Gobio gobio, Cabins taenia and Scophthalmus marimus and many other euryhaline fishes such as Oncorhynchus kisuich, 0. niloticusParallchthys dentatus and 0. masou where the two types of Ccs based on the electron density have been reported(Richmau Ill et al 1987, Wendc,aar Bonga cud 1990, Schreiber and Specker L999 and Mizuno ct at 2000). Interestingly, in coho salmon, the differentiation in two Cc based on the round and elongated shape (Richman III el al 1987) is exactly the same as observed in the present study on 11. fussilis. The basolateml tubular network at high magnification shows its close proximity with the mitochondria which in certain instances are found to encircle a cluster of mitochondria as observed in guppy Lebistes reticulalus a freshwater teleost (Pisarn 1981).This proximity between the basolateral tubular system which is the site for ion transporting enzyme Nat /K'-ATPase with that of the mitochondria which is the site for ATPase molecules may indicate the transfer the ATP molecules from the mitochondria to the ion pump i.e. Na4 /K~-ATPasc to facilitate ion transporting mechanism in these cells, In the present study, we also observed a lubulovesicular system (TVS) located at the subapical cytoplasm comprising of numerous vesicles and tubules. It is generally assumed that the function of this TVS is to transport numerous vesicles and fuse them with the apical membrane. In K fossilis a large number of PVC characterized by the presence of microvilli and microplicae with cytoplasmic vesicle, distinct golgi apparatus, lysosomes and comparatively fewer mitochondria have been observed. The occurrence of PVCs in conjunction with Ccs both in freshwater and marine teleosts i.e. stenohaline or euryhaline in nature have been observed. In the present study on H fossilis, the PVCs form an overlying extended covering on the Cc and finally joined at the apical surface side by side. Interestingly, junctions between the Ces and PVCs have been multistranded indicating thereby that these junctions ace not leaky and do not allow the passage of ions to external environment which is a physiological advantage to a FW teleost like 11. fossilis. A large number of PVCs intercalated with Ccs have been observed in several FW teleost (Evans et al 2005). We have been able to detect the presence of few accessory cells (AC) in H. fusailis maintained in TW which differs with the observatious on many other FW stenohaline teleost such as Gobio gobio and Cobitis taenla (Pisam et al 1990). Pisani el al (1990) have suggested that ACs are found in teleosts exclusively living in SW or pre-adapted to SW and that they probably are involved in the formation and modulation of papa-cellular pathway for ion excretion. This observation gets credence from fact that the ACs disappear when the teleost fishes are trans£ctrcd from SW to FW such as in Mugil capito, Fundulus heteroclitus, Anguilla anguilta in 10% SW and Salmo gairdneri (Laurent and Dunel-Erb 1980). The presence of ACs in FW maintained H fossilis (present study) and goldfish Carassius carassius (Franchini et al 1994) is intriguing and the fact that the catfish H. fossilis and goldfish show poor adaptability to SW makes the matter even more complicated. In our study on H fossilis. we observed the greater abundance of ACs following transfer to 25% SW and they occur in close proximity with the Cc showing the same characteristic features as those of Ccs except being smaller in size, having fewer mitochondria and less developed tubular system. Although ACs have been found in gill epithelium of euryhaline salmonids in FW (Pisain et al 1989) and two species of tilapia (Cioni et al 1991) and following transfer of marine teleost turbots to FW (Pisam et al 1990), the functional relevance of these cells in FW remains unexplained. In our observation on catfish, the greater abundance of ACs in higher salinity may simply be an attempt to acquire osmoregulatory capability which, however, does not seem to succeed as may be in the case of goldfish and juvenile rainbow trout which show poor adaptability in SW despite the presence of AC (Pisani or al 1990). Following transfer of H fossilis to higher salinity i.e. 25% SW, we have observed the appearance of apical pit and enhanced number of lamellar Ccs but there is also a concomitant reduction in the number of mitochondria and much less invagination in the basolateral system in Ces which may largely account for a poor adaptability of this fish in higher salinities. 106 Chapter IV: Gastrointestinal tract Chapter-IV (Gastrointestinal tract) I. Introduction: The gastrointestinal tract (GIT) in all vertebrates, with mouth at one end and anus at the other, is essential for procuring food from external to the internal environment. The mouth is beset with several internal structures whielt are largely based on the specific feeding habits such as sharp and sturdy teeth in carnivorous sharks. From the anterior mouth, food passes posteriorly into successively arranged regions such as the oesophagus, stomach and finally into the intestine. There are large differences in the morphology and physiology of the gastrointestinal regions between vertebrae groups and also between different feeding strategies within the same vertebrate group. For example, birds have gizzard to comperisate for the teeth whereas the ruminant mammals have four chambered stomach due to their habit of initial rapid swallowing and subsequently mastication_ The digestive tract wall of various vertebrates has remarkable similarity in terms of gross function as well as histological organization. The (FIT, other than mouth and pharynx, is characterized by four layers i.e. tunica mucosa, tunica submucosa, tunica muscularis and the outermost tunica serosa. Histological Organization of Gastrointestinal Tract: The innermost layer of tunics mucosa is made up of mucosal epithelium which is close to the lumen and is supported by underlying lamina propria which consists of loose cellular connective tissue. The muscularis mucosa, the last layer of this zone, consisting of smooth muscles separates the tunica mucose from the tunica submucosa. The second layer the tunics submucose is another connective tissue layer which is more compact and robust than the lamina propria and is provided with the interspersed blood vessels, nerve fibers an connective tissue cells. The tunics muscularis is made up of two layers of smooth muscles i.e. inner circular and outer longitudinal which is responsible for the propelling of the food bolus. The outer most layer is the serosa or adventitia which is suspended by mesenteries. The Gastrointestinal tract of fish, amphibians, reptiles, birds and mammals, despite a broad common structural pattern, show considerable degree of finer structural variations. Some of these variations can be attributed to the diet or feeding strategy and other to the habitat such as aquatic versus terrestrial. The salient histological variations in different groups are discussed below. 107 A. Oesophaeus: Cyclostomes: The cyclostomes, i.e. lampreys show distinctly different plan where the mouth cavity opens posteriorly into two tubes. One tube leads to dorsally located oesophagus and second into ventral pharynx, the latter is responsible for respiration. The oesophagus is lined with numerous folds and leads directly into a straight intestine due to the absence of true stomach. The posterior end of the intestine is widened to form the rectum which ends at cloaca. In the hagfish, however, the condition is much the same as observed in other vertebrates. Fish: The oesophagus in pisces is very short, thick-walled and straight mbe connected dorsally to pharynx and ventrally to stomach or to intestine in agastric fishes. The distensible nature of oesophagus is evident from the fact that most of the predaceous fishes have been found to engulf fishes as big as their own size and never suffer from overstuffing or blockage of oesophagus. in elasmobranch, numerous backward-projecting papillae appear in oesophagus lumen as well as at the first part of the stomach. The oesophagus bears longitudinal folds in freshwater fishes which are lined with multilayered squmaous epithelium containing large number of mucous cells (MCs). However, the oesophagus of marine teleosts is lined with complex and highly vascularised mucosal folds which are made up of columnar epithelium and few mucous cells. This feature is associated with the osmoregulatory function in marine telcosts. In most embryonic stages of teleost fishes, the oesophagus epithelial layer is ciliated but this ciliated epithelium is lacking in adult tcicosts. The oesophagus is made up of multilayered stratified epithelium of which the upper and lower most layers may be either squamous or cuboidal with interspersed columnar MCs in the middle region. Multicellular glands an: mostly absent but serous or cardiac glands which are normally found in stomach may also be present in the distal end of oesophagus. Taste buds are occasionally visible in tunica mucous of oesophagus. The connective tissue of mucosa is confined to the strong basement membrane. The lamina propria, a loose connective tissue gives support to tunica mucosa. Muscularis mucosa occurs between mucosa and submucosa and is represented by longitudinal smooth muscles. In loose connective tissue of submucosa, the areolar glands are scattered specially near the junction of oesophagus and stomach and are responsible for the secretion of pepsinogen and 14Cl but in elasmobranch, the submucosa of oesophagus is condensed to form a distinct structure called the Leydig's organ which is responsible for immune system. Underlying the submucosa is tunica muscularis which corresponds to the common pattern described above. The last layer is the serosa which is usually thin and covered by a layer of simple cuboidal mesothelium. 209 Amphibians: The digestive system of amphibians does not contain any significant morphologically advanced feature over what is seen in fishes. However, some unique features are noticed in amphibian histology. The wall of oesophagus is muscular, extremely short and consists of little more than a constricted area of the alimentary tract to conduct food materials from mouth to stomach. In most amphibians, like in fishes, the epithelium of mucosa layer consists of one to several layers of cuboidal and columnar cells which are usually ciliated with densely interspersed MCs. In some amphibians, a simple or branched tubuloalveolar glandular invagination consisting of MCs and serous cells is found. The lamina propria of many amphibian species is occupied with lymphatic nodules and blood vessels. The muscularis mucosa is not a regular feature in the oesophagus, as in Trituruv, but may be weakly present in Proteus and abundantly in Necturus. In submucosa, the presence and variable distribution of the alveolar gland is more pronounced and is associated with pepsinogen/ mucus which has been documented in many amphibian genera. However, in urodeles Siredon and Salmandra and in anucans Cysfignthus, Bombinator and Pip a, oesophageal peptic glands are absent but in Rain, these glands are seen like tubular acini, increasingly large and concentrated near the gastric end of the oesophagus. However, the enzyme secreted from these cells remains ineffective until it reached the stomach. Tunica muscularis is well developed but outer longitudinal layer is irregularly present but increases in width and much stronger near stomach. Reptile: The reptilian oesophagus is much longer compared to lower vertebrates i.e. fish and amphibian especially in snakes where the wall of oesophagus is provided with longitudinal folds which have the capacity of extensive expansion to help in swallowing of large objects. On the other hand, the lining of oesophagus of marine turtles is covered with comified papillae which are pointed backwardly. The histological features of oesophagus in reptiles am more or less same as described above in other groups. However, the oesophageal glands or alveolar glands are generally absent in this group but pepsinogen secreting glands have been reported in some reptilian species. In lamina propria, the lymphoid nodules are plentiful but their functional role is not well documented. The muscularis mucosa is scanty and is represented by a few smooth muscles posteriorly and may be totally absent on the anterior portion of oesophagus. The other structures i.e. submucosa, muscularis and serosa are largely comparable to the other lower vertebrates. tag Birds: In birds, the oesophagus is vastly different from other preceding classes in as much as it tends to be long and wide and opens into a unique structure called crop or ingluvies which is used for the purpose of storage the food. It is useful in the birds to secure the abundant food in short period to compete with others for the Limited amount of foods. The crop in some birds i.e. pigeon and domestic fowl develop crep glands which are not exactly glands since they are cytogenie and undergo cell proliferation under the influence of prolaclia. The length of oesophagus and extent of mucosal folding depends upon the diet. The mucosal epithelium is provided with varying degree of keratinisation that depends on the nature of the diet. Unlike in previous classes, the lamina propria is very thin, muscularis mucosa is thick with longitudinal orientation of smooth muscles fibers. Oesophageal glands which are rarely present in submucosa exist in wide variety of shape and size which may vary from simple tubular to compound tubular or simple to compound tubuloalveolar. General histological structure of crop is similar to oesophagus except for a reduced number of glands and increase in contraction of lymph nodule. Mammals: In mammals, the nesophagus is tubular, clearly demarcated from stomach and its length depends upon the mammalian neck. Interestingly, in the ruminants which have four chambered stomach, the first three chambers are considered to be the modified region of oesophagus, The mucosa of oesophagus has longitudinal long folds with large papillae, corn Vied in such mammals as sheep, goat and cat and degree of comification depends upon the food habits, being greatest in herbivorous and least in carnivorous. Branched tubular mucous glands which generally remain immersed into the lamina propria may also at times occurs at the upper part of the mucosa. The lamina propria composed of fibroelastic tissue with many lymphocytes has a core of projection of mucosal papillae. The oesophageal glands are distinct in oesophagus. In man, they contain alveoli and produce mucus whereas in other mammals they are made up of both types of glands i.e. mucous add serous which are plentifully found in all mammals except in some rodents. The muscularis is variable in the composition and number of layers, three layers occur in many carnivores whereas in pi g it is made up of four layers. A typical serosa is present. B. Stomach The stomach is basically the dilatation of GIT for the purpose of temporary storage of food. True stomach is characterized by the presence of gastric glands into the inner lining. The shape of stomach depends upon the shape of body and hence in case of animals like snakes, the stomach is extended longitudinally but occupies a more transverse position in those with 110 wider body. The end of oesophagus which connects to stomach is nearer the heart and hence called the cardiac stomach. The main middle portion of the stomach is called body or fundic stomach while the terminal part of stomach which connects to intestine through a valve, the pylorus or pyloric valve is called the pyloric stomach. The pyloric valve consists of folds of mucous lining membrane surrounded by thick sphincter muscles which regulate the passage of the contents of the stomach. In most animals, the stomachs are U-shaped or J-shaped and their muscular walls contract rhythmically to facilitate the process of through mixing and churning of food. Cyclostomes: In this class, the stomach is either absent or very poorly developed and is hardly distinguishable from oesophagus. Fish; In fishes, the morphological distinction between oesophagus and stomach is not very pronounced and the stomach may occur in greater variety of shapes in teleosts depending upon the feeding habits. The situation may vary from the total absence in such fishes as the carps, to straight spindle shaped in pike and siphon-shaped with cardiac to pyloric regions in the sturgeon and trout. In elasmobranch, however, it may be 3-shaped. The stomach is the source of a class of enzymes called endopeptidases which are highly active in acidic environment. The stomach provides the acid pH by the production of HCI which gets mixed with pepsin. The stomach mucosa covers longitudinal folds which are distinguishable from the oesuphugus by their diszppearancc in distends J position. Another important feature in the stomach mucosa is its changeover from the stratified epithelium into single layer of columnar cells which are interspersed with gastric pits. The typical mucosal epithelial cells have columnar shape and can be identified with the clear area containing the mucigen granules which are precursor for the mucous substance. The gastric MCs secrete mucus which is characteristically alkaline but in other areas of GIT, mucus tends to be acidic. The secretion of the mucus is believed to guard against autodigestion by HCI and Pepsin. The lamina propriaof the mucosa is reduced because of the presence of numerous gastric glands in this layer. A relatively thicker muscularis mucosa with longitudinally oriented smooth muscles fibers may be present. In the stomach mucosa, two types of multicellular glands occur i.e. fundic glands and cardiac glands also known as chief glands located towards the oesophageal end and body of stomach and the second type i.e. pyloric glands which are less in number and shorter in length compared to the cardiac glands. The fundic glands are simple branched tubular glands with a mouth that extends from base to a gastric pits and the body cells of these are those which produce the peptic or MCI secretion. In mammals, each of these secretions are produced by different cells but in fish one cell type produce both. The body of fundic cells are columnar with spherical nuclei usually located towards the outer portion of the cells. The 111 cytoplasm is filled with small strongly acidophilic granules which is pioneer substance for pepsin. The neck and body regions of pyloric glands are twisted and lined with cuboidal and low columnar cells and are thought to be responsible for mucus secretion. The submucosa is composed of loose arcolar tissue containing lymphatics, arteries, nerve plexes etc. The muscularis is relatively thin with two types of smooth muscles where inner circular layer is more developed in pyloric region. A typical serosa is present and is characterized by loose connective tissue, heavily vascularized and externally covered simple cuboidal mesothelium. Amphibia; All amphibians have a stomach for digestive function which is simple saclike structure. Histologically, the amphibian stomach very closely resembles to the fishes. However; surface epithelium usually consists of non-ciliated, clear bordered cells filled with mucoid secretory product and the gastric pits extending down along the mucosa are about one fifth of its thickness. The neck cells are found at the junction of gastric pits with the findic glands which are filled with true mucous. As in fish, the gastric glands play dual role of secretion i.e pepsin and IICI and are serous in nature. The pyloric glands are also same as seen in fish and cells are mucoid and resemble the surface epithelial cells, pits leading to pyloric gland are short compared to lhndic gland. The lamina propria found between the bases of the gastric glands contains lymphoid tissue and many Ieucocyless. Muscularis subrnucosa and serosa are same as observed in fish. Reptiles: In reptiles, no remarkable deviations are observed in the stomach i.e. snake and lizard have long spindle-shaped stomach which is correlated with elongated and narrow body shape. In this class, clear cut demarcation between oesophagus and stomach are also present. The crocodiles have most specialized gastric gland. Histologically, in this class, the stomach is lined with the same type of gastric columnar cells that are seen in the lower vertebrates. The gastric pits and gastric glands present close resemblance with those of amphibians. The submueosa, muscularis and serosa are same as described in previous classes. Birds: In birds, the stomach has been greatly modified due to lack of teeth and variations in the feeding habits which give rise to the development of some unique regions which are not found in any other group of vertebrates. The stomach is divided into two regions, the unique anterior region the uroventriculus which is continuous with oesophagus and has glandular lining and functions as the region for food storage, protein digestion, HCI secretion and possibly also in some fat digestion. The posterior region, the ventriculus or Gizzard is a highly muscular and act as teeth replacement. In addition to food grinding, it also plays a role in protein digestion. The glandular cells lining the gizzard secrete a tuft of homy layer which bears bumps or tubercles which aid in the grinding process. The gizzard is best developed in grain-eating birds and much less developed in insectivorous forms. In some cases, a third region called pyloric stomach has also been reported. 112 Mammals; in mammals, the stomach is modified, primarily depending upon the feeding habit, into a single chamber stomach with the same nomenclature as cardiac, fundic and pyloric region which occurs in insectivores, carnivores and man. However, in ruminating mammals it is most complex and consists of four chambers i.e. rumen, reticulum, ornasum and abomasum. The three initial chambers are known as the modified forms of oesopbagus and the fourth chamber, abomasum is known as true stomach. In stomach, the epithelial lining shows three different arrangements in different mammalian groups: i) In the first group represented by human, most carnivores, some edentates and insect eating marsupials, it consists of gastric cells and glandular columnar cells with apical clear border. ii) In the second group represented by lower primates, the cetacean, many artiodactyls, perissedactyls, the rhinoceros, the horse, some edentates, herbivorous marsupials, the upper portion of the stomach is lined with stratified squamous epithelium while lower is columnar and glandular. iii) However, in many rodents, few ntonotremes and marsupials, most of the stomach is lined with nonglandular kemtinized and stratified squamous epithelium. In mammalian stomach, unlike in previously discussed vertebrates groups, there is wider abundance of glandular structures which are much greater in number and variety in the cardiac and ftmdic part of the stomach. However, in pyloric region, they get reduced both in abundance and variety. There are near[y four different types of secretory glands/ structures i.e. mucous, chief, parietal and argentaffin cells which occur in the different regions of mammalian stomach. In the cardiac part of the stomach, cuboidal or low columnar mucus- secreting cells and occasionally parietal cells are present. In the lundic region, the gastric juice is derived From it and all four types of above described glands may be present in this region. MCs present at the opening of the pit may be called surface MCs whereas those in the neck region of the pit are named mucous neck cells which are same as those found in amphibians, reptiles and birds. The chief cells are associated with the secretion of pepsin and the parietal cells product the HO and are found in the upper region of the gastric glands. The last argentaffin cells are diffisely scattered throughout the gastric glands and their function is not clear, in pyloric region, the gastric pit is less deep than those of the fundus and the secretory cells are primarily MCs but few parietal and chief cells may also be present in the region. The above description of the mammalian stomach pertains to 'Simple" type of stomach which is not divided by internal partitions. While the different regions of the stomach may be differentiated externally but their internal lumen is completely contiguous and no regional changes take place in the internal epithelial wall. 113 In the other type of stomach found in ruminant attiodactyls, the first part which is the modification of oesophagus and is made up of three distinct regions i.e. rumen in which the swallowed food is initially stored which gets passed on to reticulum from where the food is regurgitated into mouth, masticated and finally passed on to the third chamber, ornasum. Finally the food enters the abomasum where it undergoes digestion and then passes on to intestine. The first three chambers, therefore, serve the purpose of storage and moistening of the food and are largely without any glandular secretory structures except the presence of mucous glands. The omasum is internally provided with leaf like structures practically filling the entire lumen and are covered with large comified stratified squamous epithelium with large papillae. The abomasum is the glandular structure and is regarded as the true stomach which is equivalent to that of the simple stomach found in other mammals. C. Intestine: In Gastrointestinal tract, the intestine plays the dual function of the remaining part of the digestion of the ingested food material followed by its absorption through the circulatory vessels into the different regions of the body for its ultimate utilization. The main events consist of the mixing of acid chyme with bile from the liver, pancreatic juice from the pancreas and the secretion of the intestinal glands. The intestine may become elongated and coiled or its diameter may be increased. The absorptive function of the intestine is achieved mainly by increasing the absorptive surface area which could be done either by increasing the length of the intestine as in herbivores or by developing cecae, longitudinal folds and extensive vii. The intestine is usually made up of two parts, the long but narrow small intestine in continuation with the terminal part of the stomach and wider large intestine. The degree of external demarcation of intestine into two regions as well as the localization of secretory structures associated with the elaboration of digestive juices show considerable variations in various vertebrate groups as detailed below. Cyclostomes, In this class, the intestine is straight and enlarges slightly to form a rectum which opens into a closeal depression. A longitudinal fold called typhlosole which projects in the wall of intestine is present. Fish; In fishes, the different regions of the intestine are not clearly demarcated so also the internal histology which does not show any remarkable variation except the development of various degree of longitudinal folds. In shark and lungfishes the end of the spiral valve and narrowing of the gut marks the beginning of rectum. The terminal portion of the intestine is also characterized by the increase in the proportion of goblet cells and increase in the thickness of circular layer of muscularis. The absorptive function is clearly evident by the increase in the internal surface area which shows wide variations in different groups of fishes. For example the coiling Cr looping of intestine to accommodate the increased length 114 of intestine in a limited space, development of cecae as intestinal outpocketing and long Longitudinal folds which in certain casts may also have intestinal villi as seen in the case of mammals. Many fishes may possess a combination of all the above features to increase the surface area. In fishes, while the digestive function seems established due to the presence of digestive juices but their source of elaboration is somewhat obscure. The absorptive columnar cells are typical of those in vertebrates. The absorptive capacity of the hind gut is considerably reduced but the secretion of micus increases. Amphibia : In the Amphibians, the intestine is not differentiated into small and large regions but shows a slight degree of coiling in small intestine, like in fishes, internally the two regions can be differentiated by a marked reduction in the degree of the intestinal folds in the hind region of the intestine. The amphibian intestine is also characterized by the absence of true villi which is compensated by the increase in the intestinal length and branched mucosal folds. The terminal part of the intestine opens into the cloaca which also receives the urinogenital products. Reptiles: The reptilian intestine is essentially similar to those groups described above with no clear cut demarcation into small and large intestine except in few groups where it has been reported to be thicker and opens into cloaca. However, an ileocolic valve between the small and large intestine is present which is provided with a colic caecum which has appeared for the first time in this group of vertebrates which, however, is absent in crocodiles. Birds: The intestine of this group is different from the other lower vertebrates in showing a clear cut demarcation between the small inlesline and the large intestine by the presence of external cecae at the junction or the two. Further, the small intestine is much longer in length, smaller in diameter whereas the large intestine is shorter in length and wider in diameter, the features which closely resemble the mammals. Internally, the bird intestine is characterised by the first time appearance of the crypt of Lieberkuhn occurring between the bases of the villi and acting as an opening for short single or branched tubular gland. The large intestine differs from that of the small intestine in terms of having more goblet cells and reduced height ofvilli. Mammals: The mammalian intestine, like in birds, is clearly differentiated into the small and large intestine and this difference is more marred and clear in terms of change in length and diameter. In human, the small intestine roughly measures about 22.5 feet whereas large intestine is approximately 5 feet in length. In this group, the small intestine is subdivided into three distinct regions largely on the basis of difference in the length where the first 115 region called duodenum which is continuation with stomach, slightly curved measuring about 25 cm in length, lodges pancreas in its fold and has the opening of joint duct originating from pancreas and gallbladder. The second region of small intestine named jejunum is about 1 meter in length, not well demarcated externally, joins with the last region called ileum, is the longest part of the small intestine measuring nearly 2 meter. The ileum finally opens into a wider diameter large intestine which consists of a much longer inverted U shaped colon which finally terminates into rectum, a comparatively small receptacle opening to outside through anus. At the junction of ileum, with the colon, a small diverticulum in the form of caccum/vermiform appendix is attached and internally an ileocolic valve regulating the passage of food from small to large intestine is present. Among the mammals only monetremes possess a cloaca The mucosal folds of the small intestine have the abundance of villi which are leaf shaped in duodenum, finger shaped in jejunum and club shaped in ileum and the number of villi is highest in carnivores. In mammals, gland of Brunner which is confined to the region of duodenum , associated with the secretion of mucus and bicarbonate makes their appearance for the first time. Further, the appearance of circular and oblique folds up to 8 mm in height called plicae circulars/ valves of kercking/ valvulae conniventes, maximum in height in lower duodenum and jejunum enhancing the absorptive capacity of the surface have also been documented. The large intestine, apart from above mentioned differences, is characterised by the absence of villi and plicae circulars. Pisces Gastrointestinal Tract: The different segments of the alimentary canal in fishes, like in other vertebrates, consist of mouth, buccal cavity, pharynx, oesophagus, stomach, intestine with pyloric cacea opening out into cloaca in sharks, rays and dipnoans but not in teleost which connect to outside through anus. The digestive system, in addition to GIT, is also connected with certain digestive glands i.e. the liver, gallbladder and pancreas. The morphology and functionality of the fish GIT arc dependent upon its food and feeding habits as well as the body shape. The anatomy of fish digestive system has been reviewed extensively by many workers from time to time. The notable among them are Andew (1959), Harder (1975), Kapoor et ai (1975), Ray and Moitra (1982), Zihler (1982), Kapuor and Khanna (1993), Stevens and Hume (2004) and lately by Wilson and Castro (2011). According to Baddinglon and Kuzmina (2000), there is an overall consistency in the major functions of digestive tract among different fish species which includes primarily the digestion of food stuffs through secretion of digestive enzyme and hormones involved in regulation of digestion and metabolism and osmoregulation and defense against pathogens and harmful components of environment. 116 Steven (1930) divided the fish species based on feeding habits into three conventional categories, i.e. Herbivorous, Carnivorous and Omnivorous and the morpho-functional features of the digestive tract are closely dependent on these feeding characters. There is a general consensus that the GIT length is the largest in herbivorous and smallest in carnivorous with somewhat intermediate position in omnivorous fishes. The ratio of the body length with GIT has been reported to be 0.2-2.5, 0.6-8.0 and 0.8-15.0 in carnivorous, omnivorous and herbivorous respectively (Al-Husanini 1946). However, certain exceptions need to be mentioned where all herbivorous may not necessarily have long guts which at times may even be shorter titan those of some carnivurous. It is largely due to the fact that many fish eat food which sometimes is mixed with considerable indigestible material such as mud and secondly the size of food particles-from submicroscopic plankton to fish may also influence gut configuration. Interestingly, the feeding habits may not always show consistency and may change from carnivorous to herbivorous according to requirement as seen in the roach Rutilus netilus. Many fishes vary their diet from plankton in the summer to fish in the winter, even utilizing bacteria and algae as a source of food (Barrington 1957). The fact that the GIT in 5shes get suitably modified according to the ingested fund material has been comprehensively illustrated in marine herbivorous fishes. Their GIT has been classified into four types based on the adaptation designed to degrade cell walls of algae and bacteria by acid lysis, mechanical trituration and microbial fermentation (Hom 1989). Type I category includes herbivorous fishes such as the Surgeonfish (Acanthunis nigro(uscus) which has no mechanism of trituration of food and a thin walled stomach, but relatively long intestine, type II represented by the Mullets (Mugil cephalus) have a thick- walled, gizzard like segment of stomach and the intestine is of variable length, type III which includes fishes like marine Parrotfish (Scarus mbroviolaceus) and freshwater grass carp (Ctenopharyngodon idella) have pharyngeal teeth that grind food into small particle size hence obliterating the requirement of stomach and are provided with relatively short intestine and the type IV is exemplified by the sea chub (Kyphosus sydneyanus) that has a large intestine which includes a distinct hindgut. In fishes, the mouth exhibits a variety of fascinating adaptations for capturing, holding and sorting of the food. These include the presence of teeth which may be located on the jaws, tongue, pharynx but mandibular teeth or pharyngeal teeth are the most common types. Many carnivorous fishes have sharp teeth for the capture of prey (e.g. Shark and Piranha) and in herbivorous for grazing an the algae attached to the rocks or corals as seen in Parrotfish (Steves and Hume 2004). In some fishes pharyngeal teeth are situated on the modified fifth gill arch which does not carry gills such as in common carp which are used to break up hard food by cutting in some species and crushing in others. In omnivores, the teeth are not confined to the jaws but also occur on the plate on the roof of oral cavity and used for grasping, puncturing and holding prey (Dattamunshi and Chaudhary 1996). In essence, feeding habits of the fish cause the major adaptive structural modifications primarily in teeth, stomach (if present) and in the length of intestine. 117 The oesophagus of fishes is short and straight tube connecting the pharynx to the stomach and in agastric fishes to intestine directly with certain exceptions as in eels wlterc it is relatively long, narrow, and serve during seawater residence to dilute ingested seawater before it reaches the stomach. In chondrichthyans and chondrosteans, the inner fold of the oesophageal lining is provided with conical and backward facing papillae whereas, in osteichthyans they are in the form of 6 - 50 longitudinal folds which are additionally provided with secondary and tertiary mucosal foldings (Suyehvo 1942, Buddington and Christofferson 1985, Holmgren and Nilsson 1999). In many species, a duct of swim bladder opens into oesophagus which serves as a hydrostatic organ for the adjustment of specific weight (Stevens and Hume, 2004). The literature shows a wide morphological heterogeneity of oesophageal wall among species (Kapoor et a] 1975, Reifel and Travill, 1977, hlitji 1983, Martin and Blaber 1984, Elba! and Agulleiro, 1986, Grau eta! 1992, Scocco eta!, 1998). In freshwater teleost the mucosal layer has large number of goblet mucus secreting cells but in cyclostomes mucus is secreted by the columnar mucous epithelial cells (Wilson and Castro 20{ 1). The abundance of MCs in the stratified columnar epithelium depends upon species and environmental salinity (Sullivan 1907, Yamamoto and Hirano 1978 and Meistter or at 1983). The mucus secreted from the MCs, covers the epithelial layer for the protection from the chemical and mechanical damaged and also plays a active role in the lubrication of food ([lumbert et al 1984. Grau et al 1992, Shephard 1994, Abaurrea- Esquisoain and Ostos- garrido 1996, Vegetti eta] 1999 and Albrecht eta! 2001). Highly viscous mucus produced by the pharynx as in the case of carps helps to trap small particles which aggregate into boluses and lubricate the chewing plates (Sibbing and Uribe 1985). The posterior oesophagus of some species i.e. Winter flounder Pleuronecres unrericanus and yellowtail flounder P. ferrugineur may have role in pregastric digestion (Kapoor and Khanna 1993 and Murray et al 1994a). Taste buds are predominantly present on the anterior oesophagus in most of the teleost species and are generally absent in posterior portion (Reifel and Travill 1977, Martin and Blaber 1984, Arellano et at 2001, Diaz et at 2003, Diaz et al 2006 and Raji and Norouzi 2010) suggesting that the gustatory sense extends up to this region( Moitra and Sinba 1972 and Ray and Moitra 1982). In some elasmobranch, masses of lymphomyeloid tissue (Lcydig organs) are found (Fange and Grove 1979). in general, the glands are absent in the oesophagus although some complex structures resembling glandular features have been described. Considering the fact that posterior region of the oesophagus finally connects with the stomach, the occurrence of some transitional glandular structures may not be surprising. However, whether the structures are indeed gastric glands has not be clearly established as in the case of milkfish Clranos clans (Chandy 1956) and pike Esox lucius (Reifel and Travill 1977) where these glands arc different from gastric glands. The scanning electron microscopy (SEM) of the epithelia topography of mucosa reveals fingerprint like microridges which dominate the surface with mucus exuding from small crypts (Yamamoto and Hirano 1978, Ezeasor and Stokoe 1981, Meister et a] 1993). These microridges are thought to improve mucus adherence and minimization of the impact Its damage to epithelium (Abaurrea-Esquisoain and Ostos-garrido 1996). The columnar epithelium has high NeW-ATPase immunoreactivity and is associated with osmoregulation in marine teleost (Crosel12011). The morphology of stomach of fish species, like other GIT segments, is dependent on food and feeding habits and may be classified into four general configurations (Harder 1975, Kapoor 1957, Reifel and Travill, 1978. Martin and Blaber 1984). The simplest type may include a straight stomach with an enlarged lumen, as in Esox americanres & Esox lucius. The other type may be a 'U' or `J' shaped or siphonal which is more common in Osteichthyes and Elasmobranchii and show the enlarged lumen as in Ambloplites nipertris, lxpomis rrraerochirus, Pomosis uigromaculaius, Salneo solar and Coregonua lavureiur. The third typeis Y shaped with agastric cecum in the form of a blind pouch meant for the purpose of storage of large food particles as in Ictolurug nehulosus, Mieropterus sauna ides, Pereajlaveseens alosa and Anguilla. The last category does not have any stomach as in cyprinids, gobids, blennies, scarids and many others. Klumpp and Nichols (1983) suggested that the absence of stomach indicates that the acid hydrolysis does not have a role in the breakdown of the plant cell walls and the fish pharyngeal grinding plays the role of rupturing the plant cell walls. Some parts of the digestive tract can also be used for gas exchange such as in Dalia pectoralis (Umbriidae) (Crawford 1974) and two families of bottom-dwellers catfishes inhabiting South American freshwaters that have digestive tracts modified for air breathing. Many genera from the families Loricariidae and Trichomycteridae have evolved modifications of the stomach which enable its usage as an accessory respiratory organ (Carter and Beadle 1931, Gradwell 1971, Gee 1976, Silva et al. 1997, Armbruster 1998, Satora 1998, Takasusuki et al. 1998, Oliveira et al. 2001, Cala 1987). The demarcation of oesophagus from the stomach is not clearly marked out except in the case of few elasmobranch which are provided with a distinct esogastric valve (Harder 1975, Kapoor et at 1975, Fange and Grove 1979). However, histologically the beginning of the stomach can be clearly deciphered by the abrupt change in the epithelium to the columnar epithelial cells of the stomach coupled with the appearance of gastric glands. Generally, the stomach 1s demarcated into the anterior cardiac region connected to the oesophagus which is not secretory and stores food particularly in some species that ingest large meals. The second fundic region also called the true stomach and the last, the pyloric region with the muscular wall which is thicker than the two other regions. In some species it is well developed, forming a grinding organ (gizzard) e.g. mullet (Horn 1992) that allows it to be distinguished from the adjacent fundic region. The function of gizzard in fishes is similar to birds to grid or trituration of food. the overall size of stomach is dependent on the feeding urge of the fishes which varies from very large stomach as in gluttonous fishes such as Gaddus macrocephalus and angler fish to very small as in the Salangidae and 113 Opelegnathidae. The pylorus part transports the food from the stomach to intestine which is controlled by muscular sphincter and may be absent in Borne fishes, Cataldi et al. (1987) stated that the gastric glands, which contain one type of cells, are present in the cardiac region and increase in number in fundic part, but are absent completely in the pyloric region. However. Arellano et at (2001) reported that the gastric glands of Solea seuegalensis are numerous in the pyloric and fundic region but are absent in the cardiac portion. The cells of the gastric mucosa secrete different components which arc involved in the digestion and absorption. These tnbvlar gastric cells are lined by a single type of cuboidal cells, containing acidophilic granules, indicative of zymogen granules and the oxynticopeptic cells present in the gastric glands in gastric mucosa and secrete the acid and enzymes (Barrington 1942, 1957). However, in mammals this secretion is executed by two cell types i.e. parietal or oxyntic cells and chiefs or peptic cells (Stevens and Hume 1995). The oxynticopeptide cells do not seem to be dependent on freshwater or marine environment of the fish but ultrastructully some modifications may be evident (Bomgren et at 1997). The presence of endocrine cells that secrete gastrin, somatostatin, and other hormones in the stomach mucosa has been established (Rust 2002) and Noaillac- Depeyre and Gus (1982) have reported that in the perch, Perca fuviatihs and catfish, Ameinrus nebulosus three and four different types of endocrine cells each possessing characteristic granules exist. The lamina propria contains connective tissue fibers, blood vessels, nerve plexus and mast cell sand is also rich in immune cells known as lymphocytes. The last part of the stomach ends at pylorus which is present as a sphincter of circular muscles and is a valve like structure to regulate the passage of food into intestine and to prevent backward flow into the stomach. In a number of stomachless fishes such as Tina and Aspius, the pyloms-like structure is also found which due to the absence of stomach is referred to as the oesophageal-intestinal valve (AI-Hussaini 1946). Certain structures in the form of small evaginations of the intestinal wall in the teleost fishes called pyloric cecae which appear as blind ended linger-like projections at the proximal intestine immediately posterior to the pyloric sphincter have been reported (Rust 2002). They vary in dimensions from small evaginations to branched or tuff-like structures and also in number from one to over a thousand (Stevens and Hume 1995). The pyloric cecae increase the absorptive surface area of the proximal intestine increasing the intestinal length and are also associated with the secretion of the digestive enzymes (Ddpido et al 2(04). The cecae are much developed in carnivorous fishes particularly with short intestine compared to herbivorous fishes (Clements and Raubenheimer 2005). Generally, ceace are present in gastric species but are lacking in agastric species (Buddington and Dimond 1987). The mucosa of pyloric caeca is similar to the intestine and no special cell types or glands and lesser abundance of goblet cells are present in this organ (Kapoor et al 1975, Buddington and Dimond 1987 and Houssain and Dutta 1996). 120 Many interesting surveys have been made to describe the distribution of stomachless fishes in the entire piscine group. Wilson and Castro (2011) based on revised phylogeny of (Nelson, 2006) and a through survey of literature stated that 27% families and 77% species are without stomach. The major families of the stomachless fishes as listed by Harder (1975) include cyprinidae (carp), cobilidae (leaches), cyprinodontidae (killifish), gobiesocidae (clingtish), gobioides 1perciformes (gobies), Blemidae (blennies), scaridae (parrottish) where the species of the largest order cypriaiformes is predominantly agastric (Fiertak and Kilarski 2002, Nalson 2006, Slechtova at at 2007, Chen at at 2008). Further, studies have demonstrated that lack of stomach in numerous fish is not directly related with their diet (Logothetis at at 2001). Curiously, in some agastric fishes like pufferfish Takifugu rubripes, the stomach has acquired an alternative function to food digestion whereby it is inflated as a balloon to serve as a defensive mechanism (Kurokawa at a], 2005). In some agastric fishes, the anterior intestine may bulge to form an intestinal bulb or pseudogaster as in Petromyzon and Labeo where it is used for temporary storage of food and the gastric gland and a pylorus is lacking. A spherical shaped ceacal chamber found on the anterior portion of intestine and posterior portion of oesophagus in the parrot fish Takiifugu rubripes has been found to play the same function (Al-Hussani 1946). The intestine, playing an important role in the completion of digestion and finally the absorption, is highly variable according to the feeding habits and is relatively short and straight particularly in carnivores where the length is about only 20% of the body length (Buddington and Kuz'mina 2000). However, in herbivore species, it can be over 20 times of the body length and the percentage of plant material in the diet is the major determining factor for intestinal length (Buddington at at 1997, Clements and Raubenheine 2005) where intestine changes direction and can be highly coiled to fit into the limited space available in the body cavity. In stomachless fish i.e. carps, the intestine has three histological zones (anterior, mid and posterior intestinal segments), where the anterior intestine of the fishes can be expanded into an intestinal bulbs which is different from stomach by the absence of gastric glands (Wal lace at al 2005). Externally, it may not be easily possible to decipher the three different segments of intestine except some tentative bulging visible at the presumptive beginning of the new intestinal segment. The intestine histological patterns of fishes consist of structures similar and with the same nomenclature as those found in all vertebrates (Banan Khojasteh et al2009b). The gut wall from foregut to hindgut consists of four concentrically arranged layers. The presence of a highly folded spiral valve along the longitudinal axis largely to increase the intestinal surface area is an important characteristics of the etasmobranchs which, however, is either rudimentary or totally absent in teleosts. In some species i.e. Salmo facia, mid region contains an ileorectal valve that separates it train posterior region (Steven and I-lume 2C04). Midgut which is principle site of nutrient absorption and hindgut for water and salt absorption plays an important role in immunity and functions as a barrier to pathogens and 121 other environmental toxins (Wallace et al 2005). It is believed that eosinophilic granule cells are analogous to mammalian mast cells which are extensively present in mucosa layer of teleosts where their function remains unclear. The main functions of intestinal epithelium are lipid digestion, absorption and transport and thatthe rectum is important at least partially for protein degradation (Murray or al 995). The intestinal epithelium of the simple or pseudostratified columnar type, is made up of cells named enterocytes possessing a well- marked striated border or microvilli. Goblet-type mucous cells, lymphocytes and enteroendocrine cells are scattered throughout the epithelium and rodlet cells are also found in some teleost fishes (Kapoor et a11975). The columnar epithelial cells that dominate the intestinal epithelium are relatively homogenous in appearance but show regional differentiation (Yamamoto 1966). Secretory!Absorptive Cells in Gastrointestinal Tract: Different varieties of secretary cells have been reported in the GIT of fishes. Some of them such as MCs are most widely occurring and others like ciliated cells and zymogen cells are mostly confined to the primitive group of Fishes. Some cells with two different secretions such as oxynticopeptic cells are found to be in combined cluster in Fishes which, however, get separated into two discrete types of cells in higher vertebrate groups. A brief account of these secretary cells occurring in fishes in order of their degree of abundance is given below. a. Mucous cells (MCs): Mucus is secreted from specialized mucus producing cells present in mucosal lining all along the alimentary canal and the number of mucus secreting cells varies between the buccal, oesophagus, stomach and intestine regions (Martin and Blaber 1994, Kupermann and kuzmina 1994, Tibbetts 1997, Sklan & Lupatsch 2004 and Abate at al, 2006). However, the types of mucus may vary from species to species due to different feeding habits, diet composition and the ambient conditions of different regions of gut (Olsen et al 2007, Diaz at al 2008. Dezfuli et al 2009, Manjakasy et at 2009). While there may be a predominant presence of MCs in the entire GIT, the difference primarily ties in the shape and the arrangement of these MCs. They are distinctly columnar in stomach but are mostly globular in intestine and somewhat of mixed shape in oesophageal region. These cells have been essentially involved in lubrication and mechanical and pathogenic defense of GrT. In fishes, the salivary gland are lacking and hence the oesophagus MCs could carry the same function as that of mammalian saliva in protecting the mucosa of the first part (Marchetti et al 2006). However, in buccal and oesophagus regions, the increase in the number of these cells help in the rapid passage of food and the stratified epithelial MCs protect the oesophagus Gam injuries during the passage of solid particles. In the mucus granules stained with periodic Schiff the neutral mucus secreted by these cells protect the epithelium and underlying tissue from gastric juice (Allen and Flemstrom 2005). Unusually, in gobiid fish, mucus is not detected in surface columnar epithelium (Milward 1974, Kobegenova and 122 Dzhumaliev 1991 and Jaroszewska et al 2008) suggesting that acid-peptic digestion is not significant and hence a protective mucus coat not required. The mucus secreted by goblet cells in tae intestine has many functions. It lubricates undigested materials for onward progression into the rectum, may have a possible role in osmoregulation and regulates the transfer of proteins or a fragment of them as well as of ions and fluids. The possible role of mucosubstances in intestinal absorption processes is supported by the findings of Bozic et al (2001) who observed that starvation induced an increase in the number of intestinal goblet cells in carp (Petrinec et al 2005). In many species, it is observed that the number of MCs increase from the pyloric caeca to the rectum which may imply the need for increased mucosa protection and lubrication for faecal expulsion (Murray et al 1996). b. Enteroendocrine cells; The gastrointestinal endocrine cells are distributed in the mucosa of the gastrointestinal tract and they synthesize various kinds of gastrointestinal hormones. They play important functions in the regulation of the physiological functions of the digestive tract. To understand the comprehensive functioning of the metabolic processes in GIT, it is important to study the gastrnenteropanereatic system (GEP) which would also include the secretions frcnn pancreas and also the nervous system. The existence of endocrine cells has been immunohistochemically demonstrated in the gastrointestinal tract mucosa of different fish species (Diler et at 2011). These cells have been found throughout the epithelium of the digestive system (Holmgren and Olsson 2009) including the pancreas. The secretion from the gastrointestinal net are chemically peptide or amine, detected in lamina epithelial, glands, several connective tissues, mucosal nerve ganglions and intermuscular nerve plexus. The pancreas of teleost fish is particularly interesting because its endocrine portion is anatomically distinct from the exocrine portion. The gastroenteropanereatic peptides include a variety of different hormones! factors which are grouped together on the basis of their structural and functional similarities. One important family is that of insulin (INS) which also includes IGFs. The second largest family is that of secretin which has glucagon (OLD), glucagon like peptide (GL?). gastric inhibitory peptide/ glucose-dependent insulintropic peptide (GIP), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). The somatostatin (SS) family contains many small variants of SS. The pancreatic polypeptide (PP) family include PP, neuropeptide Y (NPY), peptide YY (PYY) and fish pancreatic polypeptide . The gastrin family also included cholecystokinin (CCK) in addition to gastrin. The galanir. contained galanin alone (Nelson and Sheridan 2006). However, in another subsequent review by Takei and Loretz (2011) the families of guanylin, natriuretic peptide, calcitonin gene-related peptide, leptin, tachykinins and neuromedin U were also added. The occurrence of the above peptides in fish GEP space has been difficult to establish conclusively. Most of the biological actions have been broadly similar to those reported in mammals except the functional modifications based on 112 the dietary habits of different fishes. Hence, the biological actions of these Gt peptides in fishes have been mostly based using mammalian preparations on fish target system even though the conclusive origin of these peptides in fish GIT may still be a wide open field. e. Oxyntieopeptic cells: Contrary to the mammalian system, in fish, there are generally only one secretory cell type observed within fish gastric glands. In mammals, there are separate pepsinogen-secreting chief (peptic) cells and acid-secreting parietal (oxyntic) cells but in fishes, single oxynticopeptic or oxyntopcptic cells contain the cellular machinery for both the functions, Yao and Forte (2003) identified the gastric proton pump, H`fKtATPase through the immunolocalization in the apical region of gastric cells in Atlantic stingray by using the porcine a subunit antibody (Smo€ka et al 1994 and Gawlicka et al 2001). Expectedly, immunoreactivity was found to be absent in stomachless fishes (e.g. Fugu crysopus, Pseudolabrus japonicas and Eptatretus burger. (Yasugi 1987, Yasugi at at, 1988). The enzyme pepsinogen is found in round-electron dense zymogen granules in the supranuclear region of the gastric cells and is localized by cosinophilic stain (Barrington 1957, Kapoor et al 1975). Pepsinogen has also been localized in oxynticopeptic cells in fish using in situ hybridization in Pseudopleuronectes americanus (Gawlicka et at 2001), Pagrus pagrus (Darias or at 2007) and immunehistochemistry in Osteichthys e.g. Sebastes inemis, Gimnolhorax hikado and Oreochromis myla'ss and Chondrichthyes Narke japonica and Triakis Scylla. Pepsinogen production and secretion are associated with the golgi apparatus and rough endoplasmic reticulum in the basal region of the cell (Ezeasor 1981, Michelangeli et at 1988). d. Enterocytes: The columnar absorptive cells present in single layer with distinctive apical brush border in intestinal epithelium are called the enterocytes which are present in teleosts fish species and are found to be generally similar to those in other vertebrates. (Marchetti et al 2006). These cells, which are generally tall and narrow with elongated nuclei, a well-developed brush border and mitochondria located both in apical and basal region, play a role in absorption and secretion (Yamamoto 1966, Ezeasor and Stokoe 1981 and Cyrino et al 2008). In the posterior intestine absorptive cells with large vacuoles in the apical cytoplasm are present (Yamamoto 1966, Ezeasor and Stokoe 1980, Langille and Youson I984). Enterocytes show strong hasolateral expression of Na+/K--ATPnse which is essential in driving a number of transepithellial transport processes important for nutrient uptake and ion regulation (Genten et at 2009. Wilson and Castro 2011). The apical portion of enterocytes is differentiated by the occurrence of microvilli which look like the brush border through Iight microscopy and contribute more than 90% of the apical surface area in Tilapia aurea and T. zilli (Frierson and Foltz. 1992). The function of brush border is the absorption and digestion between the microenvironment where enzyme involved in food break down and transportation is located (Kuzmina and Gelmen 1997). The activities of a number of enzymes i.e. alkaline 124 phosphatase, dissacharidase, leucine-amino peptidase, tri and di-peptidase have been localized in border membrane in vertebrates including fish. However, pancreatic lipase and esterase, a amylase and oxypeptidase are absorbed to the microvilli. In the trout, lipid absorption in the anterior intestine and pyloric caeca has been demonstrated (Sire at at 1981). In the posterior intestine, columnar epithelial cells with large vacuoles have been identified as the site of macromolecules uptake ((lersten et al 2009). However, it is important to keep in mind that the majority of protein uptake (80%) occurs in anterior intestine (Fange and Grove 1979). e. Oesophageal cells: It is generally assumed that there are no specific glands associated with oesophagus. However, some alveolar and tubular glands in submucosal or distal cesogastric region have been observed (Kapoor eta! 1975). These were thought to be the transitional gastric glands although in milkfrsh, Chanos chanos and Esox lucius they are clearly distinct from the gastric gland and secrete mucus (Chandy 1956 and Reifel and Travill 1977). f. Rodlet cells: Another category of infrequently occurring cells include rodlet cells which are associated with many epithelial tissues, including gut lining of teleastean fishes. These cells derive their nomenclature from the presence of large rod-shaped cytoplasmic granules and are characterized by a wide fibrous layer beneath the plasma membrane and ovoid and a basally located nucleus. It has been suggested that rodlet cells may be involved in water or electrolyte transport or have functions similar to those of MCs such as lubrication, antibiotic effects and combating ectoparasites on epithelial surfaces (Manera and Deafali 2004). g. Ciliated and Zymugen cells: Both these secretory cells are considered to be the character of the primitive group of fishes even though their presence has occasionally been recorded even in tefeost fishes. The ciliated cells are provided with apical cilia and closely resemble with those of absorptive cells present in the teleost intestine. Zymogen cells are present within the intestinal epithelium and are normally found to occur in those groups where discrete exocrine pancreas is absent. GIT STUDIES ON TELEOSTS OF INDIAN SUBCONTINENT: The survey of literature reveals that a large number of teleost fishes of Indian subcontinent have been studied. There has been a gradual evolution of knowledge of the structural details depending upon the availability of the analytical tools and techniques. It is worthwhile to mention that one of the few first studies on GIT of Indian fishes was carried out by Sarbahi (1939) on the GIT morphology of Lebeo rohila. an important herbivorous fish which followed another study by Mosin (1946) on the morphology of the GIT of a 125 carnivorous fish Anabas testudinevs. This followed a long gap of nearly one decade during which no major study on the GIT of Indian fish species is found documented in literature. The subsequent period of 60s and 70s witnessed a resurgence of interest on the studies of GUT of Indian fishes by large number of different workers but such studies were mostly confined to morphology of predominantly carnivorous fishes belonging to either carp or air- breathing catfish. At this point, it is important to acknowledge the voluminous contribution made by Khanna (1961) who gave a comparative account of GIT of large number of teleost fishes of Indian subcontinent. These studies were further extended by the same group to include aspects like comparative account and histomorphology, distribution of taste buds and mucous secreting cells of buceo-pharyngeal region and morphology and histology of intestine of Indian teleosts (Khanna 1962, 1964, 1968; Khanna and Melnntm 1970, 1971; Khanna and Pant 1964). During the period of 1950.70, an overwhelming number of studies were carried out on anatomy and histology of G1T based on feeding habits i.e. carnivores, herbivores and omnivores categories and the studies of Pasha (1964 Lb,c) on three different fishes with aforesaid different feeding habits i.e. Megalops cyprinoides, Tilapia mosammbica and Mystss gufo deserve special mention. Most of the contemporary workers also focused on specific GIT segment-based studies such as on single or comparative account of oesophagus, stomach and intestine to highlight the morphological and functional sigoilicauea of each region of GIT. Luterestingly, must of these studies remained confismd to either carps or air-breathing fishes which may be due to their wider abundance and economic significance. A limited number of studies were carried out mostly on Indian major carps such as Cirrhina mrigala (Sinha and Moilra 1975), Labeo rohira (Kama) 1967), Labeo ca1basu (Sinha 1976) and Carla calla (Moitra and Bhomik 1967) describing their GIT changes concomirant with various life cycle stages of these fishes. Literature pertaining to the histochemical localization of mucopolysaccharides and some enzymes on the various regions of GIT is limited and often confined to the anterior part of the GIT. Moreover, no concerted effort has been made to localise various cell types associated with the secretion of digestive juices which facilitate the process of digestion. More Iocused studies on large number of Indian teleosts are warranted to fill the existing lacuna of knowledge on this important aspect. In order to get an idea of finer structural details of the internal surface area of Gil, Sinha and co-workers have made extensive SEM studies largely on Indian major carps and catfishes (Sinha 1981, 1983; Sinha and Chakrabarti 1985, 1986, 1989) and recently on snow trout Schizothorar curvfrons by Mir and Channa (2010). Curiously, this reviewer has not come across any study on the TEM details of the GIT of any Indian fish. It seems clear from the above details that morphohistological and anatomical features of the alimentary curial of different Indian teleosts are well documented. However, the great degree of variations in the structural details of the GIT are inevitable largely due to the great diversity in the piscine fauna and also the variations in the dietary habits of the fishes. The existing lacuna of information particularly in relation to the distribution of various kinds of mucopolysaccharides in the different regions of the Gil prompted us to undertake this study. OBJECTIVES OF STUDY: The comparative morpho-histoIogy and anatomy of the gastrointestinal tract of an air breathing fish ff. fossilis has been described by Chitray and Saxena (1962), Rastogi (1966) and Dattaurunshi and Chcudhary (1996).Sastry and Subhadm 11982) and Chakrabarti and Glmsh (1989) have studied the toxic impact of cadmium poisoning and Zinc on the alimentary canal of H fossilis. Recently, Sarkar and Ghosh (2010) have demonstrated the toxicopatho logical effects of the metani] yellow an the intestine and rectum of H. fossilis. However, no systematic study has been carried out to study the gross morpho-histology, the qualitative and quantitative distribution of glycoconjugates in various acgments of GIT. Further, no data is available on the effect of higher salinity on the changes of glycoconjugate moiety and the population of MCs in the gastrointestinal tract where they are speculated to play an important role in fresh and salt water osmoregulation. In the present study, an attempt has been made to document the detailed histomorphology, distribution of glycoproteins (GPs) component in the GIT and qualitative and quantitative changes of GPs following transfer of FL fossilis to higher salinities. The present study has, therefore, three main objectives. To study the gross morphological structures of alimentary canal in K fossilis in its native fresh water environment. 2. To study the different types of GPs present in the mucosa layer of the different segments o£ alimentary canal of II.fossilis. 3. To study the changes in GPs moiety present in the mucosa layer of alimentary canal of the catfishes, H. fossilis following transfer of these fishes from tap water to higher salinities i.e. 25%, 30% & 35% sea water. II. Experiment protocol: The experiments on GIT were performed on acclimatized catfish maintained under controlled laboratory conditions as described in Materials and Methods. The catfish H. fossilis were allocated iu four groups. The fishes of Groun 11 and HI and IV were gradually transferred to 25%, 30%, and 35% SW after residence of one week in each salinities whereasre oup i was maintained in dechlorinated TW and served as control. The fish were fed on alternate days and water was replaced daily. After 7 days of transfer five fish from each group were anaesthetized and sacrificed and whole alimentary tract were removed from oesophagus to rectum and various segments of GIT were cal and fixed in Camoy's fluid for histological and histochemical studies as detailed in Materials & Methods. 127 III. Observations: A. Gross Histology: The H. fssilis is a bottom living omnivorous fish. Based on food and feeding habits, the overall appearance of the GIT is that of a convoluted tubular structure which makes it much longer compared to that of a carnivorous fish. The GIT in this catfish, like other typical teleost, is divided into short oesophagus which is attached to J-shaped stomach and followed by intestine (Pig-4.1 and 4.2). The stomach consists of three regions, anterior (cardiac), mid (fundic) and posterior (pyloric) (Fig -0.3). The pyloric division is demarcated by pyloric constriction called pyloric sphincter (Fig-42) which is highly muscular organ with narrow lumen connected to the intestine. The cluster of finger-like diverticula, the pyloric caeca attached with the anterior intestine as observed in other teleosts are not present in this catfish. The stomach is followed by tubular intestine which is divided into anterior (duodenum), mid region (mid-intestine) and posterior (rectum). The anterior portion of intestine is wider compared to mid and posterior rogions (Fig-4.2). Like other vertebrates, the gut wall of catfish is divided in four layers i.e. tunica serosa (adventitia), tur ica muscularis, tunica submucosa and tunica mucosa. Oesophagus: In longitudinal and cross sections of catfish oesophagus, four layers are observed when stained with hematoxylin & eosin (HIE) and Massous Trichrome (Fig- 4.4 A, B). The serosa is well developed and tunics. muscularis of eesophagus is composed of two types of smooth muscles i.e. outer circular and inner longitudinal muscles which are arranged in isolated bundles scattered in the submucosa. The mean width of the outer circular muscles is 259.35 µm SD t 64.518 ranging from 167.86-328.78 pm whereas the longitudinal muscle being irregular, its width cannot be measured (Fig-4.4 A, B). The submucosa is thin and composed of loose collagen fibres and large number of granulated cells are intermingled with these fibres. The total width of suhmucosa ranges between 196.75 - 496.17 µm (mean 319.63 pm SD + 112.29). The mucosa shows closely arranged deep longitudinal folds which extend the entire length of oesophagus (Fig-4.4 A). The distinct feature of mucosal epithelium is that it is made up of stratified epithelium consisting of basal cuboidal cells, intermediate columnar mucous cells and superficial layer of flattened cells (Fig-4.4 E and F). The numerous columnar mucous cells which are present in entire mueosul epithelium constitute its major thickness which stain positive with Alcian blue/Periodic acid Schiff (pH 2.5) (Fig-4.4 F). The mucosa epithelium tests on thin but prominent basement membrane which is visible with AB (pH 2.5)/PAS stains (Fig-4.4 C and F). The mean width of mucosa is 253.99 pm SD t 93.44 (range 181.74 — 349.28 µm) whereas the mean width of mucosal epithelium is 78.144 µm SD t 25.863 (range 110.43 - 45.09 µm). The lamina propia is composed of connective tissue containing mostly collagen 5bers, nerve plexes and blood vessels (Fig-4.4 E and F) which are present beneath the mucosal epithelium. No taste buds are seen in the mucosal epithelium in this catfish. The 128 'unction of posterior oesophag s and cardiac stomach is clearly demarcated with AB/PAS (pH 2.5) where the stratified epithelial cells are modified into columnar cells in stomach and gastric glands (serous cardiac glands) appear in cardiac region (Fig-4.4 D). Stomach; As mentioned earlier, the stomach is divisible into three different regions starting From anterior cardiac, middle fundic and posterior pyloric region. Much like oesophagus, all three regions of stomach are made up of fundamentally four layers i.e. outer serosa followed by muscularis, submucosa and rnucosa. However, there are distinct differences in the histological appearance in the different layers of each segment, The interesting new features which make their appearance in stomach are the reverse position of tunica muscularis where the longitudinal muscles, unlike oesophagus, are located towards the external side immediately after serosa and the circular muscles are located inward closely followed by longitudinal muscles. Further, the compound tubular gastric glands appear for the first time in cardiac stomach which show greater abundance in fundic region and disappear in pyloric region. Yet another feature is the first time appearance of the muscularis mucosa beneath the gastric glands of mucosa which is not present in oesophagus and connects the submucosa to rnucosa (Fig-4.5 D). Also, there is an overall reduction in the width of submucosa in all regions of stomach as compared to that of oesophagus. The cross section of cardiac stomach (Fig-4.5 A and B) reveals a well developed serosa which is made up of continuous layer of endothelial cells. The width of tunica muscularis ranges between 269.26 - 325.17 pm (mean 30029 µm SD f 25.58) with a less thicker but prominent longitudinal muscles in the range of 108.23— IO.6S µm (mean 125.352 µm SD t 1S.41) and comparatively thicker inner circular muscles with the mean width of 170.39 pm SD f 19.40 (range 164.2 - 179.45pm) and the mean width of cardiac submucosa is 100.56 um SD f 27.34. The mucosa of cardiac stomach r is unquestionably different from oesophagus mucosa and is lined with a columnar epithelium with interspersed gastric pits which lead into the compound tubular gastric glands (Fig-4.5 A, B, C and D). Goblet shaped mucous cells are not observed in the mucosal epithelium and apical cytoplasm of the epithelial cells show the presence of mucosubsiance (Pig-4.5 C).The gastric glands which do not stain with PAS are attached with basal part of mucosal epithelium and are without mucosubstance (Fig-4S C and D). The lamina propia located between the compound tubular gastric glands provides support to connective and muscular tissue and also separates the gastric gland and mucosal epithelium (Fig-4.5 E and F). The total width of the mucosa layer is more (range 127.76 — 229.12um, mean 156.04 in SD ± 41.74) compared to that of oesophagus and the mucosal epithelium consists of single layer of columnar cells with basally located oval nuclei. The mean width of mucosal epithelium is 30.98 µm SD s 6.99 (range 21.85-41.24 pm) which is significantly thin compared to the oesophagus mucosa epithelium (Fig- 4.5 F and (3). The stomach lumen has well developed 7-8 epithelial folds which give rise to lateral shorter invaginations (Fig- 4.5 A). The gastric glands exude their secretions through the neck cells which open in the gastric pits and finally into the lumen (Fig-4.5 E). 129 The fundic region of the stomach is almost same as cardiac region except that the muscularis is very much thin (mean 61.51 µm SD t 10.84) compared to the cardiac muscularis (mean 300.29 µm SD ± 25.58) (Fig-4.6 A and B). The mucosal fold are more extensively developed and the gastric glands are plenty in number compared to cardiac stomach (Fig-t1.6 A). Additionally, the numerous lymphocytes are observed in lamina propia just beneath the mucosa layer (Fig-4.6 C). The pyloric region of the stomach differs from the cardiac and fundic regions in as much as that it is made up of three layers i.e. inner oblique muscles followed by circular and longitudinal muscles (Fig-4.7 A). The total mean width of muscularis layer is 578.51 pm SD t 22.17 (range 556.86 - 608.53 µm) where the circular muscles are more developed (mean 477.16 µm SD f 25.88) compared to longitudinal smooth muscles (mean 122.94 pm SD f 12.09) and oblique muscles (mean 73.96 SD t 13.46) (Fig-4.7 A and B). The pyloric mucosa of the catfish is characterized by the absence of gastric glands which are present in cardiac and fundic regions with many deep regular folds of mncesa epithelium (Fig-4.7 C and D)- Intestine: Like other regions of GIT, the intestine (duodenum, mid-intestine & rectum) of catfish consists of four layers i.e. serosa, muscularis, submucosa and mucosa. A close perusal of Figs-4.8, 4.9 and 4.10 reveals that there are no significant differences in the histology ofserosa, muscularis and submucosa in all the three regions of intestine. Also, these structures do not show any different histological features when compared with different regions of stomach except that the width of these regions is understandably much less compared to stomach regions. Further, the lamina propia, unlike in stomach regions, is not well demarcated from the submucosa region and muscularis mucosa is altogether absent. Mucosa consists of two types of cells, simple columnar absorptive cells (enterocytes) characterized by brush border and oval nucleus and scattered mucus secreting goblet cells. Interestingly, there is a tendency of progressive shortening of the intestinal folds from anterior to posterior part of the intestine which is also followed by the reduction in the number of absorptive cells and simultaneous increase in mucous cells (Fig-4.8 and 4.10). Hence, the last part of the intestine i.e. rectum is characterized by highest number of mucous cells, comparatively less number of absorptive cells and considerably reduced mucosal folds (Fig-4.l0 C and F). An interesting feature is visible in Fig-4.10 E which shows the junction of stomach and duodenum where intensely stained PAS positive cells line the lumen which is physically strengthened on either side by the thick musculature of sphincter wall. B. Glycoconjugates: One of the distinguishing features of the distribution or glycoconjugates in the mucous cells (MCs) of G11 of IL fossilis is the conspicuous absence of sulphated moiety in the acidic glycoconjugates from its entire regions. The other characteristic features of glycoconjugates in the MCs in different segment of GIT of H fossilis are as follows. 130 Oesophagus: The mucous cells in oesophagus region stain positive both with PAS and AB (pH 2.5) indicating the presence of both neutral as well as acidic glycoproteins. However, the content and distribution of neutral glycoprotein seems to be much mare predominant as compared to acidic glycoproteins (c.f. Fig-4.1l A and B). Further, while the mucous cells with neutral glycoproteins are distributed all over the mucosal lining of oesophagus, the acidic mucous seems to be confined only in the restricted region of oesophageal mucosa, even though the other regions may have small traces of the acidic moiety (Fig-4.] 1 A and B). Sections of oesophagus stained with the combination of PAS-AB (pII 2.5) show the purplish appc&ancc with a greater tinge towards the magenta colour showing larger proportion of neutral glycoprotein (Fig-4.I I C, E and F). As one proceeds from oesophagus to stomach region, there seems to be a shift from a higher proportion of the neutral glycoconjugates to somewhat uniform ratio of both neutral as well as acidic glycoproteins. This fact is clearly visible in Fig. 4.11 D and E which shows the junction of oesophagus and stomach where oesophagus part is predominantly neutral rich and stomach part shows an almost equal mix of both neutral as well as acidic moieties (see also Fig-4.12). Stomach: The regions of stomach i.e. the anterior cardiac and the middle fundic and also a fairly big part of pylofic stomach resemble the oesophagus inasmuch as it has the equal proportion of neutral and acidic glycoproteins (see Fig-4. 12 A-F). However, at the terminal end of the pyloric stomach, there is a gradual increase in the relative proportion of neutral glycomoiety which becomes more pronounced and evident at the junction, the terminal part of the stomach which connects with the duodenum (Fig-4.13 A, B and C). The perusal of Fig-4.13 G shows the location of pyloric sphincter valve, the purplish colour shows much greater tinge of magenta colour, indicating thereby the greater proportion of neutral g[ycoprotein. Intestine: The intestine which is divisible into three parts i.e. anterior duodenum, mid and the terminal intestine which may also be referred to as rectum has the features resembling with those of oesophagus, stomach and intestine excepting the terminal pyloric end. These regions of the GIT show almost equal proportion of neutral and acidic glycoproteins (Fig- 4.13 D, E and F, Fig-.14 A-F). However, the interesting feature is seen in the region immediately after sphincter wall into the anterior most portion of duodenum where there is a great abundance of acidic glycoproteins (see Fig-4.13 0 ). to the rectal part of the intestine, while the contents of acidic and neutral moiety seem almost equal, the number of MCs are greatly increased (Fig-4.14 D, E and F). C. Salinity Induced Changes: When the catfish H fn.esilis were gradually transferred from TW to 25%, SO% and 35e/ SW for 7 days, no significant change in the glycoprotein moiety was observed in any segment of gastrointestinal tract i.e. oesophagus, stomach (cardiac-pyloric), duodenum, mid intestine 131 and rectum (Fig-4.15, 4.16 and 4.17 A, B, C, D, E and F). Interestingly, some initial, yet significant, necrotic changes in the submucosa and lamina propria of mucosa and the hypertrophy in the absorptive cells of post gastric region and large amount of mucus secretion in all segments of GIT were observed in 25% SW (c.f. Fig-4.15 A to F vs Fig. 4.14). In 30% SW, extensively damaged stratified epithelial cells and profound vacuolation in submucosa of oesophagus, (Fig-4.16 A), elongation in gastric glands, hypertrophy in mueosa layer and iumcn periphery lined with mucus were clearly visible (Fig-4.16 B and C). the haemorrhage of mucosa layer in duodenum, disruption and vacuolation of lamina propria and submucosa of intestine, disintegration of columaar epithelium, accelerated mucus production in rectum were also observed (Fig-4.16 D - F). However, in still higher salinity i.e. 35 % SW, severe degenerative changes in all segments of GIT were evident (Fig-4.17 A-I). There were intermittent erosion of umcosa epithelial cells exhibiting detached mucosa from submucosa, atrophied gastric glands and degenerated muscles, resulting in the accumulation of ceh debris in stomach lumen (Fig-4.17 B and C). In post gastric region, there was a pronounced edema in the submucesa and lamina propria, fused and degenerated villi. erosion of top mucosa layer resulting in the rupture of mucosal layer (Fig-4.17 E, G and I). IV. Discussion: A. Gross Histology: The gross morphological structure of digestive tract of H. fossilis essentially shows such structural features which are largely similar to those found in other species such as Chanos chanos, Ictalurus punclatus, Oncorhynchus mykiss, Leporimus friderici, L. laeniofastialus, Clarias batrachus and Serrasalmus naltereri (Chandy & George 1959, Sis et al 1979, Yasutake and Wales 1983, Albrecht et al 2001 and Baji and Noorouzi 2010). Oesophagus, [hc first segment of the Off is a short passage anteriorly attached with mouth and posteriorly with stomach. Most of the workers have divided the stomach into either two or three distinct regions. On the studies dealing with Clarias lazera, Serbia dumerili, Oncorhynchus myklss, O.kisutch. Wallago ottu, Tilapia .spilvrus, Dentex dentex, ithamdia quelena nd Schilbe mystus, the stomach is divided into two parts i.e. cardiac and pyloric (Al-Hussain and Kholy 1953, Grau or al 1992, llelmick et al 1995, Al-Abdulhadi 2005, Carrason et al 2006, Yadav 2006, Hernandez et al 2009 and Naguib et al 2011).However, in Oreochromis niloticus (Caceci et al 1997), Ontorhyndroe ,,'kiss (Yasutake and Wales 1983), Leporinns friderici, L. laeniofosciatus (Albrecht eta] 2001) and Clarias batrachus (Raji and Nourozi 2010), the stomach, like in the present study, has been divided into three parts i.e. cardiac, fvndic and pyloric even though Dattamunshi and Choudhary (1996) have demarcated this into only two regions in this catfish. In carnivore and omnivore fishes, the stomach is mainly Y & C shaped because it is used as a reservoir and can be easily differentiated in three parts when it 132 est' w _ _ ney ' o enum -r y oric stomach ~a~h • 1N Fig- 4.1 In situ Gastrointestinal tract (GIT) of H. fossilis Cardiac stomach Fundic stomach Rectum Pyloric stomach Oesophagus Fig- 4.2 Dissected out GIT of H. fossilis from oesophagus to rectum Fundic stomach H Cardiac stor Duodenum Fig- 4.3 Magnified view of anterior GIT. Boxes indicate various stomach regions employed for sampling of tissue 132 (i) p 0 culads Serosa ~~ • -~ Serosa A1 B Stratified epithelium, S ach V Mucus M Sa layer (, la~er Gast. rk cells F'' 1\i ~; w • s~ ► ' ~ lgn~lE~din~ muscles t die Lamina propia ~ ~ l`)esophaKus $U - ''. longitudinal ' • - Muscle~bu dle Submtl~a • X50 - = .r=, C .x50 ous calls , Superficial Superfic ~•, epithelial cells Stratified epit ial ce~fs Mucqus cells epithelium - , E• - _ ~ - .~' 'f Basal ep~~~~~a ~y t cells ' . A, ' . + Basement membrane ' f• - R Nerve Oxus ~ ~. , • 7 a' r BV Lamina propia F x4 x400 - Fig- 4.4 (A) Longitudinal and (B) cross section of oesophagus of H. fossilis showing four layers i.e. serosa, muscularis, submucc and mucosa, H&E and Massons Trichrome, x50. (C) cross section of oesophagus; mucosa layer stained positive with PAS, x50. cross section of the junction of oesophagus and stomach, PAS-AB (pH 2.5), x50. Note differential structure of epithelial mucosa lal of oesophagus and stomach. (E) & (F) are the magnified view of mucosa epithelial layer showing mucous cells, superficial and ba epithelial cells and basement membrane. H&E, PAS-AB (pH 23), x400. (b, circular smooth muscles; BV, blood vessels). 132 (ii) C • ` Submucosa 7R ~` Serosa .& x100 d e i ui Fig- 4.5 (runs section of cardiac stori dcri. (A) & (B) show four clearly visible layers i.e. seru~a, rouseulans, subrr ucosa and mucosa, H&E & Massons Trichrome, x50, x100. (C) mucosa layer stained with PAS but gastric cells are PAS negative, x100. (D) magnified view of mucosa and submucosa, Massons Trichrome, x200. (E) magnified view of enclosed box in Fig. D showing neck region, Massons Trichrome, x400. (F) magnified view of mucosa layer made up of columnar cells, PAS, x400. (G) magnified view of mucosa layer made up of the columnar cells, H&E, x400. (a, longitudinal smooth muscles; b, circular smooth musdes; c, subrnucosa; d, gastric gland; e, lamina propia; f, mucosal columnar epithelium; g, neck cells; h, mucous layer). 132 (iii) Lumen Muscular mucosa F X50 x 50 b a B ric pit :. fl*# z- ' •ii OW •4k.k e • Ir r 7# IMP ~ - g ( I) Fig-4.6 Cross section of fundiac stomach. (A) & (3) show four clearly visible layers i.e. serosa, muscularis, submucosa and mucosa, 14&E & PAS, xSO. Note comparatively thin mucularis (C) magnified view of mucosa layer with gastric cells, the nucleus of columnar cells, leucocytes in lamina propria and gastric pit, H&E, x400. (D) magnified view of mucosa with lamina propria, PAS, x400. (a, longitudinal smooth muscles; b, circular smooth muscles; c, submucosa; d, gastric gland; e, lamina propia; f, mucosal columnar epithelium; h, mucous layer; i, leucocytes). 132 (iv) ~1 'k: •.; . cosa . b Muscularis x50 J a B Seros,i ~~ • Mucosal epithelium C I mnar cells Muscularis mucosa \ s_ '-T'A' _. Submucosa ~y z Px400 - D Fig-4.7 Cross section of pyloric stomach. (A) & (B) show four clearly visible layers i.e. serosa, muscularis, submucosa and mucosa. Oblique smooth muscles as part of muscularis are evident, H&E & PAS, xSO. (C) magnified view of mucosa layer with lamina propria and the nucleus of columnar cells, H&E, x400. (D) magnified view of mucosa with lamina propria. Note the total absence of gastric glands. PAS, x400. (a, longitudinal smooth muscles; b, circular smooth muscles; j, oblique smooth muscles). 132 (v) \iT • x50 Lumen -' I IR~fllil t u Its `~ hinct(K' Mb l " f rte} - mucous f~ JyJ•{~~• //~'` y.~ //~~ ~ / :Mucous ceQs - I - -. Laminate-:~ Propia - Fig-4.8 (A) & (C) the cross and (B) & (D) longitudinal sections showing the duodenum with four visible layers i.e. serosa, muscuiaris (Longitudinal and circular), submucosa and mucosa, H&E & Massons Trichrome, x50, x100. (E) the longitudinal section showing the junction between pyloric stomach and anterior intestine, PAS-AB (pH 2.5), x50. Note intensely stain mocosa layer of pylorus (F) cross section of duodenum showing abundance of mucous cells, PAS-AB (pH 2.S), KSO. (G) longitudinal section of duodenum showing the abundance of mucous cells on the mucosa, PAS-AS (pH 2.5), x50. (H) & (I) magnified view of mucosa showing absorptive cells with brash border and mucous cells, Massons Trichrome and PAS-AS (pH 2.5), x400 132 (vi) r? `. ' • ~~~~ I Muços ce1W' r •~-~ ~ ., .~ ~, lam,\ .•. ~. i x100 Ser Sa "` Pn- - -.bVsh border 4 Absor five ~s d►~Colo at cel ~.t r lynina Propia . .. : •. • o. a' : i w ~ Fig-4.9 (A) & (C) the cross and (B) & (D) longitudinal sections of mid intestine showing four visible layers i.e. seros. muscuiaris (Longitudinal and circular), submucosa and mucosa, H&E & PAS, x50, x100. (E) & (F) the magnified view i mucosa showing absorptive and mucous cells, H&E and PAS, x400. 132 (vii) r r. x50 Serosa_. ucous cT f c Submucosa um " `4 ~fn4 pr: PLI Lamina propria ?~ T x400, F Fig-4.10 (A), (C) & (D) the cross and (B) longitudinal sections of rectum with layers i.e. serosa, muscularis (Longitudin; and circular), submucosa and mucosa, H&E, Massons Trichrome & PAS, xSO, x100. (E) & (F) the magnified view of mucos showing absorptive and mucous cells stained with H&E and PAS-AB(2.5), x4OO. Note predominance of mucous cells an decrease in number of absorptive cells. (a, longitudinal smooth muscles; b, circular smooth muscles). 132 (viii) (LI r x • , 'T~ _ Neu. Glv. Mix. Gly. Wr Lonk;icuclina1 muscles bundle ilI W. Gly. ,. ,. .; Iy{ } V. Stomach . Oesophagus .h' P x400 G • x1B0 F x400 Fig-4.11 Cross sections of oesophagus of catfish H. fossifis maintained in TW (A) stained with PAS showing a great predominance of neutral glycoprotein, x50. (B) AB (pH 2.5) showing acidic glycoprotein only in some restricted region, x50. (C) combination of PAS - AB (pH 2.5) showing abundance of neutral and limited number 01 darkly stained cells of acidic glycoprotein, x50. (D) the junction of oesophagus and stomach stained with combination of PAS - AB (pH 2.5). Note the localization of mixture of acidic and neutral glycoprotein on stomacl and predominantly neutral glycoprotein on oesophagus, xSO. (E) magnified view of mucosa layer of oesophagus and stomach of red box of Fig. D. Note a well-demarcated neutral glycoprotein rich oesophagus and mixed moiety containing stomach x400. (F) magnified view of mucosa layer enclosed in green box of Fig. D. Note predominant neutral moiety, x100. (G) magnified view of mucosa layer enclosed in red box of Fig. F, showing mostly neutral anc few acid glycoprotein, x400. (Neu. Gly. , Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Mix. Gly. , Mixed glycoprotein). 132 (ix) Cardiac stomach Fundic stomach ' g ç Of ' Neu. Gly. a 1, ' ._ .;~,~s►~.T ~ : ~ , ~ i tix. Gly. dk Mix. Gly. f ' t ~ " .t A Fig-4.12 The cross sections of cardiac stomach of catfish H. fossilis maintained in TW (A), (B) & (C) stained with PAS, AB (pH2.5), combination of PAS-AB (pH 2.5) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. The cross section of fundic stomach (D), (E) & (F) stained with PAS, AB (pH2.5), combination of PAS-AB (pH 2.5) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. (Neu. Gly., Neutral glycoprotein; Aci. Gly., Acidic glycoprotein; Mix. Gly. , Mixed glycoprotein). 132 (x) Pyloric stomach Duodenum . f - i { c Neu. !lye. r •; t~~, . '.,.,fir' _ ~ • ! h'iN. S.. L • : Aci.GIy. ' R! ~; .+ ~. •• ,( • r ~ CSC.- • ~ ; • • .~•. 'r✓ ,Mix. faly. , \~ \c:L:. ." C X ~• '' • N& Gay Ad. GIy xS0 ..- Fig-4.13 The cross section of Pyloric stomach of catfish H. fossilis maintained in TW. (A), (B) & (C) stained with PAS, AB (pH2.5), combination of PAS-AB (pH 2.S) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. The cross section duodenum of catfish H. fossils maintained in TW. (D), (E) & (F) stained with PAS, AB (pH2.5), combination of PAS-AB (pH 2.5) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. (G) the longitudinal section of the junction of pyloric sphincter and duodenum stained with the combination of PAS - AB (pH 2.5) showing neutral and acidic glycoprotein, x~0•~ Pay rl sphincter shows neutral rich glycoprotein moiety. (Neu. Gly. Neutral glycoprotein; Aci. Gly. , Acidic glycoproteih 1Ni>~. , Mixed glycoprotein). Mid intestine Rectum •Neu. GIy. Neu. Gly. ii ~ c A; J. ' Aci. Gly. ci. GIy. t':~rj', !et x5 B x50 h-? - ~ ~;' E Mix. Gly. r ♦ - i k` V. V . x50, Fig- 4.14 The cross section of mid intestine of catfish H. fossilis maintained in TW (D), (E) & (F) stained with PAS, AB (pH2.5) and combination of PAS-AB (pH 2.5) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. The cross section of Rectum of catfish H. fossilis maintained in TW. (0), (E) & (F) stained with PAS, AB (pH2.5) and combination of PAS-AB (pH 2.5) showing neutral, acidic and mixture of neutral and acidic glycoprotein respectively, x50. (Neu. Gly. , Neutral glycoprotein; Aci. Gly. , Acidic glycoprotein; Mix. Gly., Mixed glycoprotein). 132 (xii) x400 .,. i l - 4• 4 . if I. 4 : •. ~ 111... A rI a~ 4 ,~ c54 X100 :` D, x100 F Fig-4.15 The gastrointestinal tract of catfish H. fossilis maintained in 25% SW for 7 days. (A) oesophagus, x100. (B) cardiac stomach, x400. (C) pyloric stomach, x100. (D) duodenum, x100. (E) mid intestine, x100. (F) rectum, x100. Note initial, yet significant structural changes like hypertrophy in submucosa and mucosa (indicated with arrows). ► l _f t _. : is a A x400 B .. C p. E •- I J ;t' I¼.1 ~ 1 e 5 ~.;.~. Fig-4.16 The gastrointestinal tract of catfish H. fossilis maintained in 30% SW. (A) oesophagus, x100. (B) cardiac stomach, x400. (C) pyloric stomach, x100. (D) duodenum, x100. (E) mid intestine, x100. (F) rectum, x100. Note the hypertrophy of mucosal layer in all regions of GIT (shown by arrows). 132 (xiii) ,4' 1& .•I 10 - A x100 ... ' B' x100 C Ii•'\ S •d' # .4 j;(/ : x400 / ' x100__ ~. •R1 aulJ ~ ` ~ ~~~•y, J l x400 + . 400 :j:r:/'~ ic1Q0, H Fig-4.17 The gastrointestinal tract of catfish H. fossilis maintained in 35% SW. (A) oesophagus mucosa layer showing significant atrophy, x100. (B) cardiac stomach mucosa and submucosa showing necrotic changes and severe degeneration in the gastric glands and lamina propia is clearly seen, x100. (C) pyloric stomach mucosa and submucosa showing necrotic changes x100. (D) duodenum mucosa necrosis and extensive hypertrophy of absorptive cells, x100. (E) magnified view of the mucosa layer of duodenum, x400. (F) mid intestine mucosa and submucosa showing hypertrophy and haemorrhage, x100. (G) magnified view of mid intestine showing edema in submucosa and mucosa, x400. (H) Rectum showing edema in the submucosa and lamina propria, x100. (1) magnified view of rectum mucosa layer showing edema in lamina propria, 400X. Changes are indicated by arrows 132 (xiv) is filled with food (Wassersug and Johnson 1976 and Clarke & Whitcomb 1980). In some herbivorous fishes, due to the absence of stomach, oesophagus is directly connected with intestine and its anterior portion which becomes bulky or swelled is called the intestinal bulb which is presumably used as a storage organ of the ingested food. This has been seen in some fishes such as Lepiducephalichthys gunlea (Meitra et al 1989), Cirrihinus mrigala, Laheo rohita (Yadav 2006), Labeo nilolicus (Naguib et al 2011). In H. jbssilis, there is aprominent constriction, the pylorus sphincter, between the pyloric portion of stomach and the intestine which may serve to control the passage its food into duodenum which is also evident in Salnro gairdneri irideus, Ambassis productus, A. natalensis, A. grnnocephalus, Oncorhynchus my/n'sr, Leporinus f iderici, L. taeniofasciatus, Pseudosciaena crocea and Schilhe mystus (Weinreb and Bilstad 1955, Martin and Blaber 1984, Ostos-Garrido et all 1993, Albrecht et al 2001, Mai et all 2005, Fawzy et al 2008) The absence of stomach in these fishes indicates that acid hydrolysis does not have a role in the breakdown of the plant cell walls and, therefore, the fish rely on the action of its pharyngeal mill to break the plant cell walls. In our study on catfish which is omnivorous, the pyloric cecae are absent which is unlike two other omnivorous fishes i.c. Leporinusjriderici and L. taeniofasciarus where they am reportedly present (Albrecht et at 2001). The fact that pyloric cecae have basically the same histological structure as the intestine suggests that one of their roles is to increase the absorption zone (Houssain and Dutta 1998). Generally, the relative ratio between the intestine and body length is 0.5— 0.4 in carnivorous, 1.3— 4.2 in omnivores and 3.7— 4.2 in herbivores which depends upon species, its feeding habits, age and sex (Kaponr et al 1975). The survey of the literature reveals that there is considerable variation in the nomenclature to demarcate different regions of intestine. Some workers have divided intestine into two portions i.e. small and large or anterior and posterior without any specific criterion. This is exemplified in Lepidocephalichthys guntea, Boteophihalmus pectinirosiris, Cirrihinus mrigala, Clarias batrachus, Serrosalmus nattereri and Synodus variegatus (Mishra et a] 1991, Zhu and Zhang, 1993, Yadav 2006, Raji and Norouzi 2010 and Fishelson et al 2012). Albrecht et al (2Q01), however, divided the intestine in Leporinus fridenici and L. eaemiofaschus into cranial, caudal and intermediate part. In the present study on the cafish H fossils, intestine has been divided into broad duodenum, mid intestine and rectum which again varies from Dattamurshi and Choudhary (1996) classification of catfish intestine into wider duodenum, thinner and tubular intestine and wider rectum, a feature of the rectum which has not been observed in the present study. The internal histology of gastrointestinal tract of different fish species, like other vertebrates, show the similar arrangement of the gut wall layers as seen in catfish H. fossils where it is composed of four layers i.e, mucosa, submucosa, museularis and semsa. This fact has been quite evident even in the fishes with different feeding habits such as in carnivores Barb us stigma (Kapoor 1957), in omnivores Leporinus friderici (Albrecht et al 2001), and in herbivores Neogobius gymnotraczelus (laroszewska - et at 2008). In H. fossilis the oesophagus mucosal layer shows deep longitudinally arranged folds which have also been reported in the fishes inhabiting divergent environment such as in marine teleost, Eel Anguilla anguilla and freshwater species, Silverside Odontesthes bonoriensis (Abaurrea- Equisoain & Ostos-Garrido 1996, Diaz et al 2006). Petrinec et at (2005) and Kozaric et at (2007) described that in Northern pike (Esox Lucius), European catfish (Silures glans) and .Atlantic bluefin tuna (Thunnus llrynnus) the longitudinal folds are differentiated into major and minor folds. Mishra et at (1999) observed in two Indian freshwater teleost fishes, Punt/us sophore and Ompok bimacvdatus. that the longitudinal folds, unlike other fishes, are wide blunt and sharp. The anterior most covering i.e. the mucosal epithelium in H. foist/is is structurally uniform, stratified and made up of basal cuboidal cells, intermediate columnar mucous cells and superficial layer of flattened cells. Such a feature has also been observed in other Freshwater fish species, viz. Anguilla japonica, A. unguilfa, Oreochromis (niIoticusx mossamb ices x zilli), Solea senegalensis and Odontesthes bonariensis (Yamamoto and Hirano 1978, Abaurrea- Equisoain and Ostos-garrido 1996, Scarce 1998, Arellano et al 2001, Diaz et at 2006). In another omnivorous fishes, Leporinus friderici and L. taeniofasciatus, the stratified and pseudo-stratified epithelium in oesophagus may protect this organ against abrasion and to enable rough items to pass along the tract more easily (Albrecht et al 2001). Girgis (1952) described different adaptations in the oesophagus epithelium of Lab cc' horie (Cuvier), herbivore bottom feeding cyprinoid where the epithelium contains goblet cells at the side and base, and stratified epithelial cells at the top. Expectedly, great number of mucous cells are found in the epithelium of dillcrent teteost species (Reifel and Travill 1977) which secrete mucus sheet over the epithelial surface. The continuous sheet connected with stratified epithelium generates lubrication for food particles during swallowing and protects the epithelial surface against mechanical damage and bacterial invasion and is also related to ionic absorption (Gmu eta! 1992 and Albrecht et al 2001) suggesting role in osmoregutation. (Humbert et al 1984, Shepherd 1994 and Aburrca- Esquision and Ostos-Garrido 1996). It has been observed in the present study that the catfish mucosal epithelium also contains large number of columnar mucous cells which secrete abundant quantity of mucus which is spread over epithelium. Based on the studies quoted above, it is logical to presume that the mucus layer over this region of GIT is performing the function of protection and helping in the process of swallowing offend during ingestion (see Fig.2 C & E). Murray et at (1994a) suggest that mucin in the posterior oesophagus may play a role in pregastric digestion. In the catfish, the mucosal epithelium rests on the basement membrane which has also been observed in many other species such as Thanaus thynnus, Wallago attu and Gambusia afnis (Kozaric et al 2007, Yadav 2006 and ivlishm et al 1999). The muscularis mucosa consisting of stratified muscular fibers and usually helping in the movement of folds has been found absent in the catfish H. fossills and other teleosts species (Gargiulo et at 1996, .Albrecht et al 2001, Weisel 2005 and Raji and Norouzi 2(10). Hence, the functional role of muscularis mucosa should be performed by some other alternative segment which is difficult to speculate at this moment. Similarly, the taste buds are also found to be absent in catfish 11 foss/Ifs like in many other species such as Odontesthes 134 bonariensis (Diaz el at 2006), Icralurus punctatus (Sis et al 1979), Engraalfs anchoita (Diaz et aI 2003), Clarias batrachus and Serrasalmus natteresi (Raji and Norouzi 2010). However, in many other species such as Barbus stigma, Gadus morhua, Pontius sophore, Ompok bimaculatus, Lepidocephalichthys guntea, Neogobius gymnotrachelus and Rhamdia quelen the taste buds are reportedly present (Kapoor 1957, Morrison 1987 Mishra, 1991, 1999, Jaroszewska et at 2008 and Hernandez et al 2009). The reported presence of taste buds in some teleosts and its absence in others may have something to do with its gustatory abilities which will obviously be dependent on the feeding habit as well as the ecological niches of the different teleosts. The muscularis, typically, consists of inner longitudinal and outer circular muscles and both are smooth muscles and a similar arrangement is observed also in the catfish oesophagus where the longitudinal muscles are arranged in isolated bundles extending into the submucosa. Such an arrangement has been found in almost all the teleosts quoted above including winter flounder Pseudpleuronectes americanus and yellowtail flounder Limandn ferruginea (Jaroszewska et at 2008). In these species, the assumed function of longitudinal muscles in oesophagus is to provide the ability to reject any unpalatable material (Murray et al 1994b). [ii the catfish, the serosa which is continuously present externally and is made up of endothelial cells is known to reduce the friction and to protect the surrounded organs, as has also been observed in Pintius sophore (Mishra ci at 1999). However, in other species like Wallago attu, Gamburia affrnis and Mystus aor it is very poorly developed (Alim et al 19)5. Mishra et at 1999, Yadav 2006). in an exhaustive review on the morphological diversity of the gastrointestinal tract in fishes, Wilson and Castro (2011) have described that the mucosal epithelium in teleost is stratified which may become more columnar towards the posterior region (cardiac stomach) depending on species. In the present study on catfish, we have also observed the typical stratified epithelial layer towards the terminal portion of oesophagus which gets noticeably modified into columnar cells in the cardiac region of the stomach. This fact is clearly visible on the perusal of Fig, 2 D which shows the junction of posterior oesophagus with cardiac stomach and a sudden change over of stratified epithelial cells into columnar cells in stomach. This is in complete agreement with Wilson and Castro (2011) observations. Like oesophagus, the structure of the walls of all regions of stomach of H fossilis resembles with those of other fishes where it is composed of bi-layered muscularis, the connective tissue of submucosa. the muscularis mucosa, Lamina propria of connective fibers with gastric glands and inner epithelial layer of mucosa (Albrecht et al 2001, Park and Kim 2001, Diaz et at 2003, Carrasson et al 2006, Naguib or at 2011 and Fiskielson et at 2012). Externally, the stomach of catfish H fossilis is divided into three regions i.e. cardiac, fundic and pyloric where the gastric glands are present in the cardiac stomach which increase in fundic region but are completely absent in the pyloric region. Cataldi et at (1988) described the similar pattern of varying abundance of gastric glands in different regions of Oreochromis 135 niloticus. In some fishes where the stomach is divided in two regions i.e. the anterior cardiac and posterior pyloric, the gastric glands are present in the cardiac portion but are totally absent in pyloric region (AI-Hussaini and Kholy 1953, Grau et al 1992, Albrecht 2001, Al-Abdulhadi 2005, Carrason et it 2006, Naguib at al 2011). On the contrary, Arellano et al (2001) have reported in Solea senegalensis that the gastric glands of stomach are numerous in the pyloric and fundic region but absent in the cardiac region. Based on the survey of literature, it is revealed that in some teleosts i.e. Haloban-achus didactylus, Rhamdia quelen, C/arias balrachus and Serrasalmus natteresi, the gastric glands are few but arc not totally absent in the pyloric region (Desantis et al 2009, Hernandez et al 2009 and Raji and Norouzi 2010). The cardiac and fundic regions of the stomach of catfish H. fossilis are characterized by the presence of compound tubular type gastric glands but these glands are totally absent in the pyloric region. This observation on catfish is in agreement with the similar findings of Al-Hussaini and Kholy (1953), Cataldi eta] (1987), Grant et at (1992), Ostos Garrido et at (1993), Satora (1998), Albrecht et al (2001), Mal et al (2005), Raji and Norouzi (2010) and Naguib et al (2011). However, Osman and Cacei (1991), Morrison and Wright (1999), Diaz (2003), Petrince et al (2005), AI-Abdulhadi (2005), Carrasson at al (2006). Kozaric et al (2007) and Pishelson et al (2012) have reported the presence of simple tubular gastric glands in the fishes studied by them unlike compound tubular found in catfish. Generally, the gastric glands secrete both enzyme and acid through one type of secretory cells which open in the gastric pits and finally into the lumen (Western and Jennings 1970, Verma and Tyagi 1974 and Ray and Moitra 1982). However, in mammals each of these secretions is produced by two different cells such as chief and parietal eels (Smith 1989). In the catfish, the lamina propia of the cardiac and lundic portion of the stomach consists of loose connective tissue that holds the gastric glands together and also it penetrates in the mucosal folds of all regions whereas submucosa is made of vascularised connect ve tissue that lies above the lamina prupria and its gastric glands, a feature which is a common characteristics of most teleosts. The muscularis mucosa is well developed in the pyloric stomach of H fossllis whereas it is poorly developed in cardiac and fundic regions. This may possibly indicate that the functional significance of combination of well developed musculature and the absence of gastric glands in pyloric region is to mix and push the food distally which also has been observed by Reifel and Travill (1978) in the stomach of eight species of teleosts. The mucosal epithelial cells of H. Jbssilis stomach which lines the lumen and pits, arc tall columnar cells with basally placed oval nucleus and numerous apically concentrated granules. These secretory granules secrete mucus which stains positive with PAS/ AD(pH 2.5) and may serve to protect the stomach epithelium from the auto digestion caused by enzyme and acid. These findings have also been recorded in many other teleosts species such as Senora dumerili, Oreochromis niloticus, E,rox lucius, Silurus glans, Dentex denlex. Thunnus thynnus, Halobairachus didaclylus, Clarias batrachus, Serrasalmus natterei and Schilbe mystus (Gram at al 1992, Morrison and Wright 1999, Petrinco et at 2005, Carrusson et at 2006, Kozaric et at 2007, Desantis et at 2009, Raji 136 and Norouzi 2010 and Naguib et al 2011). Interestingly, in contrast to oesophagus, the arrangement of museularis of catfish in stomach is reversed with the existence of thin outer longitudinal and slightly thick inner circular smooth muscles towards the external serosa which has also been observed by Reifel and Travill (1978) in eight species of toleosts. One of the unique features observed in the fundic region of stomach is a highly significant reduction in the thickness of muscularis layer, perhaps not reported in any other telcost which is concomitant with highly developed gastric glands occupying most of the thickness of GIT in this region. Such a histological feature seems to have functional significance since the major load of digestion is taking place in this region for which extensively developed gastric glands are extremely important. Simultaneous reduction of the muscularis is possibly because of the lack of any significant function as well as to keep the thickness of entire Cff in a reasonable limit. One is also tempted to suggest the respiratory role due to sudden increase in the vascularisation of this region of GIT. I-Iowever, concrete evidence needs to be adduced to confirm this assumption. The circular muscles in the pyloric region of catfish tend to be more developed than in cardiac region, assuming the appearance of globular or spindle shape gizzard found in birds. The function of the gizzard in fishes is similar to that found in birds i.e. to grind or triturate food which is facilitated in fishes by the mucous membrane 1med with a thick layer of mucopolysaccharide rather than keratin found in birds (Wilson and Castro 2011). Interestingly, in pyloric region of catfish FL fosrilis, the muscularis layer is arranged into inner oblique followed by circular and longitudinal muscles whereas I-Iemandez of all (2009), in American catfish Rhmndia quelen. observed a reverse arrangement of the pyloric region where three types of smooth muscles are arranged into inner longitudinal, oblique middle and outer circular. In the intestine of the catfish, a well developed serosa similar to that found in the stomach region, is present and is made up of loose connective tissue with blood cells which also conforms with this observation of Genten et at (2009). The fact that intestinal muscularis of catfish is differentiated into two layers of smooth muscles fibers i.e. an inner circular and an outer longitudinal has also been reported by several workers such as Martin and I3laber (1934) in Arnbassis spp., Gargiulo of al (1996) in Tilapia spp., Albrecht et al (2001) in Leporinus friderici, L. raeniofascia(as, Suicmea et at (2005) in Orthrias angorae, Hernandez et al (2009) in Rhamdia quelen and El- Bakary and El-Gamma! (2010) in Spares aumta. However, in ,Llvgil cephalus, the position of smooth muscles is opposite where longitudinal muscles are inner and thick circular muscles are outer (El- Bakary and El- Gammal 2010). It has been generally suggested that the thickness of muscularis in the intestine may be correlated with the temporary storage of food and expulsion of faecal material from the region. However, the above generalization contradicts with the observation of Gnu et al (1992) who described the existence of three layers of muscle fibers throughout the whole intestinal length of Serbia dumeriti. The absence of muscularis mucosa underneath the lamina propria in the catfish has also been documented in some other lelcosts such as Oncorhynchus rnykiss and Clarias batrachus (flan an Khojasteh et al 200% 137 and Raji and Norouzi 2010). However, some other studies have described the presence of muscularis mucosa in the teleusts such as cornmun carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichlhys nobilis) (Bevan Khojasteh et al.2009b, Delashoub el al.2010 and Banan Khojasteh et al.2011). Further, an overwhelming number of studies have reported that intestinal mucosal folds which consist of two types of cells i.e. simple columnar absorptive and mucus secreting goblet cells have a tendency of shortening from anterior to posierinr part of the intestine as thserved in Sponsc aurata, Tilapia spilurus, My/in cuvieri, Menopterus albus, Rhamdia quelen, Oncorhynchus nykiss and Ifypophthalmic/rkys nobilis (Cataldi et al 1987, Al-Abdulhadi 2005, Dai et al 2007, Hernandez et al 2009, Baran Khojasteh et a] 2009a and Delashoub et a] 2010). Similar observation has also been made in the present study on H. fossilis where the simple columnar absorptive cells (enterocytes) characterized by brush border and oval nucleus are arranged all along and lined by a single layer of mucosa epithelium with goblet cells scattered in between them. It is widely accepted that the absorptive cells play the role in assimilation of ions and fluid and mucous cells lubricate the passage of undigested food material towards the posterior segment of intestine in many fishes such as Tilapia mossumbiea, Ictalurus pvnetatus, Serbia dumerili, Pleumnectes ferrugineus, Pseudopleuronecies antericanus, So/ea so lea, Leporinus friderici, L. taeneofasciatus, Umbrina cirmsa, Anguilla anguilia and Rhamdia quelen (Narasimham and Parvatheswarao 1974, Ss et al 1979, Crau et al. 1992, Murray et al.1996, Veggetti et al. 1999, Albrecht et al 2001, Pedini et al. 2001,Domeneghini et al 2005 and Hernandez et al 2009). In the catfish rectum, the decrease in absorptive cells and simultaneous increase in number of mucous cells may be related to the increased protection of mvcosal epithelium and lubrication for faecal expulsion as has also been observed by Khanna and Mehrotra (1971), Murray et al (1994b), Petrinec et al (2005), Cinar and Senol (2006) and Raji and Norouzi (2010). B. Glycoprotems (GPs) in GPI': Studies on the function and identification of GPs in the fish are very limited. However, the fact remains that GPs plays an important role in the gastrointestinal wall viz, lubrication, protection of mucosa layer against chemicals, hypertonic media and acidity in the stomach, defense against parasites, and formation of a diffusion barrier for various ions and possibly role in transepithelial ion transport in intestine (Smith 1976, Allen 1981, Pabst 1987, Gupta 1989, Lcretz 1995 and Veggetti et al 1999). The catfish H fossrlis is omnivorous and the GPs which are present in the whole gastrointestinal tract seen to be both neutral and acidic types whereas in oesophagus and terminal part of stomach these are predominantly neutral. However, in carnivorous northen pike Esox lacius, the gut mucous substances in different regions of CIT are uniformly neutral and acid glycocenjugates with no relative predominance of either of them (Petrinec et al 2005). Interestingly, the sulphated glycoconjugates have not been observed in the gut mucosal lining of the catfish H. fossilis 138 which partly tallies with the observation of Scocco et of (1998) in oesophagus of the Tilapia spp. However, mast of the studies have shown the presence of sulphated glycoconjugates in oesophagus and intestine such as in Sparus aurata, Urnhrina cinesa (Fry). Anguilla anguilla, Pseudophoxinus anta!ycw and Qdontesthcs bonariensis (Domeneghini et al 1998, Parillo et al 2004, Domeneghini et al 2005, Cinar and Senol 2006 and Diaz et al 200 r~. It has been shown that the sulphomucin present in the mucosa layer would confer high viscosity to mucus which aids in trapping small particles (Tibbetts 1997). The absence of sulphomucin in the Gil of catfish suggests that such a functional necessity in catfish may either be diminished or not required at all. In the present study on catfish If. fossils, the oesophagus mucosa lining shows the predominant presence of neutral GP even though acid GPs is also present in small measures. Similar qualitative distribution of GPs pattern in oesophagus has also been reported in ether teteosts such as Northen pike Esox lucius, European catfish Silures glanis, Atlantic bluefm tuna Thunnus ihynnus (Petrinec et al 2005 and Kozaric or al 2007). In addition, there are number of other studies where the presence of neutral and acidic GP in the oesophagus have been reported. However, the predominant status of either of the two GPs in such studies have not been commented upon. Such species include Loach (Lepidocephalichthys guntea) (Moitra et al 1989). Tilapia spp Hybrid (niloticusx mossambicusx zil i) (Scocco at al 1998), Nile Tilapia (Oreochromis niloricus) (Morrison and Wright 1999), Eel (Anguilla anguilla) (Domeneghini et al, 2005), Silverside (Odontesdrcs bonarrensis) (Diaz et a[ 2006), Walking catfish (Clarias hatrachus) and Piranha (Serrasalmus nalterer( (Raji and Norouzi 2010). In herbivore teleosts, acidic glycoconjugates predominate over the neutral glvcoproteins in oesophagus as observed in Arrhamphua scleralepis, Tilapia spirurus and Odonlesihes bonariensis (Tibbetts 1997, AI-Abdulhadi 2005 and Diaz et at 2006) but there seems to be somewhat inconsistent pattern in carnivore fishes. A group of carnivore fishes represented by Sea bream (My Go cuvieri), Amberjack (Seriota dumerili), Shi drum (Uorbrina cirrasa) and Rainbow trout (oneorhyncfies myldss), the quantity of acidic moiety predominates over the neutral (Grau et al 1992, Parillo et al 2004, Al-Abdulhadi 2005and Marchetti or al 2006). On the contrary in Esox Lucius, Silurus glanis and Thunnus thynnua, neutral GP is predominant (Petrinec et al 2005 and Kozaric et al 2007), while in Spares aurora, Anguilla angufla, and Clarias batruchus both these glycoconjugates appear in almost equal proportion of neutral and acidic (Domeneghini et al 1998, 2005, EI-Bakary and El-Gametal 2010 and Raji and Norouzi 2010). Another curious fact is that most of the above fishes are ouryhaline and endemic to sea water even though any habitat related functional significance is not evident presently. The qualitative differences in the glycoconjugate moiety present in the mucus of oesophagus may be associated with the different functions. The absence of salivary glands in fishes may be duly compensated by the lubrication function provided by the mucus secreted by the oesophagus mucous cells. However, the predominance of neutral GP as observed in catfish 139 may further aid in lubrication function whereas the acidic glycoprotein may protect the mucosal lining from the invasion of various pathogens which may enter with the swallowed food. Significant variations in glycoconjugate composition in stomach have been described in many teleost species (Kapoor et at 1975 and Reifel and Trevil 1978). In the present study, the occurrence of GPs on the surface epithelium of stomach which stains positive with PAS and AB (pH 2.5) indicate the mixture of neutral and acidic GP. Similar high quantities of both neutral and acidic glycoconjugates in the stomach mucosa have been reported in the several teleost species such as Sparus aurata, Oreochromis niloticus, Anguilla anguilla, Esox Lucius and Thunnus thymus (Domeneghini or at 1998, Morrison and Wright 1999, Domeneghini et at 2005, Petrinec at at 2005 and Kozaric et at 2007). However, in certain teleost species, the neutral GP predominates over the acidic moiety as exemplified in, Seriola dumerili, Silurus aurata and Halobatrachus didactylus ((ran et at 1992, Sarasquete et al 2001, Petrinec et at 2005 and Pesantis et at 2009) which, interestingly, share the some feeding habits, behavior and biology. It is interesting to note that in some species like Tilapia spirurus, Mylio cuvieri, Umbrina cirrosa, Rhamdia quelen, Clarias b❑trachus and Serrasalmus nattereri totally lack acidic moiety in their stomach lining even thought he neutral glycoconjugates may be predominantly present ( Parillo et al 2004, Al- Abdulhadi 2005, Hernandez et at 2009 and Raji and Norouzi 2010). In contrast, in the fish species like Seabream Spares aurora (Arellano 1995), Solea Solea solea (Veggetti et al 1999), and Senegal sole Solea senegatensis (Arellano et at 2001), the surface gastric epithelium was histochemically unreactive, whereas the adherent mucus on gastric mucosa was weakly AB- positive. In the present study, the sulphated GP has been found to be absent in the gastric mucosa of H fossilis which is similar to that has been reported in S. senegalensis juveniles (Vieira 2000) and Solea senegalensis (Arellano et at 2001) even though it has been shown to be present in many other different fishes. (Reifel and Travill 1978 and Grau et al 1992) which may probably be related to diet. Spicer and Schulte (1992) speculated that sulphomucins may be able to form a complex with pepsin, stabilizing or buffering the enzyme. The neutral and acidic GP produced by gastric epithelial cells may serve to form a visco-elastic gel acting as a physical barrier to protect the underlying mucosa against HCI and the injury caused by enzyme produced in gastric glands (Ferraris et at 1987 and Smith 1989). Neutral mucins on the surface of gastric epithelial cells have been related to the absorption of easily digestible substance such as disaccharides and short fatty acid (Gram et at 1992). In addition, the presence of neutral glycoconjugates may serve to protect the underlying layers from chemical and physical damage during trituration processes in order to compensate for the lack of keratinized lining or another analogous structures (Gisbert or at 1999). An interesting observation of the present study relates to a sudden transition of neutral GP present in the sphincter region of the stomach leading to the duodenum into predominantly acidic moiety. Although no evidence is adduced but it is likely that this 140 transition may be useful to kill any possible pathogen occurring in the ingested diet of the catfish. In the intestinal mucosa, presence of mucous-producing goblet cells has been reported Jr many teleost fishes (Narasimham and Parvatheswarao 1974, Ray and Moitra 1982, Domeneghini et at 1998, Nachi et al 1998, Domeneghini at at 1999 and Morrison and Wright 1999). The intestine of H. fossilis shows the uniform distribution of GPs in all portions Le. duodenum, mid intestine and terminal part (rectum) which are made up of neutral and acidic GPs. Similar observations have been reported in the intestine of many other teleosts such as Lepidocephalichthys gun/ea, Arrhamphus sclerolepis, Sparus aurata, Oreochromis nilolicus, Chondrosloma non's, Clenopharyrgodon idellu, Cyprinus carpio, Leucisus cephalus, Esox lucius, Si/urus glanis, Anguilla anguilla , 7hunrrus thynnus, Clarias barrachus and Serrasalmus nauereri, (Moitra et at 1989, Tibbetts 1997, Domeneghini et at 1998, Morrison and Wright 1999, Fiertak and Kilarski 2002, Petrinec et at 2005, Domeneghini eta! 2005, Kozaric et at 2007, , El-Bakary and El-Ga nmal 2010 and Raji and Norouzi 2010). However in some fishes the acidic GPs predominates over the neutral moeity as observed in Amberjack Serbia dumerif, Shi drum Umbrina cirrosa (Fry), Flowerfish Psevdophoxinus antalyae andAmerican catfish Rhamdia quelen, (Gnu at at 1992, Parillo et at 2004, Cinar and Senol 2006, Hernandez et at 2009). AI-Abdulhadi (2005) described the presence of two different GPs moieties in intestine of two tlshes of different feeding habits. However, in an earlier study on four fishes of different feeding habits, the glycoconjugate moeities in the intestine were the same (Fiertak and Kilarski 2002) suggesting thereby that the GPs moiety is not dependent on the feeding habits of the teleost fishes. In H. fossilis, the number of mucous cells increase towards the distal part (i.e. rectum) which may be related to an enhanced need for lubrication for the smooth ejection of faeces as reported in other ]ish species (Grau et a] 1992, Murray or at 1996, Domeneghini et at 1998, Veggetti et a1 1999 and Pedini et at 2001). The sulphated glycoconjugate is not detected in the intestine of H. fossilis but Rcifct and Travill (1979) and Pedini et at (2001) observed a predominance of carboxylated glycoconjugate which also contains sulphated moiety along the entire intestine of different teleost species. On the contrary, in other teleosts the sulphated glycoconjpgates are abundant in rectum (Sengeret al 1994, Murray et al 1996, Domeneghini at al 1998, Veggetti eta] 1999, Domeneghini et at 2005). Parillo eta! (2004) hypothesized that the sulphated GP may be involved in the process of morphofunctional differentiation of the alimentary tract of Umbrian cirro izfry. Among the functional multiplicity of mucus, acid GPs have been implicated in the increased viscosity of mucus in the fish alimentary tract (Tibbetts, 1997) which lubricates the undigested materials for onward progression into the rectum. Neutral mucosubstance are is said to be involved in the enzymatic digestion of food transforming it into chime and also facilitating absorptive function (Clarke and Witcemb 1980, Anderson 1986 and Grau et a] 1992). Narasimham and Parvatheswarao (1974) suggested a possible role of intestinal mucins in osmoregulation. The studies of Ribelles et al (1995) have shown that the quality of gut mucosubstanees is directly 141 related to environmental conditions, mucosubstances, especially sulphated in the intestine, possibly regulates the transfer of proteins or a fragment of them, as well as of ions and fluids (Sire and Vernier 1992, Senger of all994 and Domeneghini at of 1998). C. Salinity Induced Changes: In a classical osmoregulatory pattern, gastrointestinal tract ((liIT) of a freshwater teleost, unlike its marine counterpart, plays a minimal role. Such a role is largely confined to intestinal uptake of Na` and Cl- from dietary source to compensate diffusional loss of these ions across the gill to an extremely hyposaline external medium. Marine teleosls, on the contrary, facing an acute osmotic water loss to highly concentrated ambient environment resort to drinking of seawater to offset the water loss. Therefore, GIT in marine teleosts play a role of desalination of ingested seawater by absorption of seawater right from the oesophagus up to the intestine. Hence, one would expect significant consequential histological alterations in the structural features of various segments of GIT as part of osweregulatory adaptation (Grosell 2011). On the contrary, in a freshwater stenohaline fish such as catfish X fossilis which survives only up to limited salinity range through the process of passive tissue tolerance (Goswami et al 1983), there is no evidence of any enhanced drinking of ambient saline medium and clearly no instance of active osmoregulation. Any substantial role of GIT in higher salinities up to 35% SW as an osntoreguatory organ is clearly not expected The observations of the changes in GIT internal histology corroborate this assumption. However, there is an overwhelming evidence to show that while there is no significant qualitative and quantitative change on the GPs moiety of the GIT mucosal lining of various segments, there are highly noticeable disruptive changes in the internal mucosal lining and even on the underlying layers of various segments. These changes which are in the form of extensively damaged stratified epithelium, necrosis in submucosa and lamina proptia, profound vacuolation of submucosa of oesophagus, elongation of gamic glands, hemorrhagic changes and enhanced mucus production. These changes become more pronounced as the salinity increases from 25% to 35% SW. The survey of literature reveals that there is extremely limited data on the C IT role in osmoregulation particularly in sienohaline freshwater fishes which have only limited salinity tolerance range. However, in euryhaline eel, some workers have studied the structural changes following osmoregulatory function of oesophagus and reported that stratified epithelium rich in mucous cells is replaced by simple columnar epithelium covered with highly vascularized microvilli (Laurent & Kirsch 1975, Yamamoto & Hirano 1978 and Meister ct al 1983). Even with the limited number of studios, some interesting generalizations can be made. The changes observed in eel oesophagus during SW-adaptation were also documented in sea bream Saris aurata, Tilapia Oreochromis macsambicus and in its hybrids which are all euryhaline species (Cataldi et al 1987) and also in Oreockromis ni/oticus when exposed to 20% SW onwards (Cataldi et al 1988). These observations add some validity to the suggestion of Lotan (1960) that mechanisms that balance the osmotic 142 pressure begins to act when external salinity goes over 70% SW. In this present study on catfish H fossilis, which has barely 35% SW tolerance limit, none of the above changes were observed giving credence to our assumption that Off is not playing any active osmoregulatory role in catfish in higher salinities. However, the fact of the matter is that there are highly significant degenerative changes in catfish GIT in higher salinities which may have been caused by the process other than osmoregulation. Literature abounds with studies on histopathological changes in several vital organs including GIT following exposure to wide variety of toxicants such as heavy metals, pesticides and organic load on aquatic fauna particularly fish. Such a load of pollutants, to varying extent, is stressful and seriously hampers the normal metabolic functioning of these aquatic species. Such changes in CIT are manifested in the form of epithelial degeneration of mucosa and atrophy and degeneration of submucosa, focal necrosis, infiltration of lymphocytes into the larninal propria and overall desquamation of mucosal epithelium (Sastry and Gupta 1979, Ghosh and Chakrabarti 1992, Banerjee and Bhattacharya 1995, Kleinow el al 1998, Braunbeck and Appelbaum 1999, Cengiz eta! 2001, Hann et al 2005, Cengiz and Unlu 2006, Ghosh et al 2006, Soufy 2007, Velmumgan et all 2007, Mohamed 2009 and Sarker and Ghosh 2010). In the present study, a careful analysis reveals that the changes observed in the catfish GIT in higher salinities quite closely resemble with. the changes observed in those fish species which have been exposed to various pollutional load. This leads to most likely conclusion that the histopathological changes seen in catfish GIT following transfer to higher salinities are more of a manifestation of salinity induced stress response rather than an osmoregulatory adaptation as observed in euryhaline teleost transferred to marine environment. MN References References A I. Abate F., Germane G. P., De Carlos F., Montalbano G., Laura R., Levanti M. B. and Germane A. (2006). The oral cavity of the adult zebrafish (Danio rerioj. Anat. Histol. Embryol, 35:299-304. 2. Ahanrrea-Esquisuai n M. A. and OstusGarrido M. V. (1996). Cell types in the esophageal epithelium of Anguilla mtguilla (Pisces, Teleostei). Cytochemical and ultrastructural characteristics. Micron. 27: 419-429. 3. Abraham M., Igor V. and Zhang L. (2001). Fine structure of the skin cells of a stenohaline freshwater fish Cyprinus carpro exposed to diluted seanater. Tissue and Cells, 33: 46-54. 4. Agarwal S. K., Banerjee T. K. and Mitial A. K. (1979a). Physiological adaptation in relation to hyperosmotic stress in the epidermis of a fresh-water teleost Barbus sophor (Cypriniftermes, Cyprinidae) A histochemical study. Z. mikrosk. Anat. Forsch., 93: 51-64. 5. Agarwal S. K, Rai A. K., Bauerjee T. K. and Mifal A. K. (1979a). Histophysiology of the epidermal mucous cells in relation to salinity in a frees water telcost, 7leteropnensles fossilis (Bloch) (19eteropneustidae, Pisces). Zool. Beitr., 25:403-410. 6. Agrawal N. and Mittal A. K. (1992 a). Structural modifications and histochemistry of the epithelia of lips and associated structures of a carp -Labeo to/ii/a. Em. Arch. Biel., 103: 169-180. 7. Agrawal N. and Mittel A. K. (1992 b). Structural organisation and histcchemistry of the epithelia of lips and associated structures of a carp- Cirrhina nviga(n. Can. J. Zool., 70:71-75. 8. Agrawal N. and Millar A.K. (1994). Structural modifications in the epithelia of lips and associated stmchval ofa Mmrel, Channa straila having predatory feeding habit. 3. Appl. rchthyol., 10:114-122. 9. Ahmed A. E., Mohamed K., Ahmed S.A. and Missend F. (2008).Anatomical, Light and Scanning Electron Microscopic Studies on the Air Breath'mg Dendretic Organ of the Sharp Tooth Catfish (ClariargoriepinncJ_ I. vet anal, 1:29 - 37 10. Ahuja S.K. (1970). Chloride cell and mucous cell response to chloride and sulphat enriched media in the Gills of Gambusiu afnisafnis ( Bard and Girard) and Calla tetra (Hamilton). J. Esp. Zool., 173:231-250. 11. Al-Abdulhaai H.A. (2005). Some comparative histological studies on alimentary tract of tilapla fish (Tilapiu spitnrus) and sea bream (Mylio cvvierril. Egyp. J. Aquatic. Res, 13: 387-397. 12. Albrecht M. P., FelTeira M. F. N. and Caramaschi E. P. (2001). Anatomical features and histology of the digestive tract of two related nectropical omnivorous fishes (Characiformes; Anostomidae). 3. Fish Biol., 58: 419-430. 13. Al-Hussaini, A. H. (1946). The anatomy and histology of the alimentary tract of the bottomfeeder .Mulloides aurnamma (Forsk.). J. Morph. 78: 121-153. 14. Al-Hussaini A. H. and Kholy A. A. (1953). On the functional morphology of the alimentary tract of some omnivorous teleost fish. Proc. Egypt. Acad. Sci., 9: 17-39. 15. Ali M. S. and Pearson J. P. (2007). Upper airway humid gene expression: A review. Laryngoscope, 117: 922-938. 16. Alim A., Abmul F., Sen N-S. and Mishra K. P. (1995). Occureuce of dub cells in the esophagus of a carnivorous silurid Mwrus uor (Hann.). Sci. & CulL 61: 193-194. 17. Allen A. (1981). Structure and function of gastrointestinal mucus In: Physiology of the Gastrointestinal Tract, Jolnson L. R. (ed.), Raven, New York, pp. 617-639. 18. Allen A. and Flenrstro m G. (2005). Gastroduodenal mucus bicarbonate barrier protection against acid and pepsin. Am. J. Physiol. Cell Physiol., 288: 1-19. 19. Allen P. J., Cech, Jr J. J., and Kuntz D. (2009).Mechanisms of seawater acclimation in a primitive, anadromous fish, the green sturgeon. J. Comp. Physiol. B, 179: 903-920. 144 20. Anderson, T. A. (1986). Histological and cytological study of gastro-intestina: tract of Luderick Cite/la tricuspidata (Pisces, Kyphosidae), inrela@on to diet. J. Morph., 48:187-206. 21. Andew W. (1959). Textbook of Comparative Histology. Oxford University Press, New York. 22. Arellapo J. M. (1995). ContribuciOn a to hisloIogia, hisloqu:niic2 e histopatelofiia de la domlo, Sparus aurata, L. Tests de Licenciatura. em Biologia Animal,Depattamento de Biologia Animas, Vegeml y Ecologia. FacWtad de Ciencias del Mar, Universidad de Cadiz, Cadiz 23. Arellanu J. M., Starch V. and Sarasquete C. (2001). Histological and hislocRemical observations in the stomach of the Senegal sole, Solea senegolensfs. Histol. Histopathol., 16:511-521. 24. Arellnno J.M., Storch V. and Saraquete C. (1999). Histological changes copper accumulation in liver and gills of the Senegal sole, So? en senegalensls. Foutux Enivrou SaC, 44:62-92. 25. Arellano J.M., Scorch V. and Saraquete C. (2004). Ultrashvcntre and histochemical study on gills and skin of the Senegal sole, Soleu senegalenrix. J. Appl. Ichthyol., 20:452-460. 26. Armbruster J.W. (1998). Modifications of the digestive tract for holding air in loricariid and sooloplacid catfishes. Cooeia, 3:663-e75. 27. Asakawa M. (1970). Histochemical studies of the mucous on the epidermis of ml, Anguilla japonica. Bull. Sap. Soc. Scient. Fish, 36: 83-40. 28. Audet C. and Wood C. M. (1993).Branchial morphological and endocrine responses of rainbow trout (Oncorhynchm npk1ss) to a long-term sublethal acid exposure in which acclimation did not occur. Can. J. Fish. Aquat. Sol, 50: 198.209, 29. Avella M. and Bornanein M. (1990). Ion t1'xes ht the gill of freshwater and seawater salmonid fish. Anim. Nutr, Trump. Process, 26: 1-3. 3D- Avella M., Berhaut .I. and Bornancin M. (1992). Salinity tolerance of two tropical fishes, Oreochromis aurens and O. ni(oiicua I. Biochemical and morphological changes in the gill epithclium.J. Fish Riot., 42:243 -254. 31. Avella M., Masoni A., Bornancin M. and Mayer-Goslan M. (1987). Gill morphology and sodium influx in the rainbow trout (Salmo gairdneri) acclimated to artificial freshwater environments. J. Exp. Zcol., 241: 159- 169. 32.Azi i S., Koehaniat P., Peyghan B., Khansari A. and Bastami K. D.(2010). Chloride cell morphometrics of common carp Cyprinus carpio, in response in different salinities Comp. Cliin. Pathol., DOI: 10.I0D7/s0058U-0lD-10(13-8. B 33. Baker J. R (1946). The hinWChcmicnl recognition orlipinc. Quart. Jour. Microsc. Sc'., 87: 441- 470. 34. Baldwin E. (1948). An Introduction to Comparative Biochemistry. Cambridge: Cambridge Univ. Press. P. 126. 35. Hanson Khojasteh S. M., Masoumi L. and Ganji Panahi P. (2011). Histornorphology of the alimentary canal in silver carp (Hypophthalmichthyv molitri). J. Vet. Res., 15: 277-282. 36. Bnnan Khojastek S. N., Sheilmzadea F., Mohammadnejad D. and Azami A. (2009a). Histological, histochemical and ultrastructural study oS the intestine of rainbow trout (Oncorhynchus myh'ss). World. Appl. Sci. J., 6: 1525-1531. 37. Banan Khojasteh S.M., Ebrahimi S., Ramezani M. and Ilaghnia H. (20096). The histological and histochemical study on the esophagus and intestine in common carp fish (C+prima carplo). 1. Anim. Biol.. 1:18-26. 38. Banerjee S. and Bhattacharya S. (1995). Hismpathological changes induced by chronic non lethal levels of elsan, mercury and ammonia in the small intestine of Channa pmclanes (Bloch). Ecotuxicol. Environ. Sal., 31: 62-68. 39. Banerjee T. and Mittat A. K. (1975).A cytochemical study of the "chloride cells" in the skin of a fresh-wat.r taeosL (Chmnm.ctriata (RI) Channidae, Pisces). Acta Histechem„ 53:126-35. 40. Banerjee T. and Mittel A. IC (1976). Histochemistry and the functional organisation of a `Live- fish' Clarias batrachur ILinn.) (Claridge, Pisces). Mikroskopes, 31: 333-349. 145 41. Bansil Rand Turner B. S. (2006). Mucin structure, aggregation, physiological functions and biomedical applications. Curr. Opin. Collcid Interface Sci., lit 164-170. 42. Bareellos L. J., Vulpatu G. L., Barreto R. E., Coldehella [. and Ferreira B. (2011). Chemical communication of handling stress in fish. Physiol. Behay., ID3:372-375. 43. Barrington E. J. W. (1942). Gastric digestion in the lower vertebrates. Biol. Rev., 17:1-27. 44. Barrington E. J. W. (1951). The alimentary canal and digestion. In: The Physiology of Fishes (M. E. Brown, ed.), Academic Press, New York, p. 109-161. 45. Bartels H. (1998). The gills of hagfishes. In: The Biology of Hagfishes, (Eds.) Jorgensen J.M., Lomholt I.R. Weber R.E, and Matte H, London: Chapman and Hall, p. 205-222. 46. Barton B.A. (2002). Stress in fishes: A diversity of responses with particular reference to changes in circulating corlicosteroids. lnteg. Camp. Biol., 42:517-325. 47. Barton B. A., Bollig H., Hauskins B. L and Jansen C. IL (2000). Juvenile pallid (Scaphirhynches albus) and hybrid pa1Iid*$rovebose (S. alhusxploloryHehus) sturgeons exhibit low physiological responses to acute handling and serve confinement. Comp. Diochem, Physio1., 126: 125-134. 48. Barton B. A., Schreck C. B. and Barton L. A (1987). Elects of chronic cortisol administration and daily acute stress on growth, physiological conditions, and stress responses in juvenile rainbow trout. Dis..Aquat Org., 2: 173-185. 49. Belanger L. F. and Hartnett A. (1960). Persistent toluidine blue metachrornssia. J. Histochem. Cytochem. Jan, 8:75-75. 50. Benfcy T. J. And Bison 3f. (2000). Acute stress response in triploid rahtbow trout (Oncorhynchus mykiss) treated with a single injection of o, p'-Dichlorodiphenyidichloruethane. Environ. Toxicol. Chemistry, 21: 1753-1756. 51. Bern M. (1993). A selective survey of tie endocrine system of the rainbow trout (Oncorhynchus nyktss) with emphasis on the hormonal regulaliun ofiun balance. Acuaculture, 100:237-262. 52. Reratssenl M. H. G., Krogluud F, Rosscland B. O. and Wendalaar Bonga S.E. (1997). Respmues uCskiu mucous cull to almnin.mr exposure at IOW pH in Atlurtie salmon (Suimo .cuter) smelts. Can J. Fish. Aquas. Sci., 54: IC39-1045. 53. Bevelnnder G. (1935). A comparative study of the bronchial epithelium in fishes, with special reference to ectrarenal excretion. 1. Morphul., 57:335-351. 54. Biswas A. IC, Seoka M., Takii K, Matta M. and Kumai U. (2006). Stress response of red sea bremn Pagrus major to acute handling and chronic photoperiod manipulation. Aquaculture, 252: 566-572. 55. Biaekstoek N. and Pickering A. D. (1980).Acidophilic granular cells in the epidermis of the brown trout. Solmo truaa L. Cell Tiss. R. 210:359-369. 56. BJanc-Livni N. and Abraham M. (1971). The influence of environmental salinity on the prolactin- and gnnadotrepin-secreting regions in the pituitary of Mugil (Teleoslel), Gen. Camp. Endoeimol., 14:184-97. 57. Bumgren P., Einarsson S. and dousson A- C. (1997). Similarities and differences in oxynticopeptic cell ulrrastruchve of one marine teleosl, Canis mop/w, and one freshwater teleost, Oncorhynchus mykiss, during basal and histamine-stimulated phases of acid secretion. Fish Physiol. Biocaem., 18: 285-296. 58. Bordas M.A., Balebona M.C., Chabrillon M., Rodriguez-Marnto J.M. and Morino M.A. (2003). influence of temperature and salinity on the adhesion to mucous surfaces of gilt-head seabream (.Sports nar(ta L.) of pathogenic strains of Vibrio arginolylicns and Listonella angui4annn. B. Eur. Assoc. Fish Pat., 23:273-280. 59. Boric F., Sreboean E. and Kozaric Z. (2001). Starvation induced pathobiology in the gut of carp (Cyprinus carpio L.). Berl. Munch. Tiersnl. Wschr., 114: 134-138. 60- Braunbcck T. and Appelbaum S. (1999). Ulnastructuml alterations in the liver and intestine of carp C}prinas carpio induced orally by ultra-low doses of endosullan. Ois. Aquas. Organ., 36: 183- 200. 145 61. Brown P. (1992). Gill chloride cell surface area is greater in freshwater adapted adult sea trout (Salmo trutta, L.) Than those adapted to sea water. J. Fish Biol., 40: 481-484. 62. Buddington R. K. and Christofferson J. P. (1985). Digestive and feeding characteristics of the chondrosteans. Env. Biol. Fish., 14: 31-41. 63. Buddington R. K. and Diamond J. (1987). Pylotic caeca of fish: a 'new' absorptive organ. An J. Physiol., 252: 65-76. 64. Buddington R.K. and Kuz'rnina V. (2000). Digestive system, In: The Laboratory Fish, Ostrander G.K. (Eds.), Academic press, p.173-384. 65. Snddington R.K., Krogdabl A. and Bakke-McKellep A. M. (1997). The intestines of caidvorons Hsh stnmtum and tunetions and the relations with diet. Acta Physiol. Scand, 161: 67- 80. 66. Bullock A. M. and Roberts R. J. (1974). The dermatology of madne telcost fish. 1. The normal integument. Oceanogr. mar. Biol. A. Rev., 13:383-411. 67. Burden C. E. (1956). The failure of hypophysectomized Funduius hereroclitws to survive in freshwater. Biel. Bull., 110: 8-28. 68. Burton D. ad Fletcher C. L. (1983).Seasonal changes in the epidermis of the winter flounder, Pseudopleuronecres americenus.J. Mar. Biol. Ass. U. K,63: 273-287. 69. Burton D., Burton MP., Truscott B. and Idler 1114. (1985). Epidermal cellular proliferation and differentiation in sexually mature male Salmo.vular with androgen levels depressed by oil. Prot. R. Son l.nnd. Ser B. Hint. Sci-, 225:I71-l2 70. Burton D., Burton M.P., Truscott B. and Idler D,R, (1935). Epidermal cellular proliferation and differentiation in sexually mature male Salmo salar with androgen levels depressed by oil. Proc. R. Soc. Load. Ser B. Biol. Sci, 225:121-128. C 71. Cabemy N. B. and Quinitio G. F. (2000). Changes in Na, K`-ATPase activity and gill chloride cell morphology in the grouper hpinephelns coloedes larvae and juveniles in response to salinity and temperature. Fish Physiol. Biochem., 23: 83-94. 72. Unreel T., El-habbaek H. A., Smith S. A. and Smith B. J. (1997).The stomach of Oreochromis niloticus has three regions. J. Fish Boil., 50: 939-952. 73. Cala P. (1987). Aerial respiration in the catfish, Eremophilus mudsIl (Trichomyctecdae, S 1uriforn es) in the Rio Bogota basin, Colombia. J. Fish Biol., 31:301-303. 74, Calabro C., Albanese M.P., Lauriano E.R., Martella S. and Licata A. (2095). Morphological, his o.herrical and immnoohr5ro-chemical study of the gill epithelium in the abyssal telcost fish Coe1r4ynchus 0aelorbyueAus. Folia Histochem. Cytokiol., 43: 51-56. 75. Cameron ML L. and Steele, J. E. (1959). Simplified Aldehyde-Fuchsin Staining ofNeurosecre- tory Cells, Stain Technologic, 34: 265-266. 76. Cardoso E. L., CEiarini-Garcia H., Ferreira R. M. A. and Loui C. R. (1996). Morphological changes in the gills ofLophiosilurus ulexandri exposed to rm-ionized ammonia. J. Fish Biel, 49: 778-787. 77. Carmana R., Garci A-Csllego M., A. Sanz, Demeaai"N A. and Ostos-Garrido M. V. (2004). Chloride cells mid pavement cells in gill epithelia of Acipenser naccarii'. ultrastmctural modifications in seawater-acclimated specimens. J. Fish Binl., 64: 553-566. 78. Carrasson M., Gnu A., Dnpazo L. it and Crespo S. (2006). A histological histochemiea: and ultrasnetural study of the digestive tract of Dentrx denrex (Pisces. Sparidae). Histol. Ilistopathol., 21(6): 579-593. 79. Carter C.S. and Beadle L.C. (1931). The fauna of the swamps of the Paraguayan Chaco in relation to its environment. 11. Respiratory adaptations in the fishes. J. Linn. Soc. Land., 37:327- 366. 80. Cataldi E., Cataudella S., Monaco G., Rossi A. and Tancinni L. (1987). A study of the histology and morphology ofthe digestive tract of the sea bream. Sparus aurata J. Fish. Biol, 30: 135-145. 147 Si. Catnldi E., Crosetti D., Leoni C. and Cataudella S. (1988). Oesophagus structure duricg adaptation to salinity in Oreochromis niloticus (Perciformes. Pisces) juveniles. Boll. Zool., 55: 59- 61 K. Cengiz E. and Unlu E. (2006). Sublethal effects of commercial deltamethrin on the structure of the gill, liver and got tissues of mosquito fish, Gambusia #nis: A microscopic study. Environ. I oxicol. PharmacoL, 21: 246-253. 83. Cengiz E. L, Unlu, F and Balci K (2001). The histopalhologieal effects of Thiodan on the liver and gut of mosquito fish, Gambusra uznis. J. Euvhon. Sci. Health. B., 36:75-85. 84. Chabrillon M., Bordas M.A., Morhnigo M.A. and Balebona M.C. (2004). Kinetics of adhesion of Lislone](u urigiilarum to the mucus of gill-head sun bmsm and the implication of surface components. Aquae. Res., 35: 403-409. 85. Chalcrabarti F. and Chosh A. R. (1989). Effects of cadmium poisoning on mucopolysaccl>eride and some cellular enzymes of the alimentary canal in Neteropnenatee fossilfs (Bloch): A histochemical study. Proc. Nat. Syn. of Cnvironmental Impacts on Animals and Aq naculture. 86. Chempy C. (1913).Granules tdduisant 1'iodure d'osmium. J. Anat. Physiol. Norm. Pathol., 49323- 343. 87. Chandra S. and Banerjee T.K. (2003). Histopatbological analysis of the respiratory organs of C(arias batrachus (Linnaeus) subjected to the stress of air-exposure. Acta Zool. Taiwan, 14:45-64. 88. Chandra S. and Banerjee T.X. (2004). Histopmholobical analysis of the respiratory organs of Channa striala subjected to air exposure. Vet. Arhiv., 74:37-52. 89. Chandy M. (1956). On the oesophagus of the milk-fish Chonos chunos (Forskal). J. Zeol. Soc. Ind_ 8.79-84. 90. Chandy M. and George M. G. (1959). Further studies on the alimentary tract of the milk-fish Chanar chonas in relation to its fond and feeding habits. J. Zool. Soc., 26: 126-134. 91. Chang C., Lee T., War H. C. and Hwang P. (2002). Effect of environmental Cr levels on C1 uptake and mitochondria-rich cell Morphology in Gills of the stenoholine Goldfish Carassias aurular. Zool. Studies, 4: 236-243. 92. Chang LC., Lee TI!., Yang C.H„ Wei Y.Y., Cliou F.I. and Hwang P.P. (2001). Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments. Physiol. Biocbc,c. Zool., 74:111-119. 93. Chang I.C., Wei Y.Y, Chou F.1. and Hwang P.P. (2003). Stimulation of cruptake and morphological changes in gill mitochondriarich cells in freshwater tilapia (Oreochromis mosvambm,$). Phvsiol. Biochem, Zool., 76:544-552. 94. Charles H.J. and Terry A.U. (1997). Changes in gill morphology of Atlantic salmon (Salmo sa(ar) smolts due to addition of acid and aluminium to stream water. Environ. Pollut., 97: 137-146. 95. Clreloache M., Iger Y. and Abraham M. (1992). Cell activity in the epidermis of fish during osmotic stress. Abstrac- Bulletin, World Fisheries Congress, Athens, Greece. 96. Chen C., Lin L. and Lee T. (2004). lonocyte Distribution in Gills of the Euiyhaline Milkfish, Chanus charms (Forsskal, 1775), Zool. Studies, 43; 772-777. 97. Chen W. J., Miya M., Saitoh K. and Mayden IL L. (2000). Phylogenetic utility of two existing and four novel nuclear gene loci in reconstructing tree of life of ray-finned fishes: the order Cyprinifurmes (Ostariophysi) as a ease study. Gene, 423: 125-134. 98. Chitray B.B. and Saxena U.B. (1942). Functional anatomy of the digestive organs of freshwater fish of India, Heteropneueles fossifis (Bloch) and Clariar batrachas (Linn.). L Morphology. Vest. Cs. Spot. Zoo], 26:148-159. 99. Ciccoti K, Macchi E., Rossi A. and Cautella S. (1993). Glass eel (Anguilla aaguiia) acclimation to freshwater and seawater: morphological changes of the di&estive tract. J. AppL lchthyol., 9: 74-81. 100.Cinar K. and Sennl N. (2006). Histological and histochcmical charecterir EM 101.Cinar K., Senol N. and Ozeul M.R. (2008). Histochemical characterization of glycopruteins in the gills of the carp (Cyprinue carpio). Ankara Univ. Fak. Derg., 55: 61-64. 102.Cioai C., de Merich D„ Cataldi E. and Catandella S. (1991). Fine structure of chloride cells in freshwater- and seawater-adapted Oreochromis niIot)crrs (L-mnaeus) and Oreochronnis macsambicus (Peters). J. Fish Biol., 39: 197-209. 103.Claiborne J.B. (1998). Acid-base regulation. In: 'Lhe Physiology of Fishes (2i' ed.), (Eds). Evans D-H. Boca Baton, F.L.: CRC. p.177-198. 104.Cla rap J. R, Allen A., Gibbon R. A. and Roberts G. P. (1978). Chemical aspects of mucus. Br. Med. Bull, 34:2541. 105.Clark G. A. (2002). Efficacy and side eaCects of freshwater bathing as treatment for amoebic gill disease: implications or water chemistry end bath duration. M. Appl. Sci. Thesis. University of Tasmania., 94- 106.Clarke A. A and Witcomb D. M. (1980). A study of the histology and morphology of the digcslive tract of the Co.. eel (Anguilla anguifla). J. Fish Biol, 16: 159-170. 107.Clements K.D. and Raubenheimer D. (2005). Feeding and Nutrition. In: The Physiology of Fish. (3`s' ed.) Evans D.H. and Claiborne J.B., (6ds) CRC Press, p,47-74. 108.Coleman R., Yaron Z. and Ilan Z. (1977). An ultraetmcmral study of the mitochondria-rich `ch]oride'celle from the gills of freshwater and seawater-adapted Tilapiu aura subjected to a pesticide. J. Fish Biol., 11:589-594. 109-Cook R., McKeown R- A., half D. W., Flagg L N- and Squires T. 3. (1979). Studies on the blood and branchial mucous cells of freshwater and marine alewives (Alosa pseudoharengus). Comp. Bic them. PhysioL, 64:245-249. I10.Copeland D.E. (1948). The cytological basis of chloride transfer in the gill of Fundalus heteroc?itus. J. Morph., 82: 201-228. 11 1.Crawford R II. (1974). Structure of an air-breathing organ and the swim bladder in the Alaska blackfsb Delia pecmrafis Bean. Can. J. Z0ol., 52:1221-1225. I l2.Culling C. F. A., Reid P. E., Clay M. G. and Dunn W. L. (1974). The histochemical demonstration of 0-acylated sialic acid in gastrointeslna. mucins. Their association with the potassium hydroxide periodic acid-schiff effect. J. Histocherr. Cytochem., 22:826.831. 113.Cyrino J. E. P., Bureau D. and Kapoor B.G. (2008). Feeding and digestive function of fishes. Science Publishers, p. 575. D 114.Dagdeviren A. Alp H. and On U (1994). New app1k lions for the zinc iodide-osmium technique. J. Anal., 184:83-91. I15.Dai X., Slim M. and Fang W. (2007). Histological end ultmstmctural study ofthe digestive tractof rice held ccl, Mmwpteros albses. J. Appl. IehthyoL, 23: 177-183. I16.Darias M. J., Murray H. M., Gallant J. W., Douglas S. E., Yufera M. and Martinez- Rndrignez O. (2007). Ontogeay of pepsinogen and gastric proton pump expression in red porgy (Pagnspagrus): Determination of stomaci functionality. Aquaculture, 270:369-373. 117.Das S. and Srivastava G.J. (1918). Response of gill to v rious changes in salinity in freshwater toleost Colia fasciatus (BI. and Seim.). Z. MiKrock Anat For3ch., 92: 770-780. 118.Dattamunshi J. S. and Choudhary S. (1996). Ecology of Heteropnevstes fossilis (Bloch)_ An air- breathing catfish of South East Asia. Narendra Publishing House, pp. 17-25. 119.Davis K B. (2006). Management of physiological stress in finfish aquaceltore. N. Amer. J. Aqua., 68: 116-121. 120.Delashoub M., Pousty F. and Barran Khojastch SM. (2010). Histology of bighead carp (Ifypophthafmirhrhys nobi(L) intestine. Global Vet., 5:302-306. 121.Desantis S., Acone F., Zizza S., Dellorio M., Fernandez J. L. P., SaraSquete C. and De Metrio C. (2069). G1yrnhistnrhemiraI study of the toadtis8 Halobatrachus dfdactylus (Scheider, 1801) stomach. Seientia Marina, 73: 515-525. 149 122.Dezfnli B. S., 3zekely C., Giovinaao G., Hills K. and Clan i L. (2009). Inflammatory response to pa-asitic helminths in the digestive tract of Anguilla anguilda (L.). Aquaculturc, 296: 1-i. 123.Dlaz A. O., Garcia A. M., Blauth De Lima F.. Pinheiro C., Draccinl drM. G, and Galarga Cuimaraes A. C. (2010).Glycoprolems in the branchial mucous cells ottelndachnerina hrevipiaa(characifonnes, Curimatidae): a histachemical study. Revises Real Academia Galega de Ciencias. Vol. XXIX. pp. 35-08. 124.Dkiz A. 0., Garcia A. M., Devincenti C. V. and Goldenberg A. L. (2003). Morphological and histochamical characterization of the mucosa of the digestive tract in Engraalis anchoita (Hubbs and Marini, )935). Anat. l-listol. Embryol, 32: 341-346. 125.Dias A. O, Garcia A. M., Escalaute A. H. and. Goldcmberg A. L (2010).Glycopreteins histochemisiry of the gills of Odomeslnes bonoriearis (Tel eostei, Atherinopsidae). J. Fish Biol., 77: 1665-1671. 12G.Diaz A. 0., Garcia A. M., Figueroa 1). E. and Goldemberg A. L. (2008). The nrucosa of the digestive tract in Micropogouias fazrnicri: a light and electron microscope approach. Anat. LGstol. Embryol., 37: 251-256. 127.Dinee A.O., Escalante A.H., Garcia A.M. and Goldemberg A.L. (2006). Histology and histochemistry of the pharyngeal cavity and oesophagus of the Silverside Odontasthes bonariensis (Cuvier and Valenciennes). Anat. Metal. EmSryoL, 35: 42-46. 128.Diaa A.O, Garcia A.M. and Goldemberg A.L. (2005). Glycoconjugates in the branchial mucous cells of C.):rroscion grmtncupn (Cuvier, 1330) (Pisces: Sciaenidae). Sci. Mar., 69: 545-553. 129.1)iaz A.O., Garcia A.M., Devincenti C.V. and Goldemberg A.L. (20D5), Ultrastructure and hostochemical study of glycnconjugates in the gils and of the white cracker (Micropognni¢r fvrnieri). Anat. Histal. Bmbryol., 34: 117-122- 130.Diaz A.O., Garcia A.M., Deviacenti C.V. and Goldenberg A.L. (2001). Mucous cells in Micropogonias Jurnieri gills: Histochem:stry and ultrasttucmre. Anat. Histol. Emhrvol, 30: 135- [39. 131.Diler D. and rinar K. (2010). Aphrmiss a aloliae .rureyanus (Neu, 1937) (CsleichDryes: Cyprinodontidae) Solungag Glikokonjugatlarmm Histalumyasal 6zellikleri, Makufebec, 1: 1- 8. L32.Diler D. and Cinar K. (2009). A Histochemical Stady of Glycoconjugates in the Gills of the Sea Bass (Dic~ntrarckus labrar L. 1758) Journal of Science, 22: 257-261. 133.Diler D., Cinar K. and Zorin S. (2011), An immenohistochemical study on the endocrine cells in the stomach and intestine regions of the Dicentrarchus labraz, L., 1758. F. U. Sag. Bit. Vet. Deg:, 25: 1-6. 134.Domeacghini C., Arrighi S., Radaelli G., Busi G. and Mascarelo G. S. (1999). Morphologicai and hislochemical peculiarities of the gut in white sturgeon. Acipenser tranrriuiarms. Bump. S. Histechem, 43: 135-145. 135.Dorneneghini C., Arrighi S., Radaelii G., Bosi G. and Veggetti A. (2005). Hstochemical analysis of glycoconjugate secretion in the alimentary canal Anguilla asEudla L Aces Histochemica, 106: 477-487. 136.Domeneghini C., Pannelli Straini R. and Veggetti A. (1998). Gut glycoconjugates in Sparas aurato L. (Pieces, Teleostei). A comparative histochemical study in larval and adult ages. Histol. HistopathoL, 13: 759.372. 137.Dopidn R., Rodriguez C., Gomez T, Acosta N.G. and Diaz M. (2004). Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Spares aurata L). Comp. Biochem. Physiol. A., 139: 21-31. 138.Downing S. W. and Novels R It (1971). The fine structure of lamprey epidermis. D. Club cells. J. Ultrasmmt. Res., 35:295-303. 139.Downing S.W., Spitzer RH., Koch E.A. and Salo W.L. (1984).The hagfish slime gland thread cell. 1. A unique cellular system for the study of intermediate filaments and intcrmediatc 6lzrncnt- microtubole inter'ction.J. Cell Biol. 98:653-669. 150 140.Doyle W. L. (1977). Cytological changes in chloride cells following altered ionic media. J. Exp. Zoo[., 199: 427-433. I41Dunel-Erb E.B., Sebest P., Chevalier C., Simon l3. and Bart H.L. (1996). Morphological changes indicated by acclimation high pressure in the gill epithelium of the freshxnter Yellow Eel. 1. Fish Biol., 48: 1018-t022. 142.Dunel-Erb S. and Laurent P. (1973). U1lmslmcture compare Ia pseudnbranchie chez les Teleosteens marins et dean deuce. Leptthelinm pseudobranchfaf.1. Micros. (Paris), 16: 53-74. 143.Dutar It M., Riehmands C.R. and Zeno T. (1993). Effect of dia4nnn on the gibs of Bluegill Sunfish. Lepomis macrochirus. J. Environ. Pathol. Toxico:. Oncol., 12:219-227. E 144.E1-Bakary N. E. R. and El-Cammal H. L. (2010). Comparative histological, histochcmicai and ultmdruotural studies on the Proximal intestine of Flathead Grey Mullet (Mugil cephalus) and Sea Bream (Spares aurata). Wet. App. Sci. J., 8(4): 477-455. 145.EIhaI M. T- and Agulleiro B. (1986). A hislochemical and intrestroocmel study of the gut of Span+s aurarur (Teleostei), 3. Submicros. Cytol., I&335-347, 146.Elger M. (1987). The branchial circulation and the gill epithelia in the Atlantic hagfish, .Myxine ghvnnosa L. Anal. Embryol., 175:489-504. 147.ERiolt D. G. (2000). Integumentary System, In: The Laboratory Fish, (Eds.) Ostrander G.K., Academic press, p.271-315. l41fErkmen B. andKolankaya0. (2009).The Relationship between Chloride Cells and Salinity Adaptation in the Euryhaline Teleost, Lebistes renculatcs. J. Ani.Vet.A dv., g: 888-892. 149.Erlmea B. and Kolankaya D. (2000). Effect ofwater quality on epithelial morphology in the gill of Copoat, linca living in two tributaries ofKizilirmak River. Turkey Bull. Envirn. Cont. Toxicel., 64: 418-425. 150.Estehane M. A. (2012). An overview of the immunological defenses in fish skin. JSRM Immunology, p.1-29. 151.Evans U.H. (1987). The fish gill: site of action and model for toxic effects of environmental pollutants. Environ. Health Perspect, 71:47-58. I52.Evans D.H., Peirmariri P. and Chile K. P. (2005). The multifunction and Fish Gill: dominant site of gas exchange, osmoregulation. acid-base regulation and excretion of nttrogenous waste. Physiol. Rev., 85: 97-177. 153.Evans D.H., Plermarinl P.M and Putts, W.T.W. (1999). runic tmnspurt in die fish gill epitheltrtn. J. Exp. Zeal., 283: 641-652. 154.Ezeaser D. N. (1981). The fine structure of the gastric epithelium of the rainbow trout, Salmo gairdneri Richardson. J. Fish Bid., 19: 611-627. 155,Ezeasor D. N. and Stokoe W. M. (19S1). Light and electron microscopic studies on the absorptive cells of the intestine, caeca and rectum of the adult rainbow trout, Salvo gairdneri, Rich. J. Fish Biel., 18:527-5i'. 156.Ezeasor D. N., and Smkee W. M. (1980). Scanning electron microscopic study of the gut mucosa of the rainbow trout Salmo gabdneri Richardson. J. Fish RioL, 17: 529-539. F 157.Fange R. and Grove D. (1979). Digestion, In Fish Physiology (Vol. 8), Hoar W.5, Randall D.]- and Brett J.R. (Eds.), Academic Press, New York pp. 16:260. 158.Fast M.D., Ross N.W., Mustafa A., Sims D.E., Johnson S.C., Conboy C.A., Speare D.J., Johnson G. and Burka J.F. (2002a). Susczptibility of rainbow train Oamrhynchuv mykiss, Atlantic salmim Salmo saint and coho salmon Oncorhynchus kisutchto experimental infection with sea lice Lepeophtheimx salmons Dis. Aquat. Org, 52:57-68. 159.Fast M.D., Sims D.E., Burka J.F., Muslafa A. and Roo N.W. (2002b). Skin morphology and Immoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon. Comp. BLochem. Physiot., I32A645- 7. 151 160.Fawxy L.A., Nagvib S. A. A. and El-Ghafar F. A. A. (2008)Comparative study on the intestine ofSchilbe mystas and Labeo nilofcus in correlation with their feeding habits. Egypt. J. Aquat. Bio]. & Fish., 12: 275 -309. 161. Ferguson H,W., Morrison D., Ostland V.E., Lumsden J., Byrnc P. (1992), Responses of mucus-producing cells in gill disease or rainbow trout (Oncorhynchus mykiss). J. Comp. Pathol, 106:255-265. 162.Fernandes M. N. and Perna-Martins S. A. (2001)Epithelial gill cells in the Armored catfish, Hyyvslwaa' CF. pfecusiumus (Luricariidar:i. Braz. J Biol., 61(1): 69-78. 163.Ferraris R. P., Tan J. D. and de la Cruz M. C. (1957), Development of the digestive net of milktsh, C'panos churws (Forskal): histology mid hislochemutry. Aquaculturc, 61:241-257. 164.Fieldcr D. S., Allan C. L., Pepperall D, and Patrice M. P. (2007).The effects of changes in salinity on osmoregulalion and chloride cell morphology of juvenile Australian snapper, Pogrus auratus . Aquaculture, 272; 1-4. 165.Fiermk A. and Kilarski W. M- (2002). Glycoconjugates of the intestinal goblet cells of four cyprinids. Cell. Mol. Life Sci., 59: 1724-1733. 166.Ffshelson L., Colani D., Russell B., Ca01 B. and Goren M. (2012). Compurwtive morphology and cytology of the alimentary tract in lizard fishes (Teleostei, Aulopiformes, Synodoatidae), Acta Zoo. (Stockholm), 93: 308-318. I67.Fleteher T.C. (1978). Defence mechanism in fish, In: Biochemical and biophysical perspective in marine biology. (Vol. IV) (Eds), Matins D.C. and Sargent J.R., Academic Press, London. I68.Fletcher T.C., Jones Rand Reid L. (1976). Identification of glycoproteins in goblet cells of epidermis and gills of plaice (Pleurnneclee pinmssa L.) tlrunder (P!atichihys flesus (L.)) and rainbow trout (Selma gan- neri Richardson). I Iistochem. J., 8:597-608. .69.Fontaine Y. A., Pisam M., LeMoal C. and Rambourg A. (1995). Silvering and gill "mitochondria-rich"cells in the eel, Anguilla anguilla. Cell Tissue Res., 281:465-471. 170.Pasket J. K, Bern H.A., Machen T.G. and Conne, M. (1993). Chloride cells and the hormonal control of teleost fish osmoregulation. J. Exp. Riot., 106: 255-281. 171.Foskett J.K., Logsdon C.D., Turner T., Machen T.E. and Bern H.A. (1981). Differentimion of chloride extrusion mechanism during sea water adaptation of chloride extrusion mecli anism during sea water adaptation of telecast fish, the cicblid Sarotherodon mossamPicus. J. Exp Biol., 93: 209- 224. 172.Frenehin A., Alessandrini F. and Fantin A. M. B. (1994). Gill morphology and .ATPase activity in the goldfish Carossius carussius var. auratns exposed to experimental lead intoxication. Italian J. Zool., 61: 29-37. I73.Franklin G.C. (1990). Surface n1vostroctumh changes in the gills of Sockeye salmon (Teleostei: OwoMrynhus nerka) during seawater transfer comparison of successful seawater adaptation. J. Morphol., 206: 13-23. I74.Frierson E. W. and Foltz J. W. (1992). Comparison and estimation or absorptive intestinal surface areas in two species of cichlid fish. Trans. Am. Fish. Soc., 121:517- 523. 175.Fukuda Y. (2001). Fish disease and pamsiic protozoans. J. Animal Ptotozooses, l5: 8-21. G 176.Garcia-Romeo F. aid Mescal A. (19701. Sur la mise on evidence des cellules a ehlorurc de la branchie des poissons. Arch. Anal Microsc. 59,289-294. 177.Garg T. K., Deeps V. and Mittel A. K. (1995). Surface architecture of the opercalar epidermis and epithelimn lining the inner surface of the operculum of a walking catfish Clarfns bafrachur. Japan. J. Ichthyol, 42: 131-185. 178.Gargiulo A. M., Da1PAglio C., Tsoku Z., Ceccarelli P. and Pedini V. (1996). Morphology and histology of the oesophagus in a warm water tilapiine fish (Telcostci). J. Appl. lehrhvol. 12: 121- 124. 152 179.Gawliclm A., Leggiadro C. T., Gallant J. W. and Douglas S. E. (2001). Cellular expression of the pepsinogen and gastric proton pump genes in the stomach of winter flounder as determined by in situ hybridisation. J. Fish Biel., 58: 529-536. LSO.Gee J. H. (1976). Buoyancy and aerial respiration factors influencing the evolution of reduced swim-hhdder volume of some Central American catfishes Trichomycteridae, Callichthyidae, Loricariidae, Astrob1op!dae). Can. I. Zoo!., 54:1030-1037. 181.Geaten F., Trewinghc F. and banguy A. (2009). Digestive System. In: Atlas of Fish Histology, Science Publishers, 75-91. 182.Ghaffar M. A., AmaH M. and Mansor-Clyde M. (2006). Fine structure of gills and skins of the amphibious mudskipper, Prr uphlhu!nus chrysospilus bleaker, 1852, and n non- amphibious goby, Frrranigobius reichei(Bleeker, 1853). Acta lchthy. Et Piscatoria., 36: 127-133. 183.Ghosh A. R. and Chakrabarti P. (1992). A scanning electron microscopic probe into the cellular injury in the alimentary canal of Numplenss nolopterus ( Pallas) after cadmium intoxication. Ecotoxicvl. Environ. Saf.,23: 147-160. 184.Ghosh A. R., Chakraborti P. and Pal S. (2006). Impact of diesel oil effluent in the mucosal surface of the alimentary canal of Orcachromir ntlotfca (Linnaeus): A 5cannIug electron microscopic study. I. Environ. Dial., 27: 129-134. 185.Girgis S. (1952). On the anatomy and histology of the alimentary tract of a herbivorous bottom- teeding cyprinoid fish, Luhen home (Cuvier). J. Morph., 90:317-362. 1X i Gisher1 it., Saraspiete C., Willis P. and Castell6-Orvay F. (1999). Histochemistry of the development of the digestive system of Siberian sturgeon during ontageny. J. Fish Biol., 55:596- 616. 187.Gnna, 0. (1979). Mucus glycoproteins of teleosteaa fish: A comparative histochemica: study. HistochenvcalJ., 11:709-718. 188.Coss C. C., Laurent P. and Perry S. F. (1994). GE1 morphology during hypercapnia in brown bullhead (tcialurus nebul9sus): role of chloride cells and pavement cells in acid-base regulation. S. Fish Bio I.,45: 705-718. 189.Gaswami, S.V., Parwez 1. And Sundarsraj, B.I. (1983). Some aspects of osmoregulaton in a stenohaline freshwater catfish, Heterupnevstesfossilis (Bloch), in different salinities. J. Fish Biel., 23: 475-487. ]90.Gradwell N. (1971). A photographic analysis of the air breathing behaviour of the catfish, Plecosfwnus punctutus. Can. J. Zoo]., 49:1089-1094. 191.Graham J. B. (1997). Air-breathing fishes. Evolution, diversity and adaptation. New York: Academic Press. p. 1-10 I92.G ratzek J. B. and Reinert R. (1984). Physiological responses of experimental fish stressful conditions. Nalt Cancer Ins[ Monogr., 65: 187-193. 193.Gran A., Crespo S., Sarasgnete M. C. and GonzAlez de Canales M. L. (1992). The digeAivc tract of the amberjack Serbia dumerrli Risso: A light and scanning electron microscope study. J. Fish Biol., 41. 287-303. 194.Grosell M. (2011). The role of the gastrointestinal tract in salt and water balance. In: Multifunctional Out of Fishes, Grosell, M., Farrell, A. P. And Bremner, C. 7. (Eds.: Fish Physic., 30:1-55. 195.Grutter A. S. and Pankhust N. W. (2000). The effect of capture, handling, confinement and ectoparasite load on plasma levels of cortisol, glucose and lactate in the coral reef fish Hemigymaus melapterns. I. Fish Biel., 57:391-401. 196.Gnner Y., C)zlen 0., Cagirgan H., Atlantis M., KizakV. (2005).Effects of Salinity on the Osmoregulatory Functions of the Gills in Nile Tilapia (Oreochromis niloircus). Turk J. Vet. Anim. Sci., 29: 1259-1266. 197.Gupta, B. J. (1989). The relationship of ntucoid substances and ion and water transport, with new data on intestinal goblet cells and a model for gastric secretion. Symp. Sec. Exp. Biel., 43: 81-110. 153 H 198.Handy RD. and Eddy F.B. (1991). The absence of mucous on the secondary lamellae of unstressed rainbow treu:, Oncarhynchars mykiss (walboum). J. Fib Biot., 3$: 153-155. 199.Handy R.D., Eddy F.B. and Roamain G. (1989). In vino evidence for ionoregulatory role of rainbow trout mucus is acid, acid/ aluminum and zinc toxicity. I.Fish Biol., 35: 737-747. 200.Hanna M. Shaheed I. and Elias N. (2005). A contribution on chromium and lead toxicity in cultured Oreoehromis nf(oriars. Egypt L Agnat. Biel. Fish., 9: 177-209. 201.Harder W. (1975). Anatomy or Fishes, E. Schwcizerbart'sche Verlagsbuchhandlung, Stuttgart. p. Pt 1. 612; Pt 2,132. 202.Harmon T. S. (2009). Methods for reducing stressors and maintaining water quality associated with live fish transport is tanks; a review of the basic. Rev. Aqua., 1; 5-66. 203.Hnrper C. and Wolf J. C. (2009). Morpbologic effects of the stress response in fish. ILAR Journal, 50: 387-396. 2C4.Harris .1. F., Watson A. and Hunl S. (1973). Histechernical analysis of mucous cells in the epidermis of brown troln, Salmo bntm L. J. Fish Rio!., 5:345-351. 205.Harrisoa J. P. Auger D. W., Paterson K. L. and Rowley P. S. A. (1987). Mucin histochemishy ofsubmandibular and parotid salivary glands of man: light and electron microscopy. Histochem. J., 19: 555-564. 206.Aayat M. A. (1970).Principit and I'eclmiques of Electron Microscopy: Biological Applications, vol. 1. New York: Van Nostraad Reinhold Company. 207.Helmiek C. M., Bailey J. F., LaPatra S. and Ristow S. (1995). Histological comparison of infectious hematopoietic necrosis viers challenged juvenile rainbow trout Oncorhynchus mykiss and coho salmon 0. kisutch gill, esophagus/cardiac stomach region, small intestine and pyloric caeca. Dig. Aqua.. Org., 23: 175-187. 208.Hemalatha S. and Banerjee T. K. (1997). Histopathological analysis of acute toxicity of zinc chloride to the respiratory organs of the air-breathing catfish Reteropnevslesfassims (Bloch). Veler. Archiv.,67:1-24. 209.Henrickson R. C. and Metaltsy A. G. (1968).The fine structure of teleast epidermis. Ill. Club cells and other cell types. J. Ullrestruct. Res., 21:222-232. 2: D.Hernandez D. R., Perez Gianeselli M. and Domilrovic H. A. (2009). Morphology, Histology and Histochemistry of digestive system of South American catfish (Rhamdia quthn). Inc J. Morph., 7: 5-I ii. 211.Herrler G., Rott R, KlenkH.D., MullerIJ P,Sbulda ££.and Schauer R (1985). Thereceptor destroying enzyme of influenza C virus is aeuraminate-0-acetyl-esterase.EMBOJ, 4: 15D3-1506. 212.Herrlur G., Rntt R, Munk H. D., Muller H. P., Shulda A. K. and Schauer R. (1985). The receptor destroying enzyme of influenza C virus is neureminate-O-acetyl- esterase. Embo. 1., 4: 1503-1506. 213.Hilary M., Lease-Hansen-James A., Bergman-Harold L. and Meyer-Joseph S. (2003). Structural changes in gills of Lost River suckers exposed to elevated pH and ammonia concentrations. Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol., 134:491-500. 2I4.Hirai N., Tagawa M., Kaneko T., Seikai T- and Tanaka M. (1999).Distribu:ional Changes in Branchial Chloride Cells during Freshwater Adaptation in Japanese Sea Bass Lateolabrax jupanicas. Zool. Sci., 16: 43-49. 215.Hirj i K. V. (1983). Observations on the histology and histochcmislry of the oesophagus of the perch Peeca f rMatr!is L.A. Fish Biot., 22:145-152. 2L6.Hiroi J. and McCormick S.D. (2007). Variation in salinity tolerance, gill Na'/K'-ALPase, Na' !K+ 2Cf cofransporter and mitocaondria rich cell distibution in three salmonids Salve(rnus namaycw.vic, Snlvehrseus fontinalis and Salmo solar. J. Exp. Biol., 210:1015-1024. 21T.Hoar W.S., Randall D.J. (1984). Fish physiology (vol.X). New York Academic Press, NY. 218.Holmgrea S. and Nilsson S. (1999). Digestive system. In: Sharks, Skates and Rays. John Hopkins University Press, Baltimore. p. 144- 173. 154 219.Holmgren S. and Olsson C. (2009). The neuronal and endocrine regulation of gut function. In: Fish Neuroendocrinology. Academic Press, p. 467-512- 220.Hari K., Fusetani N., Hashimoto K., Aida K. and Randall J.E. (1979). Occurrence of a grrmrnistin-like mucus toxin in the clingfish Diademiehthys lineatns. Toxicon., 17: 415-025. 221.Horn M. H. (1989). Biology of marine herbivorous fishes. Oceanogr. Mar. Blot. Annu. Rev., 27: 167-272. 222.Horn M.H. (1992). Herbivorous fishes: feeding and digestive mechanisms. In: Plant-Animal Irderuuticas in the Marine Benthos. Systematics Assosciation (Spacial Volume 46), John D.M., Hawkins S.J., Price J.H, (Eds)., Oxford: Clarendon Press., p. 339-362. 223.HOSoler F. E., Ruby J. R. And Mcllwain T. D. (1979). The gill arch of mullet, Miigh cophuius 11. Modification in surface ulmistruclural and Na,K-ATPase content during adaptation to various salities. J. Lxp. Zool., 208:399-406. 224.Hossler F.E. (1980). The gill arch of the mullet, Mugil cephmws III. Rate ofresponse to the salinity change. Ain. J. Physiol., 238: 160-164. 225.Houssain A. M. & Dutta H. M. (1998). Assessment of structural and functional similarities and differences between caeca of thebluegill. J. Fish Biol., 53:1317-1323. 226.Houssain A.M. and Dutta H.M. (1996). Phylogeny, ontogeny, structure and fimction of digestive tract appendages (Caeca) in teleost fish. In: Fish Morphology. Balkema Pub., Brookfield VT, 59- 7G. 227.Hnghes G. M. and Gray I. E. (1972). Dimensions and ultrastructure oftoadfuh gills Biol. Bull., 143: 150-161. 228.Hughea G. M. and Munshi J. S. D. (1968). Fine structure of the respiratory surface of an air- breathingfish, the climbing perchAnabas restndineous (Bloch). Nature, 219:1382-1384. 229.Hughes G. M. and Mnnshi J. S. D. (1973). Nature of air-breathing organs of the bidiun fishes, Chunna, Amphrprsous, Clarias and .Succobronchus as shown by electron microscopy. J, ZeoL (Land.). 170:245-270. 230.Hughes G. M. and Munshi J. S. D. (1978). Scanning electron microscopy of the respiratory surface of Saccobranchus (=Heleropneusres) fossilis (Bloch.). Cell Tissue Res., 195.99-109. 231. Hughes G M. and Munshi J. S. D. (1979). Fire stmctne of fish gills of some Indian air- breathing fish. J. Morph., 160: 169-194. 232.Hughes G. M. and Muashl J. S. D. (1986). Fine structure of gills of some Indian air-breathing fishes. J. Morphol., 160:169-194. 233.Hughes G. M. and Munshi J. S. D. (1986). Scanning electron microscopy of the accessory respiratory organs of the snake-head fish, Channa .miata (Bloch.) (Channidae, Chamsiformes). J. Zool. Load., 209:305-317. 234.Hughes G. M. and Ojha J. (1985). Critical study of the gill of a free-swimming river carp. Calla calla and air-breathing perch, Anabas tesrudineus. Proc. Indian Nam, Sci. Mad, 8,51: 391-404. 235.Hughes G. M., Singh B.R., Guha G., Dube S. C. and MunshiJ.S.D. (1974). Respiratory surface areas of an air-breathing siiuroid fish, Soccobanchus (Heteropneustes) fossil is, in relation to body size. J. Zool. Land, 172:215-232. 236.Hughes G.M. (1984). General anatomy of the gills. In: Fish physiology. (Eds), Hoar W.S. and Randall D.J, XA.New York: Academic., p.1-72. 237.Haghes G.M., Uube S. C. sad Munshi J.S.U. (1973). Surface area of the respiratory organs of clhnbing perch, Anabas lasludines. J. Zool. Land, 170227-243. 238.Humasou 1., (1967). Animal tissue techniques- 4th cd. San Francisco: WH Freeman. 239.Humbert W., Kirsch R. and Meister M. F. (1984). Scanning electron microscopic study of tlx oesophageal mucous layer in the eel, Anguilla anguida L. J. Fish Biol., 25: 117-122. 240.Hwang P.P. (1988a). Multicellular complex of chloride cells in the gills of freshwater teleosts. J. Morphol., 196: [5-22. 241.Rwang P.P. (1968b). Ulumswctural study oa multicellular complex of chloride cells in teleusts. Bull. Inst. Zool. Acad. Sin., 27:225-233. 155 242.Hwang P.P. and Hirano R. (1985). Effects of environmental salinity on intercellular organization and functional structure of chloride cell in early stages of teleost development. I. Exp. Zool, 236: 115-126. 243.Iger Y. and Abraham M. (1997). Rodlet cells in the epidermis of fish exposed to stressors. Tissue & Cell, 22 431-43R. 244.1ger Y., Abraham M., Ilntan A., Fnttal B. and Rahamim R. (1988).Cellnlar responses in the skin of carp maintained in organically fertilized water J. Fish Biol, 33: 711-720. 245.Iger Y.. Abraham M., Datan A., Fattal B. and Rahamim E. (1994) Cellular responses of the skin and changes in plasma cottisol levels of tout (Onchorhvnthus my iss) exposed to acidified water. Cell Tissue Res., 278: 535-542. 246.1sisaig 5. and Karaidsi H. (1998). Fine structure of the chloride cell in the Gill Epithelium of Bronchydario renio (Gyprinidae, Teleostes).'Ir. J. of Vet. and .4ni. Sci., 22: 231-436. 247. Islam A. (1951). Onthe histology of tie skin of certain teleost fishes. Pak. 3.Sci., 5:37-38. 24S. Iwama G. K., Afonsu L. O. B., Vijayan M. M. (2004). Stress in fish. AquaNet workshop on fish welfare, Campbell River B.C., Canada. 27 September. J 249.Jammewska M., Dab rowski K, Wilczynska B. and Kakareku T. (2008). Structure of the gut of the meet goby Neogobius gymnotrachelus (Kessler, 185/).1. Fish Bfol, 72; 1773-1786. 250.Jentoft S., Aastveit A. H., Torjesen P. A. Andersen 0. (2005). Effects of stress on growth, cortiso} and glucose in non-dnmesdcated Eurasian perch (Percu fiuviatilis) and domesticated rainbow trout (Oncnrhynchus ueykis.v). Comp. Riochem. Physiol. Part A, 141: 353-358. 251.Jezierska R. and Witeska M. (2004). The effect of metals on fish gill functions- Gas and ion change (review). Fresenius. Envvon. Bullet., 13: 1370-1373. 252.Jirge, S.K (1970). Mucopolysaccharide histochcmistry of th stomach of fishes with different food habits. Folia Histochem. Cyeochem.,8: 275-280. 253..1ohnson D- W. (1973). Endocrine control ofhydromineml balance in Leleosts. Am. Zoo[., 13: 799- 818. 254.Jones R. and Reid L. (1978). Secretary cells and their glycop otieus in health and disease. Br. Med. Bull., 34: 9-16. 255.Jones B. and Reid L. (1993a). The effect of pH on alcian Blue staining at epithelial acid glycoproteins L Sialomucins and sulphomucias (singly or in simple combinations). Histuchem. J., 5:9-18 256..Iones R. and Reid L. (1993b). The effect of pH on alcian Blue staining et epithelial acid glycoproteins II. Humaabranchial submucosal gland. Histochem. J. 5, 19-27. 257.Jozuka K. (1967). Chloride regulation by the gill of the fresh- water teleost, Carassius auratus. Ann. Zoo]. Jap., 40:205-210. K 258.Kamal M.Y. (1967). Studies on the food and alimentary canal of the Indian major carps B Labeo rrhita (Ham) mid IIT Cirrhina mrigala (Mani.) Indian J. Fish., 14: 24-47. 259.Kapoor B. G. (1957). The digestive tube of an omnivorous cyprinoid fish, Barbs stigma (CUV and VAL). Jap. J. Ichthyol., 6: 48-53. 260.Kapoor B. G. (1966). Histological notes on the skin of the head of a cyprinoid fish, Coda cnflo (Hamilton). Zool. Ann, L76: 264-171. 261.Kapoor B. C. and Khanna B. (1993). The potential spcanm of the gut in le1sost fishes. Any. Fish. Biol., 1: 221-226. 262.Kapoor B. G., Smit H. and Verighiaa I.A. (1975). The alimentary canal and digestion in teleost. Adv. Mar. Biol., 13:109-239. 156 263.Karnaky K.S. Jr. (1986). Stmcture and function of the c1i oride cell of Fundulus heteroditus and other teleosts. Am. Zeal., 26: 209-224. 264.Karnaky K.S. Jr. (1992). Teleost osmoregulation: changes in the tight junction in response :o the salinity of the environment. Im Tight Junctions, edited by Cereijido M. Roes Raton, PL: CRC, p.175--185. 265.Karnaky H.J. Jr. (1998). Osmotic and ionic regulation. In: The Physiology of Fishes (2nd ed.). (Eds.) Evans D.H. Boca Raton, F.L. CRC. p.157-176. 266.Karnaky K.J.Jr., Ernst S.A. and Phllpott C.W. (1976).'feleost chloride cell. 1. Response of pupfish pprinadan variegates gill Na`, K'-AIPase and chloride cell fine structure to various high salinity environments. S. cell Biol., 70: 144-156. 267.Karnaky K. J. Jr, Kinter L.B., Kinder W.B. and Stirling C.E. (1976). releost chloride cell. I1. Aumradiographic localization of gill Ne,K-ATPase in killifish Fundulus heternclirus adopted to low and high salinity environments. J Cell Biol., 70: 15 7-177. 258.Katoh F., Shimizu A., Uchida K. and Kaneko T. (20(l0).Shift of chloride cell distribution during early life stages in seawater-adapledki(lifish, Fundui¢s hrderoclimx. Zool. Sri., 17; 11-18. 259.Keys A.H. and Wilmer B.N. (1932). "Cl loride-secreting cells" in the bills of fishes with special reference to the common eel. J Physiol., 76: 368-37S. 270.KIranna S.S. (1961). Alimentary canal in some teleostean fishes. 1. Zoul. Sac. India., 13:206-219- 271.Khanna S.S. (1962). A study of bucco-pharyngeal region in some fishes. ltd. J. Zoot., 3: 21-48. 272.Khanna SS. (1964). Histology of the digestive tract of a teleost, Clarias bawachus (L.). 7. Zool. Sec. India., 16:53-58. 273.Khanna S.S. (1968). The structure and distribution of the taste buds and the mucus secreting cells in the buccopharynx of some Indian teleosts (Pisces). Studies, DSB Goverment College, Nanital (India)., 5:143-148. 274.Khanna SS. and Mehrotra B.K. (1970). Histomorphology of the buccopharyr x in relation to feeding habits in teleost. Proc. Nat. Aced. Sci. India., 40:61-40. 275.Khanna, S S. & Mebrotra, B. K. (1971). Morphology and histology of the teleostean intestine. Anat. Anz. Bd., 129:1-18. 276.Khanna S.S. and Pant M. C. (1964). On the digestive tract and the feeding Imbils of some teleostean fishes. Agra Univ. Journ. Res. (Sc.). 13:15-30. 277.Kikuchi S. (1977).Mitachandria-Rich (Chloride) Cells in the Gill Epithelia from Four Species of Slenohaline Fresh Water Teleosts. Cell Tiss. Res. 180: 87-98. 27S.Kirsch R. and Laurent P. (1975). L'oesophage, organ effecteur de I'osnmr6gulatlon chez un t8leostecn eurylmlin, l'anguille (Anguilla anguilla L.). C.R. Aced. Sol. Paris 280:2013-2015. 279.Kirscher L. B. (1978). External charged layer and Nat- regulation. In: Alfred Berzon Symp.X[. Osmotic and volume regulation (Eds by Jorgensen C. B. & Skadauge E.) Munksgaard, Copenhagen. 250.King J. A. C., Abel D. C. and Dillona D. R. (1989). Effects of salinity on chloride cells in the euryhaline cyprinodontid Fish Rivulus marmoratus. Cell Tissue Res., 257: 367-377. 281.KIeinow IC. M., James M. 0., Tong Z. and Venugopalan C. S. (1998). Bioavailability and biotransformation of benzo (a)pyrene in an isolated perfosed in situ catfish intestinal preparation. Environ. Health Perspect., 106: L55-166. 282.KInmpp R.W. and Nichols P.D. (1983). Nutrition of the Southern Sea garfish Hyporhunrphus melancehir: gut passage rate and daily consumption of two food types and assimilation ofseagrass components. Mar. Ecol. Prog. Series, 12: 207-216. 283.Kobegenava S. S. and Ddumaliev M. K. (1991). Morphoftenctional features of the digestive tract in sonic Gabioidei. Vopr. lkhtiol., 31: 965-973. 284.Kozaric L, Kuzir S., Nejedli S., Patrinec Z. and Srebocan F. (2004). Histochemical distribution of digestive enzymes in hake, Mcrl)lccills rucrfueehu L., 1758. Vet. Arhiv., 74(4):299-308. 157 285.Kozaric Z., Kuzir S., Petrinec Z., Cjureevie E. and Eaturina N. (2007). Hislochemistry of complex glycoproteins in the digu Slive Itad L mucusa of Atlantic blue fin tuna (Thunru s thynnas I-) Veterinarski Arhiv, 77: 411-452. 286.Kullz D., Jurss K. and Jonas L. (1995). Cellular and epithelial adjustments to altcrcd .salinity in the gill and opercular epithelium of a cidrild fish (Oreochronis mossambicus). Cell Tissue Res., 279: 65-73. 287.Kalt7 D. and Jurss K. (1993). Biochemical char-acterivation of isolated bronchial mitochondria- rich colts of Oreathrom is mossambicus acclimated to freshwater or hyperhaline seawater. 1 Comp. Physiol. [B], 163:406-412. 22g.Kumari U., Nigam A. K., Mittel S. and Mittal A. K. (2011). Antibacterial properties of the skin mucus of the freshwater fishes, Rita rila and Channa pnnciatus. Fur. Rev. Med. Pharmacol. Sci., 15:781-786. 289. Kumari U., Yashpal M., Mittal S. and Mitlal A. 1C (2011). Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Ritarita (Silurifnrmes, Bagridae). Tissue and Cell, 41:271-280, 290.Knmari U., Yashpal M., Mittal S. and Mittal A. K. (2005). Morphology of pharyngeal cavity, especially the surface ultrastructure of gill arches and gill rackers in relation to feeding ecology of the catfish Him rita.1. Morphol, 265:197-20& 29 LKumari U., Yashpal M, Mittal S. and Mittal A. K. (2009). Surface ultrastructure of gill arch and gill rackers in relation to feeding of an Indian major carp Cirrhinus mrigala. Tissue and cell 41:318-325. 292.Kuperman &1. and Kuzmina V-V. (1994). The ultrastmeture of the intestinal epithelimn in fishes with different types of feeding. J. Fish. Biol., 44: 101-193. 293.Kuppulalshmi C., Prakash M. and Gunasekaran C., 111animegalai G. and Sarojini S. (2003). Antibacterial properties of fish mucus from Channa punctatur and Cirrhinus mrigalu. Ear. Rev. Med. Pharmacol. Sei, 12: 149-153. 294.Kuzm ina V. V. and Gelman I. 1. (1997). Membrane linked digestion in fish. Rev. Fish. Sc., 5: 99-129. L 295.Lai S. K, Wang Y. Y. and Hanes J. (2009). Mucus-penetrating ranoparticlea for drug and gene deliveryto mucosal tissues. Adv. Drug Deliv. Rev., 61: 158-171. 296.Langille R. M. and Youson J. H. (1984). Morphology of the intestine oFprefeeding and feeding adult lampreys, Peoronyzon marinas (L): the mucosa of the posterior intestine and hindgut. J. Morph., lS2: 137-152. 297.Laurrut P. (1982). Structure of the vertebrate gills. In Gills, (Eds). Houlihan D.F., Rankin 7.C., and Shultleworth T.T., Cambridge Univ. Press London and New York p.25-43. 298.Laurent P. (1984 Gill internal morphology. In. Fish Physiology. (Eds), Hoar W.S. and Randall D.J., XA New York; Academic„ p.73-183. 299.Laurent P. and Dunel-Erb S. (1980). .Morphology of gill epithelium in fish. Am. J. Physlol. Regul. Jntegr, Comp. Physiol., 238: P.147-8159. 30O.Laurint P. and Kiraeh R. (1975). Modifimliuns sIructufulcs do l'oesuphagc lices a 'osmuregu lotion chez ranguille. C. r. hebd. Sanc. Mad. Sci., Paris, 380 D: 2227-2229. 30hbaureal P. and Perry S.F. (1990). EtTocts of cortisol on gill chloride cell morphology and ionic uptake in the fresh watertrou; Sahno gairdneri. Cell Tissue Res., 259:429-442. 302.Laureat P., Chevalier C. Wood C. M. (2006). Appcaeunce ofcuboidal cells in relation to salinity in gills of Fundnlns he1efoo!nus, a species exhibiting branchial NM but not Cl- uptake in freshwater. Cell Tissue Res., 325: 481-492. 303. Laurent P., Coss C.G. and Perry S.F. (1994). Proton pumps in fish gill pavement cells? Arch. lit Physiol. Bio:him. Biophys., 102:77-79. 04 Lanreat P., Rohe H. and Dunel-Erh S. (1985). The role of environmental sodium chloride relative to calcium in giL morphology of freshwater salmonid fish_ Cell tissue Res. 740: 675-692. 158 305. Leatherland and Lam (1969). Effect of prolactin on the density of mucous cells on the gill Llaments of the marine from (rrachunv) of the three spine stickleback, Gasrerosteus aeu1ea.us L. Can. J Zool, 47: 787- 792. 306.L.edy K., Giamhcrini L. and Pilmo P.C. (2003). Mucous cell responses in gill and skin of brown troutSnlmo Inrtta fario in acidic, aluminium containing stream water. Dis. Aquat. Organ., 56: 235- 240. 307.Lee T. U., Hwang P. P., Feng S. H. (1996x). The gill structure and bronchial mitochondria-rich cells of the ntedaka, Oryzias loupes. Acta Zoologica T'aiwanica 7: 43-50. 308.Lee T.N., Hwang P.P. and Feng S.H. (1996b). Morphological studies of gill and mitochondrla- rich in the stenohaline cyprinid teleosts, Cyprinus carpio and Carassius uurnl is, adapted to various bypotonic cnvironmcnts. Zoo!. Studies, 35:272-278. 309. Lee T.H., Hwang P.P., Shieh V.E., and Lin. C.H. (2000). The relationship between "deep-hole" mitochondria-rich cells and salinity adaptation in the euryhaline teleast. Oraochromis mossambicus. Fish Physiol. Biochem., 23:133-140. 3I O.Lee W., Huang C. Y. and Lin H.C. (2008). The Source of Lamellar Mitochondria-Rich Cells in the Air-Breathing Fish, Trichogoster leeri. J. Exp. Zool., 309A: 198-205. 311.Leino P.R.L., Wilkinson P., Anderson S.G. (1987). Histopathological changes in the gill of pearl dace, Smatihts margarita and fathead minnows, Pemephalse promelos, from experimentally acidified Canadian Ikes. Can.J. Fish Aquat. Sci, 44 (Suppl. 2). 126-134. 312.Lemoine A.M. and Olivereau M. (1971). Presence d'acide N-ac6tylneuraminlque dons Ia peau d'etude de I'osmoregulation-Z. Vergl. Physiol., 73, 22-23. 3 I3.Leonard J. B. and Summers R. G. (1976). The ultra-structure of the integument in the American Eel (Anguilla rosrrata). Cell Tissue Res., 171: 1-30. 314.Lev R. and Spicer S. (1964). Specific staining of sulphate groups with alcian blue at law pH. J. HiU stochem. Cytochem., 12: 309. 315.Lewis R-W. (1970). Fish cnbneous mucus. A new sauce of skin surface lipid. Lipids, 5:947-949. 316.Liem K. F. (1989). Respiratory gas bladders in Teleosts: functional conservatism and morphoingical diversity. Am. Zool., 29:333-352. 3I7.Lin T. H., Feng S. H., Lin CU., Hwang Y. H., Huang C. L. and Hwang P.P. (2003). Ambient salinity modulates the expression of sodium pums in bronchial mitochondria-rich cells of Mozambiquetilapia, Oreochromis mosrambicvc. Zeal. Sc., 20:29-36. 318.Lin Y.M, Chen C.N. and Lee T.H. (2003).'t'he expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water. Comp. Birchen. Physiod Part A, 125: 489-497. 319.Loetz C.A. (1995). Electrophysiology of ion transport in teleost intestinal cells. In: Cellular and Molecular approach to fish ionic regulation. (Eds). Wood C.H. and Shnnlewnrth T.J, Acadmic Press, London p. 25-26. 320.Logothetis E. A., Horn M. H. and Dickson K. A. (2001). Gut morphology and function in Atherinops afmis (Teleostei: Atherinopsidae), a stnmachless omnivore feeding on macroalgae. 7. Fish Biol., 59: 1298-1312. 321.Loretz C. A. (1995). Electrophysiology of ion transport in teleost intestinal cells. In: Cellular and Molecular Approaches to Fish Ionic Regulation, Wood C. H. and Shattelworth T. J. (eds. , Academic Press, London, pp.25-56. 312.Lotan R., (1960). Adaptability of Maple ni(otica to various saline conditions. Bamidgeh, 12:96- 100. 323.Ly tileiinea T, Pylkkol P-, Ritola O. and Lindstrom-Seppa P. (2002). The effect of acute stress and temperature on plasma cortiscl and ion concentrations and growth of Lake Inari Arctic charr,.Salvelinus alpinus. Envimn. Biol. Fish., 64: 195-202. M pr 324.Mai IC, Yu H, Ma H, Dun Q., Gishert E., Zambonino Infante J. C. and Cahu CL. (2005). A histological study on the development of the digestive system of Pseudasciaena crocea larvae and juveniles. J. Fish Biol., 67: 1094-1106. 159 325.Maillet M.(1963).Le reactif au tetroxyde d'osmium-iodure de zinc. 7 Mikrosk. Anat. Forsch., 70: 397. 326.Mallatt J. and Paulsen C. (1986). Gill nitrastrueture of the Pacific hagfish Eplatretu., .slouti. Am. 1. Anat., 177: 243-269. 327.Manera M. and Dezfuli B. S. (2004). Rodlet cells in teleosts: a new insight into their nature and functions. J. Fish Biol., 65:597-619. 328-Manjaksay J. M., Day R. D., Kemp A. and Tibbetts L R. (2009). Functional morpbelogy of digestion In the stomach3ess, piscivorous needle fishes 7ylosarus gavioloides and Srongytura leiura ferax (Teleostei: Belonifonnes). J. Morph., 270: 1155-1165. 329.Marchetti L., Capacchietti M., Sabbieti M. C., Accili D., Materazzi C. and Menghi G. (2006). Histology and carbohydate histochemistry of the alimentary canal in the rainbow trout Oneorhynchusmykiss. J. Fish. Biol., 6&1S-1 821. 330.Maricchiolu C. and Genovese L. (2011). Some contributions to knowledge of stress response in innovative species with particular focus on the use of the anesthethetics. Open Mar. Biol. J., 5: 24- 37. 331 Marshall W.S. (1979). Effects of salinity acclimation, Prolactin, growth hormone, and cortisol on the mucous cells of Leptocottus armatns (Teleosti; Cottidae). Gen. Comp. EndocrhioL, 37:358-368. 332.Marshall W.S. (2002). Na', Cl, Ca"' and Zni' transport by fish gills: retrospective review and prospective synthesis. J. Exp. Zaol, 293:264-283. 333.Marshall W.S. and Bryson S.E. (1998), Transport mechanisms of seawater teleost chloride cells: an inclusive model ofa multifunctional cell. Conip Biochem. Physiol. APhysiol., 199: 97-106. 334.Marshall W.S. and Nishiuka R.S. (1980). Relation of mitochondria-rich chloride cells to active chloride transport in the skin of a marine teleost. J. Exp. ZooL, 214: 147-156. 335.Marshall W.S-, Bryson S.F, Burghardl J.S. and Verbost P.M. (1995). Ca transport by opercular epithelium of the fresh water adapted euryhaline teleost, FuuCulvs heteroditus. J. Comp. Physiol. Biochem. Syst Environ. Physic1.,165:268-277. 336.Martin T..1. and Blaber S. J. M. (1984). Morphology and histology of the alimentary tract of Ambassidae (Cuvier) (Teleostet) in relation to feeding. J. Morph., 182:295305. 337.Masoni A.andlsaiaJ. (1960),Mise en c`vidence do rble des cellutes a chlorine dsus le transport des macranwlEcules organiques. In: Epithelia) Transport in the Lower Vertebrates. B.Lahlou, 8ds. University Press, Cambridge, p. 71-80. 333.Masson P.J.(1929). Some histological methods: trichrome stainings and their preliminary technique. J.Tc,L. Methods, 12: 75-90. 339.Matey V., Richards J. C., Wang Y., Wood C. M., Rogers J., Davies R., Murray B. W., Chen X.Q., Da J., and Brauncr C. J. (2008).The effect of hypoxia on gill morphology and ionoregulatory status in the Lake Qinghai scaleless carp, Gymnocypris prlewalskii. J. Exp. Binh, 211: 1063-1074. 340.Mattheij J. A. M and Sprongers d. A. P. (1969). The site of prolactin secretion in the adenohypophysis of the stenohaline teleost Anoptichthya jordani, and the effeco; of this hormone on mucous cells. Z. Z. Zellforsch., 99:411-419. 341.Matfheij J. A. M. and Stroband II. W. J. (1971). The effects of osmotic experiments and prolactin on the mucous cells in the skin and the iunocytes in the gills of the teleost Cich(osoma biocellatum. Z. Zellforseh..121:93-10]. 342.Mayberry L. F., Berry L. F, Bristol J. R., Sulmanovic D., Fojan N. and Petrinec Z. (1986).Rhandaspora /helnhani: epidemiology of and snigmtion into Cyprinus carpi bulbus atterios as. Fish Patho.,2I, 145-150. 343. McCahea C.P., Pascoe D, and Mckavanagh C. (1987). VistochemiSal observations on the salmonids salmo salar L. and salmo truna L. and the ephemeropteran Sue/is rhodani (Pi, Cl) and Ecdyommuv Yenosus (Fakir.) Following a stimulated episode of activity in an upland stream. Hydrobiologie, 153: 3-12. 344.McCormick S. D., Sundell K., Bjornson B. 'I'., Brown C. L. and lfirol J. (2003),Infuence of salinity on the localisation of Na1iK~-ATPase, Na'(K'2C t otranporter (NKCC) and CFTR anion 160 channel in chloride cells of the Hawaiian goby (&enogobius hawaiiensis). J. Esp. Biol., 206: 4575- 4583. 345.McCormick S.D. (1990). Fluorescent labelling of Na,K`- ATPase in intact cells by use of a tluorescent derivative ofot>abain: saliniland chloride cells. Cell'I'issue Res., 26U:529-533. 346.Mekim J. M. and Lieu C. J. (2001). Toxic responses of the skin. In: Target organ toxicity in marine and freshwater teleoss. (Eds.) Schlenk D. and Benson W. H., Taylor and Francis press, p.165-241. 347.McManus J. F. A. (1948). Hismlogieal and hisrochemical uses of periodic acid. Stain Technol. 23, 99-108. 348.MeMsous, J.F.A. (1946). The histological demonstration of niacin after periodic acid. Nature, 158: 202. 149.Meister M. P., Humbert W., Kirsch R. and Viviea-Raels B. (1983). Structure and ultrastruchere of the oesophagus in sea-water and fresh waterTeleosts (Pisces). Zoomorphology, 102: 33-51. 350.Michelengcli P., Ruiz M. C., Dominguez M. C. and Parthe V. (19SS). N.nmsna1inn-like differentiation of gastric cells in the shark &ranchus grireus. Cell Tiss, Res., 251:225-227. 35!.Milwnrd N. E. (1974). Studies on the taxonomy, ecology and physiology of Queensland mudskippers. PhD thesis- University of Queensland, p.1-276. 352.Mir 1.M. and Chauua A. (2010). A Scanning Flectran Microscopic Examination of the Intestinal Tract of the Snow Trout Sehizothoa.o cowmons Recket. J. Fish. Aquas. Sci., 5:386-393. 353.Mishra K. P-, Alim A-, Ran N. V- A. and Ahmed M. F. (1999)- Functional hiytotogy of oesophagus of some tolerate in relation to food preference. Trans. Zoo1. Soc. East India, 3(2):27- 32. 354.Mishra K. P., Rao N. V. A., Alim A. and Ahmed M. F. (1991). Functional peculiarities of the alimentary canal of Lepidocephalichrh s guatsa (Hamilton). J. Inland Fish. Soc. radix, 23(1): 59. 63. 355.Mitrovie D. and Perry S.F. (2009). The etTects oftlrermally induced gill remodelling on icnocyte distribution and branchial chloride fluxes ire goldfish (Carasrius meatus). J. Exp. Biol., 212: 843- 852. 356.Mittal A. K. and Banergee T.K. (1974b). Structure and keratinization of the skin of a fresh- watertelsest Noloplerus notoptezus (Notopterdae, Pisces). 3. Zeal. (Land.), 174:341-355. 357.Mittal A. K. and Hanergee T.K. (1975). Histochemistry and structure of the skin of a murrel, Channa struts (Bloch, 1797) (Channifotmes, Channidac). I Epidermis. Can. J. Zool., 53: 833- 843. 358.Mittal A. K and Banergee TIC. (1976). Functional organization of the skin of the "green-puffer fish" Tetrnndon Jlaviatilis (Hair.- Buck.) (Tetraadomidae,Prsces). Zoomorphologie, 84: 195-209. 359.Mittal A. K., Agarwal S. K. and Banerjee T. IC (1976a). Protein and carbohydrate histochennstry in relation to the kerat(nization in the epidermis of Barbus sop/or ' lq(Cyprinidae, Pisces). 1. Zeal. Lord., 179: 1-17. 36D.Mittal A. K., N'hitear M. and Agarwal S. K.(198I . Fine structure and histochemistry of the epidermis of fish Monopterus cuchia J. Zuol. London. 191:1D7-125. 361.Mittal A. K. (1968). Studies on the structure of the skin of Rita rita (Rant.) (13agridae, Pisces) in relation to its age and regional variations. Indian J. Zootomy., 9:61-78. 362.Mittal A. K. and Agarwal S. K. (1977). Histochemistry of the unicellular glands in relation to their physiological significance in the epidermis of Monopterus crrchia (Cyprinidar, Pisces). J. Zool. (Loud.), 182:429438. 363.Mittal A. K. and Banergee T.K. (1974x). A histochrrnical study of the epidermal keratinizztion in the skin of a freshwater toleost Vagarius bagarina (Ham.) (Sisoridae, Pisces). Mkrr kopie, 30:337- 348. 364.Mittal A. K. and Carg T. K. (1)94). Effect of an anionic detergent - sodium dodecyl sulphate exposure on club cells in the epidermis orClariar batrachus. J. Fish Biol., 44: 857-875. 161 365.Mitlal A. K. and Munshi J. S. D. (1970). Structure of the integument of a freshwater teleost Bagarius bagarms (Ham.) (Sisoddae: Pisces), 1. Morph., 130: 3-10. 366.Mittal A. K. and Munshi J. S. D. (1971). A comparative study of the structure of the skin of certain air-breathing flesh-water teleosts. J. Zool. (Loud.), 163: 515-532. 367.Mittal A. K. and Munshi J. S. D. (1974). Stmctre and keratinlsmion of the skin of a freshwater teleost. Notoplerus nolopeerur (Pallas) Notopteridae, Pisces. J. ZooL 174: 341-355. 368.Mittal A. K. and Whiter M. (1979). Keratiniration of fish skin with special reference to the catfish Bagarius bagarius. Cell Tissue Res, 202:213-230. 369.Mittal A. K., Fujimori 0., Ueda Y. and Yamada K. (1995). Carbohydrates in the epidermal mucous cells of a freshwater fish Mnslocembelus (mastacemhelidae, Pisces) as studied by eleciron- microscopic cylocherrical methods. Cell Tissue Res., 280: 531-539. 370.Mittal A. K., Rai A. IC, Banerjee T. K And Agarwal S. K. (1976b). Lipids in the skin of a catfish Heferopneusres fossilis (Bloch) (Hetempneustidae, Pisces)- histochemiral investigation, Histochemistry, 48:177-185. 371.)4littal A. K., Ueda T., Fujimori O. and Yamada K. (1994a). Histochemical analysis of glycopmmins in the epidermal mucous cells and saccifarm cells of an Indian swamp eel Monopterus cuchia (Hamilton) (Synbranchiformes, Pisces). Acta Histochem. and Cytochem, 27(3): 193-204. 372.Mittal A. K, Ueda T., Fujimori O. and Yamada K. (1994b). Histochemical analysis of glycoproteins in the unicellular glands in the epidermis of an Indian freshwater fish Masracembelus panca6as (Hamilton). Histochemical J, 26: 666-677. 373.Mittal A. K., Whitear M. and Bullock A.M. (1981). Sacciform cells in the skin of teleost fish. Z. MiWOSk. Anat. Forsch. Leipzig, 95:559-585. 374.Mittal A.R. and Nigam C. D. (1986). Fish skirt smfoce lipids: phospholipids. J. fish Biol., 29: 123-138. 375.Mittal S., Kumari U., Tripathi P. and Mittal A. K. (2010). Scanning electron microscopy of the operculum of Gaera !aorta (Hamilton) (Cyprinidae: Cypriniformes), an Indian hill stream fish. Aust. J, Zaol., 58:182-188. 376.Mittal S., Mittal P. and Mittal A.K. (2004). Operculum of peppered loach. Lepidncephu(ichthys gantea (Hamilton, 1822) (Cobitidae, Cypriniformes): a scanning electron microscopic and histochemical investigation. Belgian J. Zool. 134: 9-13. 377.Mittal S., Pinky, and Mittal A.K. (2002). Characterization of glyca- proteins in the secrelory cells in the operculuco of an Indian hill streamfish Gaialamm (Hamilton) (Cyprinidae, Cypnm£ormes). Fish Physiol. Biochem., 26:275-288. 378.M¢ano S., Urn K., Okubo T., Childs Y., Misaka N., Adachi S. and Yamauchi K. (2000). Ularastructural changes in gill chloride cells during smoltitication in wild and hatchery-reared masu salmon Oncnrhyachus mueou. Fisheries Sol-, 66:67D-677. 379.Mobamed IC. A. S. (2009). 1-listopathological Studies on Tdapia zeilif and Solea vu/guns from Lake Qarun, Egypt. World J. Fish Mar. Sci, I : 29-39 380. Moitra A., Singh O. N. and Munshi J. S. D. (1989) Microanstumy and cytochernistry of the gastro-respiratory net of an air-breathing cobitidid fish, Lepldocepha(ichks ganlea. 7. lchthyol. Res., 36: 227-231. 381.Moitra S. K. and Bhowmik M. L. (1967). Functional histology of the alimentary canal of the young Carlo cado (ham.), an omnivnmus surface feeding fish of Indian ireshwnter. Vestnik Cs. Spot. Zool., 31: 41-50. 392.Mallra S. K and Siaho G. M. (1972). Studies on the morphohistology of the alimentary canal of a carp, Cagunius chugunio (Ram.) with reference to the name of taste buds and mucous cells. S. Inland Fish. Soc. India., 3:44-56. 383.Moitra S., Ku uari K. Veeuapani and Sen. S. (2009). A scanning electron microscopy study of the gills of a euryhaline teleost, Seilpinna phases Hamilton. The Hioscan, 4:471-474. 3&4.Morgan M. and Tavel] P. W. A. (1973). The structure of the gill of the trout, Safmo gairdneri (Richardson). Z. Zellfnesrh., :42: 147-162. 162 385.Moron S. E., Andrade C. A. and Fernandes M. N. (2009).Respense of mucous cells of the gills of tratra (Hoplias malabaricass) andjeju (Hoplerythrhws umtaeniafi,) Teleostei: Erythrinidae) to hypo- and hyper-osmotic ion stress. Neotrop, Ichihyol,, 7491-498, 386.Morrison C. M. (1987). Histology of the Atlantic cod, Gadus morhua: an atlas. Pail one. Digestive tract and associated organs. Can. Speer Pub?. Fish Aquat. Sci., 9g:1-219. 387.Morrison C. M. and Wright Jr. J. R. (1999). A study of the histology of the digestive tract of the Nile tilapia. J. Fish Biol., 54:597-606. 388.Mosin S.M. (1946). The morphology and histology cfalimentary tract of 4nabas resrudrueus. 7. Germania Univ., t2: 66-75. 389. MovabediniaA.A., Savar7A_ MoruvvutiH.sKoochaninP., MarammaziJ.G.and NafisiM. (2009). The Effects of Changes in Salinity on Gill Mitochondria-Rich Cells of Juvenile Yellowfin Seabrcam, Ac¢nNmpagnu' talus, I Diol. Sci., 9:710-720. 390.Mowry R W. (1963). The special value of methods that colour both acidic and vicinal hydroxyl groups in the histoehemic¢l study of m¢cins with ccvised dh'eefior for the colloidal 'von stain, the use of alcian blue SGX, and their combination with the periodic acid-Schiff reaction. Aim. NY Arad. Sci. 106, 402-423. 391.Moyle P. H. and Ccch J. J. 1996). Fishes. In: An introduction to Icthyology, III Edn., Prentice- Hall, Upper Saddle River, New 7erey. 392.Munday H.L., Zilberg D. and Findlay V. (20U1). Gill disease ofmarine fish caused by infection with Neoparamueba pemngviderair. J. Fish Dis. 24: 497-507. 393.Mrsnshi J. S. D. (1964). Chloride cell in the gills of freshwater telosts. Quantitative Microscopy Sci_, 105: 179. 394.Munshi J. S. D., and Hughes G. M. (1992). A rbreathing fishes afrndia. heir structure function and life history. Rotterdam: AA Balkema. 395.Munshi J.S.D. (1980). The structure and function of the respiratory organs of air-breathing fishes of India Sectional (Zool. Entomol. and Fisheries) Presidential Address. In: Proceedings of the 67th session, Indian Sci. Con&. Association. Part ll. p. 1-70. 396.Munxhi J.S.D. (1996). Structure and function of the air breathing organs of Hsreroropnsusler fascilis. Advan. Fish Res, 1: 99-138, 397.Munshi J.S.D. and Singh A. (1992). Scanning electron microscopy evaluation of effects of low pH on gills ofC6anna (Bloch), J. Fish Biol., 41:83-89. 398.Munshi J.S.D., Ojha J. and Stabs A. L. (19S0). Marphometrics of the respiratory organs of an air-breathing catfish, Clarias balrachus (Linn.) in relation to body weight Proc Indian. Nat. Sci. Acad., B, 46(5):621-635. 399.Munshi J.S.D., Ojha J., Ghosh T.K., Roy P.K. and Mishra A.K. (1984). Scanning electron microscopic observation on the structure of gill rackers of some freshwater teleostean fishes, Proc. Indian Natn. Sci. Aced., 13., 50:549-554. 400.Munshi J.S.D., Olson K. R., Ojha J. and Ghosh T.K. (1986). Morphology and vascular anatomy of the accessory respiratory organs of the air-breathing climbing perch, Anabas testudineus (Bloch). Am. J. Anal,, 176:321-331. 401 .Munshi J.S.D., Fandey B.N, Pandey P.K. and Ojha J. (1978). Oxygen uptake through gill and skin in relation to body weighs of an air-breathing siloroid fish; Saccobrancims (=lfeteropneustes) fossilia. J. Zoos. (London), 134'171-ISO. 402.Murray H. M., Wright G. M. and Goff G. P. (1994a) A study of the posterior oesophagus in the winter flounder, Pleseronectes amerlcanus, and the yellowtail hounder, Neuron ectes ferruginevs: morphological evidence for pregastric digestion? Can. J. Zool., 72:1191-1198. 403.Murray FL M., Wright G. M. sad Goff G. P. (1994b). A compar tive histological and bistochemical study of the stomach from three species of pleuronectid, the Atlantic halibut, Hippoglossus hy,poglossus, the yellowtail flounder, Pleuronectes ferruginea, and the winter flounder, Pleuronectes amerlcanus. Can. J. Zool, 72: 1199-1210. 163 404.Murrey H. M., Wright C. M. and Goff G. P. (1996). A comparative histological and lustochemiaal study of the post-gastric alimentary canal from three species of pleuronectid, the Atlantic halibut, the yellowtail founder and the winter flounder. 1. Fish. Biol., 48: 187-206. N 405. Nachi A. M., Hcmmndetblaszquez F. J., Barbieri R. L., Leite R. C., Ferri S. and Phan M. T. (1998). Intestinal histology of a dehitiverous (iliophagous) fish Prochiadus scmfa (Characiformes, Prochilodontidec). Almales des Sciences Naturelles-Zoologie et Biologic Anim., 19: 81-88. 406.Naguih S. A. A., E!Slrahaka H. A. and Ashour F. (2011). Comparative I{istological and Ullrastmutural Studies on the Stomach of Schilbe mysrur and the Intestinal Swelling of Labeo niloricus. J. Amer. Sci., 7(8):251-263. 407.Narasimham C. and Paratheswarao V. (1974). Adaptation to osmotic stress in a fresh-water euryhyaline teleost, Tilapia mossambica. X. Role of mucopo:ysaccharides. Acta Hismchem., 51: 37-49. 408.Naskar R., Veenapani, Moitra S., Kumari K., Sen N. S. and Ahmed M.F. (2009). Surface ultrastructural changes in the gills of an Indian stenohaline catfish, Clarias bahachus (Lion.) under acute acid and aluminium strew. The Ecoscan., 3:221-226. 409.Nebel C., Rowestand B., Negre-Sadargues G., Gmnseet E., Aujoulat F., acal J, Bushomme F. and Charmantier G- (2005). Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrrtt: involvement of gills and urinary system. J. Exp. Biol, 298, 3859-3871. 4 IC.Nelson J. S. (2006). Fishes of the World, p. 6C 1. John Wiley & Sons, Hohoken, 411.Nelson L. K and Sheridan M.A. (2006). Gas1roenteropitereulie hormones and metabolism in fish. Gen. Cramp. Endocrine]., 148: 116-124. 412.Ncgus V. E. (1963). The function of mucua. Arta Oto-Lary'ngol., 56:2(1-214. 413.Nigam A. K., Knmari U. Mittai S. and Miltal A. K. (2012). Comparative analysis of innate immune paran..eters of the skin mucous secretions from certain freshwater teleost5, inhabiting different ecological niches. Fish Physiol. Biochent_ 38: 1245-1256. 414.Nishioka H. S., Lai K.V. and Bern H. A. (1985). Epidermal "white cells" in coho salmon. Aquaculture, 45:393-394. 415.Niva B., Ojha J. and Munshi J.S.D. (1981). Morphometrics of the respiratory organs of an estuarine goby, Boleop&hrtrrn bodduerB. Japan. J. Iehdhyu1., 27:316-326. 416.Noaillac-Depeyre J. and Gas N. (1982). Ullrastructure of endocrine cells in the swinach of two telenst fish, Perenitvvithlis L. and Ameixrus nebularus L. Cell Tissue Res., 221: 657 78. 417.Nolan D. T., Reilly P. and t onga S. E. W. (1999). Infection with low numbers of the sea louse (Lepeophtheirus salmons) induces stress-related effects in post smolt Atlantic Salmon (Salmo salar). Can. J Fish. Aquatic Science, 56: 947-959. 418.Nonnotte G., Nounotte L. and Kirsch R. (1979). Chloride Cells and Chloride exchange in the Skin of a Sea-Water Teleost, the shanny (Dlennius pholis U) Cell'1'iss. Res., 17: 387-396. 419.Nurthcott M. IL and Beveridge M. C. Al. (1988). The development and s:mcture of pharyngeal apparatus associated with filter feeding in tilapias (Orsachmm(s ni(oliens). J. Zoo[. Landon, 215: 133-149. 0 420.O'Byrne-Ring N., Dowling K., Cotters D., N9,e1an K. and MacEeilly U. (2003). Changes in mucous cell numbers in the epidermis of Atlantic salmon at the onset of smoltification. J. Fish Dial., 63: 1625-1630. 421.Ogawa Si, Vagasaki M. and Yamauhi F. (1973). The effect of prolactin on water influx in isolated gills of goldfish, Carassius aurunuc L. Comp. Biochem. Physiol., 44A: 1177-1183. 422.Ojha J. and Mishra A. K. (1987). Micmmorphology and cytochemistry of the bronchial glands of the freshwater mullets, Rhinomugil corsala and Sciamagi! cascasia Pree. Indian Acad. Sci. (Anim. Sei.), 96: 417-424. 423.Ojha J. and Munxhi .1. S. D. (1974). Histohctuicel and hietraphysiohgical observations on the specialized bronchial glands of a fiesb-water mud-eel Macrogna!$s aculemam (Bloch) (Mastacembefidae, Pisces). Mikroskopie, 3G. 1-16. 164 424.Ojha J., Rooj N. C. and Munshi J.S. D. (1982). Dimensions of the gills of the gills of an Indian hillstream cyprinid fish, Garra lamtaa. Japan. J. lchthyol., 29: 272-278. 425.Oliveira de C., Taboga S. R., Smarra A. L. S. and Bonilla-Radriguez G.O. (2001). Microscopical aspects of accessory air breathing through a modified stomach in the armoured catfish Liposorcus anisilsi (Siluriformes, Loricariidac). Cytobios, 105:153-162. 426.Oisen R. E., Hansen A. C., Rosenlund G., Hemre G.-L, Mayhew T. M., Knudsen D. L., Eroldogan O. T., Myklebust R. and Karlsen O. (2007). Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.) fl-Health aspects. Aquaculture, 272: 612-24. 427.Olson K. R. (1996). Scanning electron microscopy of the fish gill. In: Fish morphology: horizon of new research. Eds., Dana munschi, J. S. and Dote, H. M. 428.OIson K. R. and Fromm P. O. (1973). A scanning electron microscopic sandy of secondary lamellae and chloride cells of rainbow trout (Salmo gairdneri). Z. Zellforsch., 143:439-449. 429.O1son K.R. (2000). Respiratory system In: The Laboratory Fish, (This.) Ostrander G.K., Academic press, p.151-156. 430.OIson K.R., Conklin D.J., Weaver L. Jr., Duff D.W., Herman C.A., Wang C. J.M. (1997). Cardiovascular effect of homologous bradykinin in rainbow trout. Mn. J. Phsiol. Regul. Integr. Comp. Physiol.. 272: RI 112-RI 120. 431.OIson KR., Munshi J.S.D., Ghosh T.K. and Ojha J. (1986). Gill microcirculation of the air- breathing climbing perch, Anabas teshtdineus: relationship with the accessory respiratory organs and systemic circulation. Am. J. Anat., 176:305-320. 432.Ortiz J.B., Gondlez de Canales M.L. and Sarasquete C. (1999). Cauntificacidny alterations histopatologicas producidas for conccntrtciones subletales de cobre on Fundlus heteroditus. Ciencias Marinas., 25: 119-143. 433.Ortuoo J., Esteban M. A. and Meeguer J. (2002). Effect of four anaesthetics on the innate immune response of gilthead seabream (Spares aurata U. Fish & Shellfish Immun., 12:49-59. 434.Osman A. H. K. and Caceei T. (1991). Histology of the stomach of Tilapia nilotica (Linnaeus, 1758) fromthe RiverNile. J. Fish Biol., 3S: 211-223. 435.Osms-Garrido M.V., Nunez-Torres M. 1. and Ahaurrea-Equisoain M. A. (1993). Histological, histochemical and ultrastructural analysis of the gastric mucosa in Oneorhynchus mykiss. Aquaculture, 115:121-32. P 436.Pabst R. (1987). The anatomical basis for the immune function of the gut. Anal. Embryol., 176:135-144. 437.Pajak B. and Danguy A. (1993). Characterization of sugar moieties and oligosaccharide sequences in the distal intestinal epithelium: of the rainbow trout by means of lectin histochemistry. J. Fish B iol., 43: 709-722. 438.Parashar RS. and Banerjee T.K. (1999b). Response of the venial respiratory organs of the air- 7 breathing catfish Hereropneustesfossihis (Bloch) to extreme stress of desiccation. Vet. Archv, 69: 63-68. 439.Parashar R.S. and Banerjee T.K. (1999e). Response of the gills of the air-breathing catfish Heleropneustes fossilis (Bloch) to acute stress of desiccation. J. Exp. Zool, India, 2: 169-174. 440.Parashar R.S. and Baoerjee T.K. (2002). Toxic impact of lethal concentration of lead nitrate on the gills of air-breathing catfish Hereropnrautesfossifis (Bloch). Vet. Archie., 72: 167-183. 441.Parashar R.S. and Banerjee, T.K. (1999a). Histepathological analysis or sublethal toxicity induced by lead nitrate to the accessory respiratory organs of the air-breathing teleost Heteropneusiesfossilis (Bloch). Pol. Arch. Hydrobid., 46: 194-205. 442.Parillo, F., Gargiulo A. M. and Fagioli O. (2004). Complex carbohydrates occurring in the digestive apparatus of Umbrina cirrosa (L.) fry. Vet. Res. Comm., 28: 267-278. 443.Park C. M., Reid P. E., Owen D. A., Volz D. and Dunn W. L. (1987). Histochemical studies of epithelial cell glycoproteins in normal rat colon. Histochem. J. 19: 546-554. 444.Park J. Y. (2002). Morphology and histochemistry of the skin of the Korean spined loach, lkvookimia koreensis (Cobitidae), in relation to respiration. Folia Zool. 51: 241-247. 445.Park J. V. and Kim 1. S. (2001). Histology and mucin histochemistry of the gastrointestinal tract of the mud loach, in relation to respiration. J. Fish Biol., 5S: S61-872. 165 446.Park J. Y., Kim I. S. and Kim S. Y. (2003). Structure and histoehemistry of the skin of a torrent catfish, Liobagrus mediadiposalls. Environ. Biol. Fishes., 66: 3-8. 447.Park J. Y, Kang E. J. and Kim Y. C. (2002). Structural and histochemical study on the skin of Korean bullhead, P.seudohugrusfulridraco. Korean J. ichthyol, 14: 247.253. 448.Park J. Y., Kim 1. S. and Kim S. Y. (2002). Histology of skin of the Amphibious fish, PeriopMna[mus modesins. Korean J. Biol., 4:315-318. 449.Park J. Y., Lee Y. J., Kim 1. S. and Kim S. Y. (20036). Morphological and cytochemical study on the skin of Korean eel goby, Odonrambly op us lacepedti (Pisces, Gobildae). Korean J. Biol. Set., 7:43-47. 450.Pdrk .1. V., Lee V. J., Kin, 1. S. and Kim S. Y. (2003x). A comparative study of the regional epidermis of an amphibious mudskipper fish, Boleophthahnus pectinirostris (Gobiidae, Pisces). Foliia ZcoL, 52:431-440. 451.Park J.Y. (2002). Structure of the skin of an air-breathing mudskipper fish, Periophtha(muy magnuspinnwus. 3. Fish Biol., 60: 1543.1550. 452.Park J.Y., Kim 1. S., Lee Y. J. and Kim S. Y. (2004). Histology and Morphometrics of the epidermis of the Fins and sucking disc of the mudskipper, Periophtholmus modes/us (Pisces, Gobiidae). Korean J Biol. Sci., 8:111-115. 453.Parwez I., and Goswami S.V. (1985). Effects of prolactin, adrenocorticotropin, neurohypophysial peptide, cortlsol and androgens on some osmoregulatory parameters of the hypuphysectomized catfish, He[eropneusles fossilis (Bloch). Gen. Comp. Endocrinol., 58: 51-68. 454.Parwer I., Goswami S.V. and Sundararaj B.I. (1984). Effect of hypophysectomy on some osmorcgulatory parameters of the catfish. Heiropneusles fossilis (Bloch). 1. Exp. Zeal., 229: 375- 381. 455.Parwez I., Goswami S.V. and Sundararaj G.I. (1979). Salinity tolerance of the freshwater catfish Heteropneustes fossiiis (Bloch). Indian J. Exp. Biol., 17:810-811 456.Parwez 1., Nayyar M., Shcrwani F.A., Parwcz H. (2001). Salinity tolerance and the role of cortisol during osmotic adjustments of an air-breathing catfish Clarias bairachus. J. Aqua., 9: 9-17. 457.Pasha S.M. Kamal. (1964a). The anatomy and histology of the alimentary canal of an omnivorous fish, Mystus gtdio. Proc. [rid. Acad. Sci., 59B:211-221. 458.Pasha S.M. Kamal. (1964b). Anatomy and histology of the alimentary canal of a herbivorous fish, Tilupia mosambica. Proc. Ind. Acid. Sc., 59B:340-349. 459. Pasha S.M. Kamal. (1964r). The anatomy and histology of the alimentary canal of a carnivorous fish, Megalops cyprinoides. Proc, Ind. Acad. Sei., 60B:107-115. 460.Patil R N., Madnaik U.S. and Bodare RD. (2012).Histochemical analysis of mucosubstsnces in the gill epithelium of Channa punclatus exposed to lethal concentration of malathion.Indian Streams Reserzch Journal., 1:1-4. 461.Paul V. 1. and Banerjce T. IC (1996b).Ammonium sulphate induced stress related alterations in the respiratory epithelium of the airbreathing organ of the catfish HeteropneustesJossilis (Bloch).J. Biusci.,21: 519-526. 462.Paul V. 1. and Rauerjee T. K. (1996a). Analysis of ammonium sulphate toxicity in the fresh water catfish catfish Hereropneustes fossUts using mucocyre indexing. Pol. Arch. Hybrobiol., 43: 111- 125. 463.Paul V. L and Banerjee T. K. (1997). Histupathological changes induced by ambient ammonia (ammonium sulphate) on the opercular lining of the catfish Heteropnerstes fossilis. Dis. Aqua[. Org., 28: 151-161. 464.Pearse A.G.E. (1968). Histochemistry: Theoretical and Applied, 3a Ed., J. & A. Chrurchill, Ltd., p.918. 465.Pedini V., Scocco P., Radaelli G., Fagioli O. and Ceccarelli P. (2001). Carbohydrate histuchemistry of the alimentary canal of the shi drum, Umbrina cirrosa L. Anat. Distol. Embryol., 30:345-349. 466.Peek W. D. and Yous in J.H. (1979). Ullms1mehrte of chloride cells in young adults of the anadromous sea lamprey, Pefromyzon martinis L., in fresh water and during adaptation to see water. J. Morphol., 160: 143-164. 166 467.Perry S.F. and Wood C.N.M. (19S5). Kinetics of bronchial calcium uptake in the rainbow trout effects of acclimation to various external calcium levels. J. Exp. Bid., 116:411-433. 468.Perry S.F., Coss C.G. and Laurent P. (1992). The interrelationships between gill chloride cell morphology and ionic uptake in four freshwater teleoats. Can. J. Zool., 9: 1775-1786. 469.Perry SF. (1997). The chloride cell: structure and function in the gills of freshwater fishes. Anna Rev. Physiol,59: 325-347. 470.Perry. S.F. and Laurent. P. (1993). Environmental effect gill stmcbue and function. In: Fish ecophysiology. (Eds), Rankin J.C. and Jensen F.B., Chapman and Hall, London, p.231-264. 471.Petrinec Z., Nejedli S., Kuzir S. and Opucak A. (2005). Mucosubstances of the digestive tract mueosa in Northern pike (Flax Lucius I..) and European catfish (Si4tnts giants L.) Veter. Arluv., 75: 317-27. 472.Phllpott C. W. and Copeland D. E. (1963). Fine structure of chloride cells from three species of Fandulus. J. Cell Biol., IS: 389-404. 473.Phramsuthirak P. (1977). Electron microscopy of wound healing in the skin of Casterosteur aculeatus. J. Fish Riol., 11:193-206- 474.Pickering A. D. (1974). The distribution of mucous cells in the epidermis of the brown trout Salmo hutta (L.) andthe char Salve/inns alpinus (L.). J. Fish Biel, 6: 111-1 t S. 475.Pickering A. D. (1977). Seasonal changes in the epidermis of the brown trout Salmo trutta (L.). J. Fish Biol., I D: 561-566. 476.Pickering A. D. (1981). Stress arid Fish. London: Acadmic Press. p. 367. 477.PIckering A. 1). and Fletcher J. M. (1987). Sacoiform cells in the epidermis of the brown trout, Salmo trutm, and the Arctic char, Salvetinus alpinus. Cell Tissue Res., 247:259-265. 478. Pickering A.D. and Macey D. J. (1977). Structure. Hismchemistry and the effect of handling on the mucus cells of the epidermis ofthe char Salvelinuc alpines (L.). J. Fish Biol, 10: 505-512. 479.Pickering A. D., Pottingcr T. G. and Christie P. (1982). Recovery of die brown trout (Saint. trukal from acute handling stress: a time-course study. J. Fish Biol., 20: 229-244. 480.Pinky, Mittel S. and Mittal A. K. (2007). Glycoproteins in the epithelium of lips and associated structures of a hill stream fish Gan-a lamla (Cyprinidae, Cyprinoformes): a hislochemical investigation. Anat. Histol. Embrvol, 37: 101-113. 481.Pinky, Mittal S, Ojha J. and Mittal A. K. (2002) Scanning electron microscopic study of the structures associated with lips of an Indian hill stream Gsb Cara Jamb (Cyprinidac, Cypriniformes). Eur. J. Morphol., 40: 161-169. 482.Pinky, Mittal S:;`YashpakM..Oiha J. and Milm1 A. K. (2004). Occurrence of keratinization in the structures associatediwith-lips:nf a hill strean fish Gan- lamla (Hamilton) (Cyprinidae, Cyprinifnrmes) 1. Fish Riol. 65:1165-I 172. 483.Pisam M, CarolT A., and Rambourg A. (1987). Two types of chloride cells in the gill epithelium of a freshwater-adapted euryhaline fish: Lebtsres reticulatus; their modifications during adaptation to saltwater. An J. Anat, 179: 4D-50. 484.Pisam M. (1981). Membranous systems in the "chloride cell" of teleostean fish gill: their modifications iu response to the salinity ofthe enviruimrenL Anat, Rea., 200:401414. 485.Pisam M. and Rambourg A. (1991). Mitochondria-rich cells in the gill epithelium of teleost fishes: an ultrastructuml approach. lot. Rev. Cytol., 130:191-232. 486.Pisam M., Boeuf C., Prunet P., and Rambourg A. (1990). Ultrastructural features of mitochondria-rich cells in stenohaline freshwater and seawater fishes. Am. 1. Anat, 187: 21-31. 487.tisam M., Le M.C., Auperin B., Prunet P. and Rambourg A. (1995). Apical structures of "mitochondria-rich" alpha and beta cells in euryhaline fish gill: their behaviour in various living conditions. Anat. Rec., 241:13-24. 4g8.Pisam M., Massa F., Jammet C. and Prunet P. (2000). Chronology of the appearance of beta, A, and alpha mitochondria-rlclt cells in the gilt epithelium during onmgenesis of the brown trout (Salina ottm). Mat. Rec., 259:301-311. 489.Pisam M., Proust P., and Rambourg A. (1989). Accessory Cells in the Gill Epithelium of the Freshwater Rainbow7ruut Salmogairdneri. Am. J. Anat, 184:311-320. 167 490.Pi5am M., Pranet P, Reeuf G. and Rambanrg A. (198S). Ulnastructueal features of chloride cellsin the gill epithelium of the Atlantic salmon, Salmo .alar, and their modifications during smoltifcxtion. Am. J. Mat., 183:235-244. 491.Playle R.C. and Wood CAM. (1991). Mechanisms of aluminium extraction and accumulation at the gills or rainbow trout, Oncorhynchus mykiss (Walhamn), in acidic soft water. J. Fish Biol, 38: 791-805. 492-Podkowa D. and Gonlakowska-Witalinska L, (1993). The structure of the airbladder of the catfish Pungasnis hypophlhafmuv Roberts and Vidthayanon 1991 (previnnsly P. srdchl Fowler 1937). Folia Biol. (Krakow), 46:189-196. 493.Portz D. E., Woodley C. 117-, Cech Jr J. J. (2006). Stress-associated impacts of short-tern holding on fishes. Rev. Fish Biol. Fish., 16: 125-170. 494.Powell M.D., Speare D. J. and Burka J. F. (1992). Fixation of mucus on rainbow trout (Onchorhynchus reykiss Walbaum) gills for light and electron microscopy. J. Fish Biol., 41:813- 824. 495.Powell P., Parsons H.J. and Nowak B.F. (2001). Physiological effects of freshwater bathing of Atlantic salmon (Salmo salar) as a treatmeatfor amoebic gill disease. Aquaculture, 199: 259-266. 9 496.Quinn M. C. J, Veillette P. A,, Young C. (2003). Pseudobranch and gill Nam. K`-ATPase activity in juvenile chinook salmon, Oncorhynchus tshmvytscha: developmental changes and effects of growth hormone, cortisol and seawater transfer. Coup. Biochen,. PlrysioL A. Mol. imegr. Physiol-135: 249-26. 497.Quiniou S.M-A., Bigler S., Clem L.W. and lily 3.E. (1990). Effects of water temperature on mucous cell distribution in channel catfish epidermis: a factor in winter saprolegniasis. Fish Shellfish lmmunol., 8:1-I I. R 49&.Rai A. K. and Mittal A. IC. (1983). Histochemical response of alkaline phosphatase activity during the healing of cutaneous wounds in a cat-fish. Fxperientia, 39: 520-522. 499.Rai A. K. and Mittal A. K. (1991). On the activity of acid phosphate during skin regeneration in Heteropnenstesfatsiis. Bulletin of Life Sciences,. 1:33-39. 50D.Rai A. K., Srivastava N., Nigam A. IC., Kumari Ti., Mittal S. and Mittal A. K. (2012). Response of the chrommophores in relation to the healing of skin wounds in an Indian M&or Carp, Labeo rohita (Hamilton), Tissue Cell 44(3): 143-50. 501.Rajan M. T. and Banerjee T. K. (1991a). Histopathological changes induced by acute toxicity of mercuric chloride on the epidermis of a fresh water cat-fish, Heteropneustes foss/Its (Bloch). Ecotoxicol. Environ. Saf., 22: 139-152, 502.gajan M.T. and Banerjee T.K. (1991 b).hstopafhoIogical changes induced by acute toxicity of mercuric chloride on the epidermis of freshwater catfish Hereropnecsres fossilis (BIoch),EcotpxieoI. Environ. Saf., 22:139-52. 503.Raansamaptray B., Knnfia C.P. and Das B. K. (20D9), Antibacterial:activity of crude mucus extract ofQppokpabda against common bacterial fish pathogen. Environ. Bcol., 4:1657-1659. 504.Rajbansgi V. K. (1977). Zhe architecture of the gill surface of the catfish, Heterooncustes fossifis (Bloch): SEMstudy. J, fiph Biol, 10:325-329. 505.Raji A. It and Norgp~i E. (2010). Histological and histochemical study on the alimentary canal in Walking catfish (C4i!s batrachus) and piranha (Serrasa(mon nntterenj. Iran. J. Veter. Res., 11(3): 255-261. 506.Ralpha J. R. and Benjamin M. (1992). Chditdruitin and keratin sulphate in the epidermal club cells of tcleo I J. Fish Bial., 40:473-475. 507.Ramphal V. and Pyle M. (1983). Evidence for mucros and sialie acid as receptors for Pseudomotms aeruginosa in the Iowa respiratory tract. Infect. Immun., 41: 339-344. 168 508.Randall D. J, Brauner C.J., Thurston R. V. and Neuman S.E. (1996). Water chemistry at the gill smtace of fish and the uptake ofxenobiotics. In: toxicology of aquatic pollution, (Eds), Taylor E.W., Cambridge University Press, Cambridge. p. 1-16. 509.Raslogi ILK. (1966). the morphology and histology of the alimentary canal of Heteropneusres fossi(is (Bloch). Agra Univ. Res. (Si.), 16: 63-72. 510.Ray A. K. and Moitra S. K. (1982). On the morpho-histology ofthe alimentary tract in the Indian Climbing Perch. .nabas lastudineus (Bloch), in relation to food and feeding habits. Gegeabaurs, Morphol, Jab-b., 128: 778-798. 5I l.Reid L. and Clamp (1978. The biochemical and histochemical nomenclature of mucus. Br. Med. Bull., 34[ 5-9. 512.Reifel C. W. and Travill A. A. (1978). Structure and carbohydrate Itistoehemistry of the stomach in eight sneciec of telensts. J. Morph_, 158(2): 155-16R_ 513.Reifel C. W. and TravUI A. A. (1979). S6vcture and carbohydrate histocbemistry of the intestine in ten telertctean species. J. Morph., 162:343.360. 514.Reiieff C.W. and Travill A. A. (1977). Structure and carbohydrate histochemislry of the esophagus in tea 1elecstean species. J. Morph., 152: 303-14. 515.Rhodes J. M., Black RR., Gallimore R. and Savage A. (1985). Histruchemical demonstrationof desialatien and desulphation of normal and inflammatory bowel disease rectal mucus by faecal extract. Gut, 26: 1312-1318. 516.Ribelles A., Carrasco M. C, Rostty M. and Aldana M- (1995). A histochemical study of the biological effects of sodium dodecyl sulfite on the intestine of the gilthead seabream, Spann aurcta L. Ecotox, Environ. Saf., 32: 131-138. 517.Richmanm N. H., Diaz S. T. D, Nisbinra R. S., Prunet P. and Bern H. A. (1987). Osmoregulatory and endocrine relationships with cblcride cell morphology and density during smoltification in echo salmon (Oncor{ynchus kisutch). Aquaculture, 60:265-285. 51S.Roberts R. ,1, Bell M. and Young H., (1973). Studies on the skin of phice (Plearnectes ptutessa L). B. The development o£]arval plaice skin. J. Fish Biol, 5: 103-108. 519.Roherts S. D. and Powell M. D. (2005). The viscosity and glycoprotein biochemistry of salmonid mucus varies with species, salinity end the presence of amoebic gill disease. J. Comp. Physiol. B, 175:1-I1. 520.Roherts SD. and Powell M.D. (2003). Comparative ionic of salinity flux and gill mucous cell histochemistry: Effects of salinity and disease status in Atlantic salmon (Salmo salar L.). Comp. Biochem. Physiol. part A., I34: 525-537. 521.Rosetherg A. and Schengrund C. L. (1976). Biological Roles of Sialic Acid. New York: Plenum Press. 522.Roy D. (1988). Statistical analysis of anionic detergent induced changes in the goblet mucous cells of opercu r epidermis and gill epithelium of Rita rita (Ham.) (Bagridae: Pisces). Ecotoxicol. Ecvlron. Saf., 15:260-271. 523.Roy P. K. and Munshi A S. D. (1991). Malathion-induced structural and morphometde changes ofa fresh-water major carp, Cirrhfnus mrigala (Ham.). J. Environs Biel,12: 79-87. 524.Roy P.K. and Munshi J.S.D. (1986). Scanning electron microscopic evaluation of effects of saponin on gills of the climbing perch Ambits tesmdineus (Bloch) (Anabanfidae, Pisces),Indian J. Exp. Hiol.,24:511-516. 525.Rnst M.B. (2002). Nutritional physiology. InFish nutrition. (3rd Edition.; Halver J. E. and Hardy R. W. (Eds.), Academic Press, Amsterdam. p. 367-052. S 526.Seadatfar Z.and Shahsavani D. (2009). Morphology and changes of chloride cell of Rutdus or rulilus Caspicus (Cgprinidea, teleost) in Caspian Sea. Vet. Res. Conm ent, 33:979-986. 527.Saboia-Morace S.M.T., Hernandez-Blazquez F.J., Mom D.L. and Bittenenurt A.M. (1996). Mucous cell types in the branchial epithelium of the euryhalino fish Poecilia vrvipara. J. Fish Rio., 49:545.548. 52g.5adovy Y., Randall J.E. and Rasotto M. B. (2005). Skin structure in six dragenet species (Gobiesoci£ormes; Callioaymidae): inter-specific differences in glandular cell types and mucus secretion. J. Fish Biol.. 66:1411-1418. 529.Sarasquete C, Gisbert E., Ribeiro L. Vieira L. and Dinis M. T. (2001). Glycoconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilllrad sea bream, Spann aurata, Senegal sole, Solea senegalensis and Siberian almgeon Acipen¢er baeri development. Ear. J. Ristochem.,45: 267-278. 530.Sarasqucte C, Gonzales de C. M.L, Arcllano .T., Munoz Cueto J.A., Ribeiro L. and Dinis, M.T (1998). Histochemical study of skin and gill of Senegal sole, Sole seaegalensis 'srvae and adults. Histol. Hislopathol., 13: 727735. 531 Sarbahi D. 5. (1939). The alimentary canal of Labeo zohiza (Ham.) J. Roy. Asia. Soc. Bengal Sul., 5: R7-]16. 532.5ardella B.A. and Kultz D. (2008). Osmo- and ionoregulatory responses of green sturgeon (.4crpensermedirostris) to salinity acclimation. J. Comp. Physiol,179; 383-390. 533.Sardet C., Pisam M. and MaetzJ. (1979). The surface epithelium ofteleostean fish gills: cellular and Functional adaptations ofthe chloride cell in relation to salt adaptation. J. Cell Biol., 80:96-117. 534.Sarkar R. and Ghosh A. R. (201D). Histochemical characterization of mncin in intestine and rectum of Jleternpnevstes fossitis (bloch) induced by metanil yellow. The Bcascan, 4(4): 97-300. 535.Sassi S., Kaneko T. and Tsuhamoto K. (1998). Extrabranchial chloride cells in early life stages ofthe Japaneu: eel, Anguilla japo,ilaa. Tchthyol. Res., 45:95-98. 536.Sasai S., Katoh F., Kaneko T. and Tsukamoto K. (2007). Ontogenic change of gill chloride cells in leptocephalus and glass eel stages of the Japanese eel, Anguilla japonica. Mar. Biol.. 150:487- 496. 537Swlvy K. V. and Gupta P. K. (1979). The effect of cadmium on the digestive system of the teleost fish, Heleropneustes fossilis. Environ. Res., 19:221-230. 5 -,R.Sastry K!.', Subhadra K. (1992)- Ftfect of cadmium an some aspects of ec rbuhydmle metaMliem in a freshwater Catfish, Heleropnvstw fossilis. Toxicol. Lett, 14:45-55. 539.5atchell G. H. (1984). Respiratory toxicology of fishes. In: Aquatic loneology, (Eds.) Weber L. 3.. Vol. 2. Raven Press, New York p. 1-50. 54C.Sato M. (1978). Light mid transmission electron microscopy of the granular cell in the skin epidermis of a cottid Pseudoblerarius cotloides. Japan. J. lchthyol, 24: 231-138. 541.S2to M. (1981). Fine structure of mucous cells in the skin epidermis of the Arctic Lamprey, Lampetra Japonica. Japan. J. ichthyol. 29:69-76. 542.Satara U. (1998). Histological and ultrastmctmal study of the stomach of the air- breathing Ancismus mukispinis (Siluriformes, Teleostei). Can. ). Zool., 76: 83-86. 543. Saxena M. and Kulshrestha S. K. (1981). Histochemical studies of mucosubstances in the epidermis ofa nonscaly IeleostMysins (Mystas) vittatus BI. (pisses-bagridae). Histochemistry, 72: 155-160. 544.Schauer R. and Kamerliug J.P. (1991). Chemistry, Bioclmmistryand biology of sialicacids., In: Glycoproteinsll(Eds)Montreuil I.,- Vliegenthadl.F-C and Schachter H., Amsterdam:Elsevier,p241- 400. 545.Scherdtfeger W.K. (1979). Morphometrical studies of the ultrastruclurs of the epidermis of the guppy, Poccila vuc-uialc. Peters, following adaptation to seawater and treatment with prolactin. Gen. Comp.Endocrinol., 38: 478-483. 546.Sehrager 1(1970). The chemical composition and function of gastrointestinal mucus. Gut, 11: 450-456. 547.Schreiber A. M. and Specker S. L. (1999). Metamorphosis in the summer flounder Paralich1hys dentatus: changes in gill mitochondria-rich cells. J. Exp Biol., 202:2475-2484. / 548.SehulteB. A. and Spicer S. S. (1985). Histochemical methods for characterizing secretary and cell surface sialoglycoconjugates. J. Hislochem.Cytochem., 33:427-438. 549.5chwerdtfeger W. K and Bereiler-Hahn J. (1978). Transient occurrence of chloride cells in the abdominal epidermis of the guppy, Poecilia reticulate Peters, adapted to sea water. Cell Tice. Res., 191: 463-471. 550.Scocco F., Aceili D., Meughi G. and Ceccarelli P. (1998). Unusual glycocoajugates in the oesophagus ofatilapine polyhyhrid. J. Fish Biol., 53: 39-48. 551.Selye H. (1936). A Syndrome produced by diverse nocuous agents. Nature, 138: 32-34. 552.Selye H. (1973). The evolution of the stress concept.. Am. Sol., 61: 692-699. 553.Senger H., Starch V., Reinecker M. Kloas W. aid Hooke W. (1994). The development of functional digestive and metabolic organs in turbot, Scophthalmas marimns. Mar, Biol, 119: 471- 486. 554.5hane Bit. and Powell M.D. (2003). Comparative ionic flux and gill mucous cell histochernistry: e17ect ofsulinitiy and disease status in Atlantic salmon (Sahno salarL.). Comp. Biuchem, Physiol. PartA: Mole. Integr. Physiol., 134: 525-537. 555.Shephard K.L. (1994). Function for fish mucous. Rev. Fish Biol., 4: 4D1-429. 556.Shepherd K. L. (1981). The influence of mucus on the diffusion of water across Esh epidermis. Physiol. Zool., 54:224-229. 557.Sherwani F. Laud Parwez 1. (2008).Plasma thyroxine and cortisul profiles and gil, and kidney Nt'iKC-ATPase and SDH activities during acclimation of the catfish Referopneastes fossi7is(Bloch) to higher salinity, with special reference to the effects of exogenous cortisol on hypo-osrnoregulaeory ability of the catfish. Zool, Sci., 25:164-171, 558.Sherwani, F.A. and Parwez, 1. (2000). Effects of stress and food deprivation on the catfish, Hcteropneustesfossilis (Bloch). Indian J. Exp. Biol, 3g: 379-384. 559.ShikanoT. and Fujio Y. (1998). Relationships of salinity tolerance to immunoloeuurationofNa, K- -ATPase in the gill epithelium during seawater and freshwater adaptation of the guppy, Poecilix reticulate. ZooL Sci, 15: 3541. 560.Shikann T. and Fujio Y. (1999). Changes in salinity tolerance and hranchial chloride cells of newborn guppy during freshwater and seawater adaptation. J. Exp. Zocl., 284: 137-146. 561.Sbirai N. and Utida S. (1970). Development and degeneration of the chloride cell during seawater andfreshwater adaptation of the Japanese Eel., dnguillo Japonica Z. Zellf0rscb., 103:247-264. 562.SLuttlrworth T.T. (1989). Overview of epithelium ion-transport Mechanisms. Can. J. Zool., 67: 3032-3038. 563.Sibbing F. A. and Uribe R. (1985) Regional specialisation in the oro-pharyngeal wall and food procesiag in carp (Cyprinus carpio U. Neth. J. Zool., 35:377-422. 564.Sibbing F. A. and Urihe R. (1985). Regional specialization in the oropharyngeal wall and food processing in the carp Cyprim s carpio- Netherlands J. Zool., 35: 377-422. 565.Silva J. M., Hernandez Blazquez F.J. and Julio H. F. Jr. (1997). A new accessory respiratory organ in fishes: morphology of the respiratory purses of LoricariitNrys platymetoparr (Pisces, Loricartidae). Mn. Sci. Notion Zoel. Paris, 18:93-103. 566.Silva P., Salomon R.J. and Epstein F.H. (1997). Transport mechanisms that mediatethe secretion of chloride by the rectal gland of Squalus acanthias. J. Exp. Zool., 279: 504-508. 567.Singh C.P. and Kapoor B.G. (1967). Histolohical note or the skin of the head of cyprinidLaheu calbasu (Ham.). Vest. Cst. Spol. Zool., 76:211-216. 568.Singh C. P. and Kapoor B. G. (1968). Contribution on the histology of head-skin of a carp, Cirrhino reba (Ham.). Vest. Cst. Spol. Zool., 32:272-274. 569.Siagh A. K. and Banerjee T. K. (2008). Toxic effect of Sodium arsenate (Na2HAsO4.7HzO) on the skin epidermis of air-breathing catfish Clarias hatraciras (L.). Veterinauski Arhiv., 78: 73-88. 570. Singh B.(1990). Light microscopy, SEM, morphomelrics and histochemistry of the gills of some commercially important fishes of India. PhD. Thesis, Bhagalpur University. 571.Singh R. R., Pradad M. S., Yadav A. N. and Kiran U. (1991). The morphology of the respiratory epithelium of the air breathing organ or the swamp-eel, ,No wpteras alhus (Zuiew). A SEM study. Zool. Anz., 226:27-32. 171 572.5ingh R.N. (1966). On the gill-structure ofcobitid fish- Lepidacephalichdrysgmneea(Ham.)_ Japan. J.IchthyoL, 14: 103-106. 573.Singh B.R., Yadav AN., Ojha.F. and Munshi .f.S.D. (1981). Gross structure and dimensions of the gills of an intestinal air-breathing fish, Lepidocephalichihys gunrea. Copcia, 1981:428-439. 574.Singh N.H. (1986). The structure mid function of the respiratory organs of a freshwater air- breathing fish, Colisajascraras. Ph.D. Thesis, Bhagalporuniversity. 575.Singh S. IC and Mittal A. K. (1990). A comparative study of the epidermis of the common carp and three Indian major carp. J. Fish Biol., 36: 9-19. 576.Siuha G.M. (1976). Comparative morphology, anatomy and histology of the alimentary canal of an Indian freshwater major carp, Lateo ca(basu (Ham.) during the different life-history stages in relation to food and feeding habits. Anat. Anz., 139: 348-362. 577.Sinha G.M. (1981). Scanning electron microscopy of the inteslute in an indiac freshwuter major carp, Cirrhlnus mrigala (ham.) J. Inland. Fish. Soc. India., 13: 51-56. 578.Sinhn C.M. (1983). Seaming electron microscopy of the intestinal mucoea of an Indian fre liwarcr adult major carp, Labeo rohira (Ham.). 2. Mikrosk: anal. Farsch. Leipzig, 97:979-992. 579.Sinha G.M. and Chakrabarti P. (1985). On topological characteristics of the mucosal surface in buccophar)nx and intestine of an Indian freshwater major carp, Carla calla (Ham.): A light and scanning electron microscopy study. Zool. 16. Anat., 113: 375-389. 580.Sinha G.M. and Chakrabarti P. (19861 Scanning electron microscopy studies on the mimosa of the digestive tract in Mystux aor (Harr.) Prac. Indian Nan. Set. Acad. 52: 267-273. 581.Siuha G.M. and Chakraharti P. (1989). Micro-architecture of the gut epithelium in Lcbeo calbanr (Ham.) : Scanning electron microscopy study. Arch. Biol. Bruxelles., 100:283-294. 582.Sinha G.M. and Moitra & K (1975). Morpho-histology of the intestine in a freshwater major carp, Cr,himss mrigala (11am.) during the different life history stages in relation to °hod and feeding habits. Aria. Anz, 137:395-407. 583.Sire M. F, Lutton C. and Vernier J. N. (1951). New views on intestinal eheorpticn of lipids in teteastean fishes: an ultrastrienud and biochemical study in the rainbow trout. J. Lipid Res., 22: 81-94. 584.Sire M. F. and Vernier J. M. (1992). Intestinal absorption of protein in teleost fish. Comp. Binchem. Physiol., 103A: 71-781, 585 Si R.F., Ives P. J., Junes D. M., Lewis D. H. and Haensly W. E. (1979). The microscopic anatomy of the oesophagus, stomach and intestine of the channel catfish, lctalurus purwlalas. J. Fish BioL, 14:179-86. 586.Sklan D., Prag T. and Lupatsclr 1. (2004). Structure and function of the small intestine of the tilapia Oreochromfs niloricus X Oreochromis mucus (Teleostei, Cichlidae). Aquacult. Res., 35: 350-357. 587.Slechtova V., Ruhlen J. and Tan H. H. (2007). Families ofCobitoidea (Telenstci; Cypriniformes) as revealed from nuclear genetic data and the position of the mysterious genera Barbucca, Psiforhynchas, Serpenticohitis and Vaiflaniella. MoI. Phylogenet. Evol., 44:1358-1365. 588.Smith R. J. F. (1986). The evolution of chemical alurm signals In fishes. In Chemical signal of Vertebrates, Vol. IV, (Eds), Duvall D., Muller-Schnarse D. and Silverstein R. M, New York: Plenum, p. 303-320. 589.Smith IL J. F. (1992).Alarm signals in fishes. Rev. Ph Biol. Fish., 2:33-63. 590.Smith L S. (1976). Digestion inteleost fishes. Bull- lap. Soc .Sci. Fish., 45:409-449. 591.Smitir L.S. (1989). Digestive functions in teleost fishes. In: Halver J. E. (ell.) Fish nutrition. San Diego, USA: Academic Press, p.331-421. 592.SmoIka A. J., Lacy E. R., Luciano L., and Real a E. (1994). Identification of gastric H, K ATPase in an early vetebrate, the Atlantic stina~ay Dasyots sabina. J. Histochem. Cylochem., 42:1323-1332. 172 593.5olanki T. G. and Benjamin M. (1982). Changes in the mucous cells of the gills, buccal cavity and epidermis of the nine-spinet! stickleback, Pamgitius pungrtius L., induced by trausfening the fish to sea water. J. Fish Hio., 2!: 563-575. 594.Soufy 13., Sultman M., Lk-Manakhiy E. and Gaafa A. (2007). Some biochemical and pathological investigations on monosex Tilapia following chronic exposure to carbofuran pesticides. Global Voter., 1: 45-52. 595.Spicer S. S. and Lillie R. D. (1960). Saponification as a mean of selective reversing the me[hylat on blockade of tissue basophilia.1. 1Cisiochem. and Cymchem, 7:123- t25. 596.Spicer S. S. and Meyer D. B. (1960). Histochemical differentiation of acid mucapolyseceharides by mean of combined aldelryde fuulnia-alcian blue staining. Amer. J. Clip. Patb., 33:433-460. 597.Spicer S. S. and Schulte B. A. (1992). Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry. basic Appl. Histochem., 32:307-320. 598.Spiccr S. S. and Warren L (1960). The hislochemislry of sialic acid containing mucoproteins. 1. Histochern. and Cytochem.,8:135-137. 599.Spicer S. S., Horn R. C. and Lcppi T. J- (1967).Histochemistry of connective tissue mumpelysecc6arides.pp 251-303. he The connective Cissnes.Intecuationa] Academy of Pathology Monograph No.7. Williams and Wikins, Balthimore. 60C.Srivnstava N., Kumari U., Amita K. IL, Mittal S. and Mittal A. K. (2011). Histochcmicul analysis of glycoproteins in the gill epithelium of an Indian major carp, Crr hinua mrigala. Acta histochemica., 114:626-35. 60l.Stertid E., Harris P. D. and Bakke T- A. (1998). The influence of GymdatC'7us vnlorie.cMalm6et•o, 1957 (Me.ngepeu) on the epidermis of Atlantic salmon, Snlmo salar L, and book trout. Salve! iuusfontruafs (Mitchill): experimental studies. J. Fish Diseases, 2: 223-229. 602. Steven G. A. (1930). Bottom fauna andthefood of fish. 5. Mar. Bird. Ass. U.K., 16:677-706. 603.Stevens C. E. and Hume I. D. (1995). General characteristics of the vertebrate digestive system. hr Comparative Physiology of the vertebrate digestive system. (2'd edition). Cambridge University press. 604.Stevens E. D. and Hume 1. D. (2004). Comparative Physiology of the Vertebrate Digestive System, Cambridge University Press, Cambridge. p. 400. 605-Stevens F. D. and Hume 1. D. (2004). Comparative Physiology of the Vertebrate Diges:ivc System, Cambridge University Press, Cambridge. p. 400. 606.Sturla M., Masini M.A., Preto P., Grattarnla C. and UvaB,(2001). Mitochondria-rich cells in gills and skin of ar. African lungfish, Protnpfirus onnecteris. Cell Tissue Res., 303:351-358. 607.Snbnamanian S., MacKinnon S. L. and Russ N. W. (2007). A companlive study on innate immune Parameters in the epidemtal mucus of various fish species. Comp. Biocem. Physiol. 148: 256-263. 608.Suiemez M. and Ulus E. (2005). A study of the anatomy, histology and ultrastmcture of the digestive tract of Orrhrias angorae Steindachner, 1397- Folia Biologic. (Krakow), 53:95-100. 609.Stlivan M. X. (1907). The physiology of the digestive tract of elasmobranchs. Bull. Bureau Fish, 27: 1-27. 6I0.Suprasert A., Fujioka T. and Yamada K. (1986). Glyeoconjugates in the secretory epithelium of the chicken mandibular gland. Hislochem. J., 18:115-121. 611.Suprasert A., Fujioka T. and Yamada K. (1987). The histochemistry of glycoconjugates in the colonic epithelium of the chicken. Histochemistry, 96:49U497. 612.Sasithra N, Jothivel N, Jaykunar P- and Paul V.L (2007). Toxicopathological impact of cadmium chloride on the Accessory rerpiratary organ of the air-breathing catfish Beteropnenstes fossils. Iran. J. Environ. Health, Scien. Enginc.,4: 1.8 613.Suyebiro Y. (1942). A study on the digestive system and feeding habits of fish. Jap. J. Zool, 10: 1-303. 614.Suzuki N. (1992). Fine structure of the epidermis of the mudskipper, Perioohthalmua modes/vi (Cubiidue). Jpu. J. Ichthyo. 33:379-396. 173 615Suzuki Y. and Kaneko T. (1986). Demonstration oldie mucous hemagglutinin irthe club cells of eel skin. Develop. Comp. Immunol, 104:509-518. T 616.Takashima F. and Hibiya T. (1995).An Alas of Fish Histology. Normal and Pathological Features. 2nd ed.Kodensha Ltd., Tokyo. 617.Takasusuki J., Fernandes M.N. and Seven i W. (1999). The occurrence of aerial respiration in Riinelepis strigoea during progressive hypoxia. J. Fish 13iol., 52169-379. GlL.Takei Y. and Loretz C.A. (2011). The gastrointestinal tract as an endocrine) neuracndocrinefparacrine argans: organization, chemical messengers am physiological targets. In: Multifunctional Gut of Fishes Grosell M., Farrell A. P. and Hrs user C. J. (Eds.), Fish Physio, 30:261-316. 619.Takeyasu K., Tnm kin M.M., Renaud K.J. and Fambrough D.M. (1988). Ouabain-sensitive (Na`,Kl-ATPase activity expressed in mouse L cells by transfection with DNA encoding the a- subunil of an avian sodium pump. 3. Biol. Chem., 263: 4347-4354. 620.Thomes P. M., Pankhurst N. W. and Bremner H. A. (1999). The effect of stress and excrcisc on post-mortem biouhemislry or Atlantic salmon and rainbow trout. J. Fish Biui., 54: 1 I77-1196. 621.Threadgold L.T. and Houston A.H. (1964). An electron Microscope study of the "chloride cell" of sa(mo solar L. Exp. Res., 34: 1-23. 622.Tibbetts I. R. (1997). The distribution and function of mucous cells and their secretions in the alimentary tract of Arrhamphur scleraiepis Kre$tii. J. Fish Biol, 50: 809-820. U 623.Uchida K., Kaneko T, Miyazaki H. Hasegawa S. and Hirano T. (2000). Excellent salinity lulerance of Mozambigce tdapia (Oreoclrrovais ,nci arnbicua). Elevated chloride cell-activity in bronchial and operc ular epithelia of the fish adapted to concentrated sea water. Zool. Sci., 17: 149- 160. 624.Uchida K-, Kaneko T., Ya man chi K. and Hirano Y. (2000). Morphometrical analysis of chloride cell activity in the gilt filament and lamellae and changes in Na', K" -A1Pase activity during seawater adaptation in chum salmon fry. J. Exp. Zeal., 276:193-200. 625.Uchida K., Kaneko T., Yamauchs K. and Hirano T. (1994 Morphometrical analysis of chloride cell activity in the gill filaments and lamellae and changes in Na{,K~-ATPase activity during seawater adaptation in chum salmon fry. .1. Exp. Zool., 276: L93-200. 626.Uchida K., Kaneko T., Yamagncki A., Ogasawara T. and Hirano T. (1997). Reduced hypoosmoregulatpry ability and alteration in gill chloride cell distribution in mature chun salmon (Onsorhyoshns kela) mi gating upstream for spawning. Mar. Biol., 129:247-253. 627.Ura K., Mizunn S., Oku10 T., Chida Y., Misaka N., Adachi S. and Yamauchi K. (1997). hnunOhistochemisa1 study on changes in gill Nat, K'-ATPase a-subunit during mollification in wild masu salmon, Oncnirnchuv masou. Fish Physfol. Biechem., 17:397-403. 626.Ura K., Soyana K., Omoto N., Adachi S. and Yamauchi K. (1996). localization of Na,K'- ATPase in tissues of rabbit and teleosts using an antiserum directed against a partial sequence of the a-subunit. Zool. Sci., 13: 219-227. 629.C1ida S, Kamiya M and Shirai N. (1971). Relationship between the activity of Na'-K~-activated adenosinetripbosphatase and the number of chloride cells in eel gills with special reference to sea- water adaptation. Comp. Biochem, Pliysiol. A. Mol. Integr. PhysioL, 38:443-447. V 174 630. Varsamos S., Diaz J.P., Charmantler C., Flik G., Blasco C., and Comics R (2002). Bronchial chloride cells in sea bass (Uicenlrarchus tabrax) adapted to flesh water, seawater, and doubly concentrated seawater. J. Exp. Zool., 293: 12-26. 631.Varute A. T. and. Dirge S. K (197]).Histoc1iemical analysis of mucosubstances in oral mucusa of momhbreeding Cichlid fish and seasonal vcriatiuns in them. Hisrochern., 25: 91-102. 632.Veggetti A., Rowlerson A.. Radaelli G., Arrighi S. and Doinenegtiiri C. (1999). Post-hatching development of the gut and lateral muscle 'a the sole, Solea solea (L). J. Fish Biol., 55 (Suppl. A): 44-45. 633.Velmurugan B., Selvanayagam M., Cengiz E. and Unlu E., (2007). The effects of fenvalerste on different tissues of freshwater fish Cirrbin+a mrigala. J. &w'son. Sci. Health (B), 42: 157-163. 634.Venkaiah Y. and Lakshmipathi V. (2000). Biochemical composition of epidermal secretions and poisonous spine of two freshwater catfishca. Asian Fish. Sci., 13:183-109. 635.Verma S. H. and Tyagi M. P. (1974). A comparative histological study of the stomach of a few teleost fishes (Port 1). Gcgonbrnis Morph. Jahrb, 120: 244-253. 636.V4eira L. (2000). Histologia e hisloquimica do tube digsstivo e 6rganos anexos de p6s-la-ase juveals de Solea senega)ensis (Kaup, 1858) alimentados core diierentes dieters. Relat6rio do estAgio do curse de Licenciatma em B iologia Marinha a Peaces. Univ. do Algawe. Faro Portugal. 637.Vigliano F. A. Aleman N., Quiroga M. 1 and Nieto J. M. (2006). Ultrastructural Characterization of Gills in Juveniles of the Argentinian Silverside, Odontesthes bonariensis (Valenciennes, ]R35) (Teleestei: Atheriniformes)_ Anal. Histnl. RmTryol., 35:76-83. 638.Virahhadrachari V. (1961). Structural changes in the gills, intestine and kidney of Enophts macufatus (Teleostei) adapted to different salinities. Quart J. Mier. Sci., 102: 361-367. w 639. Wallace K N., Miller S., Smith IL. 1L, Lorent K. and Pack M. (2005). Intestinal growth and differentiation in zebrafish. Mech. Dev., 122:157-173. 640.Walsh P.J. (1998). Nitrogen excretion and metabolism. In: The Physiology of Fishes, (Eds.) Evans D.H. Boca Raton, F.L. CRC. p.199-214. 641.Wassersug E. J., Johnson B. K. (1976). A remarkable pyloric caecurn in the evermanellid genus Coccordia with notes on gut structure and function in alepisamoid fishes (Pisces, Myctophiformes). J. Zool., 179: 273-289. 642.Watrin A. and MayerGostan N. (1956). Simultaneous recognition of ionocytes and mucous cells in the gill epithelium of turbot and in the rat stomach. J. Exp. Zool., 276:95-101. 643.Wcgner N. C. Sepulveda C. A. and Graham J. B. (2006). Gill specializations in high- performance pelagic teleosts, with reference to striped marlin (fllrnprunas audac) and wahoo (Acauthocybiam so(andrt). Bull. Mar. Sci., 79:747-759. 644.Weinreh E. L and Bilsrad N. M. (1955). Histology of the digestive tract and adjacent structures of the rainbow front, Safmo goirdneri iridens. Cupeia, 3: 194-204. 645.Welsel G. F. (1957). The integument of the paddlefish, Polyodon sparhvfa. J. Morphol., 145: 143- I50. 646.Weisel G. F. (1979). Histology of the feeding and digestive organs of the shovelnose sfitgeon, Scaphir# 'nchvs plarorynchus. Copeia, (3): 518-525. 647. Weisel G.F. (2005). Anatomy and histology of the digestive system of the paddlefish (Polyodon spatlnla). 1. Morphol., 140: 243-255. 64R.Welker T. L., Lim C., Yildrim-Askoy M. and Keisiuc P. B. (2007). Effect of buffered and unbuffered tricane methanesulfnnate (MS-222) at different concentrations on the stress responses of channel catfish, letalarus punetalas Rafinesque. J. Appl. Aquae., 19:1-18. 649.Weadehaar Bnnga S. E. and Meis S. (1981). Effects of external asmolality, calcium and prolactin on growth and differentiation of the epidermal cells of the cichid teleost Snrotherodon mocsambicus. Cell Tissue Iles., 221: 1U9-123. 175 650.Weadclaar Bonga B.E., Flik C., Balm P.H.M., and van der Meij J.C.A. (1990).The ultmstmcture of chloride cells in the gills of the teleost Oreochromis mossambic,s during exposure to acidified water. Cell Tissue Res., 259: 575585. 651. Weadelsar Bongo S.E., Lock RA.C. (1992). Toxicants and osmoregulation in fish. Neth. 1 Zool., 42:478-493. 552.Western J. It H. and Jennings J. B. (1970). Histochemical demonstration ofhydmchloric acid in the gastric wholes ofteleasts using an in vivo Prussian blue technique. Comp. Biochem. Physiol., 35: S79-8S4. 653. W harton S. A., Weis W., Skeliel J. J. and Wiley D. C. (1989). Structure, function, and antigenic ily of me hemaggluim inofnf ucnaa viras.ln: The Influenza V huses(Eds.) Krug R. M., Newyork: PlenumPress,p. 153-L73. 654.Whitear M. (1970). The skin surface of bony fishes. J. Zool. (Loud.),160: 437-454. 655.Whitear M. (1989). Merkel cells in lowerveitebrates. Arch. Histol. Cytol., 52:415-422. 656.Whitear M. and Lane & B. (1983).O1ibovillous cells of the eFidermis: scnsory elements of lamprey skin. J. Zool., 220: 139-151. 657.Whhcar M. and Mittal A. K. (1983). Fine structure oftlre club cells in the skin of ostariophysan fish. Z. Mikrosk. Anal- Forscb. (Lcipz), 97: 141-157. 63€.W hitear M. (1971).The free nerve endings in fish epidermis. J. Zool. (Loud.), 163:231-236. 659.Wliiear M. (1986). The skin of fishes including cyelostemes. In: Biology of the Integument Vol 2, (Eds), Bereiter-Hahn J., Matoltsy A.G., Richards K.S., Springer, Berlin Heidelberg New York, p. 8-e4. 660. W hfear M. and Mfttal A_ iL (1984).Surface secretions of the skin of ffennius (Gpnphrys) phnlu I.. J. Fish Biol., 25: 317-331. 661.Whitear M. and Moate It (1998). Cellular diversity in the epidermis of Raja ctavata (Chondrlchthyes). J. Zool. 246:275-285. 662.Wilkins N.P. and .lanesar S. (1979). Tcmpoml variations in the skin of Atlantic salmon Salmo .valar L. J. Fish 13iol., 15:299-307. 663.Wilson J. M. and Castro L. F. C. (2011). Morphological diversity of the gaslrointestinal tract in fishes. In: Multifunctional Gut of Fishes, (Eds.) Groselu M., Farrell A. P. and Drauner C. J., Fish Physic., 30:1-55. 664.Wilsoa J. M., Laurent P., Tufts B. L., Demos D. J., Donowitz M., Wayne Vogl A. and Randall D. J. (2000).NaCluptake by the bronchial epithelium in freshwater tefeost fish: an immunological approach to ion-transport protein localization. J. Exp. Biel., 203: 2279-2296. 665. W ilmn J.M. and Laurent P. (2002). Fish gill morphology: inside out I Exp Zool., 293: 192-213. 666.Wilson R.W, Bergman H.L. and Wood C.M. (1994). Metabolic costs and physiological consequences of acclimation to aluminium in juvenile minvaw trout, Oncorhyncas mykiss (Walbaum), gill morphology, swimming performance and aerobic scope. Can Fish Aquat Sc'., 51: 536-544. 667.Wilsona J. M., Morgana J. D., Vogld A. W. and Randalla D. J. (2002). Bronchial mitochondria-rich cells in the dogfish Sgnalacv acanthus Comp. Biothem. Pbysiol. Part. A, 132: 365-374. 668. Witters H., Berckmans P. and Vaogenechten C. (1996). Immunolocalizetion of Nat, K-ATPase in Om gill epithelium of rainbow unm, Oneorhynchas mykiss. Cell Tissue Res, 283:461-468. 669.Wood C.M., Bergman H.L, Laurent P., Maina J.N., Narahara A. and Walsh P.J. (1994). Urea producfran, acid-base regulation and their interactions in the Lake MMagadi Tilapia, a unique teleost adapted to a highly alkaline environment J. Exp. Biel, 189:13-36. V 176 670.Yadav A. N. and Singh B. R. (1989). Gross structure and dimensions of the gill in an air- breathing estuarine goby, Pseudapocryprer lanceofatur. Japan. J. Ichthyol., 36: 252-259. 671.Yadav A.K., Lal B., Verma N., Bhattacharya N., Khare S. and Singh H. (1983). Effect of Hypertonie Saline Medium on Gill mucous cell density and serum electrolytes concentration in the catfish, Clarias batrachnr. Life Sci. Adv, 2:151-153. 672.Yadav A.N., Prasad M.S. and Singh B.R. (1990). Gross smucture of the respiratory organs and dimensions of the gills in mudskipper, Per(aphihalmoson.sehlosseri. J. Fish Dial., 37: 383-392. 673.Yedav B. N. (2006). Food, feeding habits and alimentary canal, In: Fish & Fisheries, Daya Publication [louse, p 22-40. 674.Yamamotu M and Hirano T. (1978). Morphological changes in the esoplageal epithelium of the eel, Angrillajaponica, during adaptation to seawater. Cell Tiss. Res., 192:25-38. 675.Yamamoto T. (1966). An electron microscopic study of the columamepithelial cell in the intestine of fresh water teleosts goldfish (Carassius aurcivs) and rainbow trout (Salmo ir(deus). 2. Zekkfirsch., 72:66-87. 676.Yumamoto T., Kuwait K. and Oshima S. (1011). Distribution of mucous cells on the body surface of Japanese flounder Paratichthys olivaceus. J. Fish Biol., 78: 848-859. 677.Y10 X. and Forte J. G. (Z003). Cell biology of acid secretion by the parietal cell. Annu. Rev. Physiol., 65; ]03-131. 678.Yashpal M., Kumari U., Mittal S. and Mittal, A.K. (2006). Surface architecture of the mouth cavity of a carnivorous fish Rha rim (I lamilton, [ 822) (Siluriformes, Bagidae). Belg.J. Zool. 136 (2), 155-161. 679.Yasugi S. (1997). Pepsinogen-like immunoreactivity among vertebrates: occurrence of comrnon antigenicity to an anti-chicken pepsinogen antiserum in stomach gland cells of vertebrates. Comp. Biochem. PhysioL, 86B: 675-680. 680.Yasugi S., Matsunaga T. and Mauna T. (1988). Presence of pepsinogens immanorcac[ive to anti-Cmbryonio chicken pcpsinogea antiserum in fish stomachs: possible anccshal molecules of chymosin of higher vertebrates. Comp. Biochem. Physiol., 91A: 565-569. 681.YasutakeW. T. and Wales J. H. (1983). Microscopic Anatomy or Salmonids. An Atlas. Academic Press, US Department of Interior, Fish Wildlife Services No. 50, Washington DC, USA. 682.Yeshikawa J.S.M., McCormick D., Young G. and Bern H.A. (1993). Effects of salinity on chloride cells and Na*, K'-ATPasc activity in the teleost Gillichthys miiabilis. Comp. Eiocbem. Physiol. A Mol. Integr. Physiol., 105:311-317. Z 683.Zaccone C. (1972). Comparative histoch-mical investigation on the mucous cells of the branchial epithelium of Mugil cephalus, and Anapticithyas jordani. Acta Histoclsem., 44: 106-115. 684.Zaccone G. (1973). Morphochemical analysis of the mucous cell, during the development of the respiratory tract in Millienisia sphenops. Acts Histoshem., 47: 233-243. 685.Zaccone G. (1981 a). Effect or osmotic stress on the chloride and mucus cells in the gill epithelium of the freshwater teleost Darbus filamentosus (C,pniuifommes, Pisces). A structural and histochemical study. Acta Histochem, 68: 147-159 686.Zaccone G. (19Rtb). Studies on the structure and Histochemistry of the epidermis in the Air breathing teleost Masmcembelus armatus (Mastacembelidae, Pisces) Z. Mikrosk-ant. Forsch. Leipzig., 95(5s): 809-826. 687. Zaccone C. (1983). Histnchemical studies of acid proleoglycross and glycoproteins and activities of1rydrolytic and oxidoreduelive enzymes in the in epidermis of the fish Bferrnius,runguinn1enrus Pallas (Teleostei, Blenaiidae). Histochemisby,76: 163-75. 688.Zaccone C., Fasulo S. and Aiuis L. (1994). Distribution patterns of the parzneuronal endocrine cells in the s[dn, gills and the airways of fishes as determined by immumhistochemical and histological methods. Histochem. J., 26:607629. 689.Zaccone G. and Licata A. (1981). Histoenzymorphology and fine structure of the ionocytes in the head epidermis of a marine teleost Xyrichrhys novacuta L. (Labridae, Pisces), Arch. Biot, (Bruxelles), 92:451-465. 177 690.Zayed A, E. and Mohamed S. A. (2004). Morphological study on the gills of two species of fresh water fishes: Oreociuromis niloticus and Clcrias gariephnus Ann. Anat., 186: 295-304. 691.7.hu Y. and Q. Zliang (1993). On feeding habits and histological structure of digestive tract of the Mudskipper, BcIeophtha!rHus pectrnirostris, in lnterfidai Zone of Jiufeng River estuary. L Ocean. Taiwan, 12:225-232. 692.Zihler F. (1982). Gross morphology and configuration of digestive tracts of Ciclilidae (Teleostei, Perciformes): phylogenetic and functional significance. Neth. 3. Zool., 32:544-5'11. 693.Zimmer C., Reuter C. and Schauer R. (1993). Use of influenza c-virus for detection of 9- Oacetylated sialic acids on immobi:ized conjugates by esterase activity. Eur. J. Biochem., 204: 209-215. 694.Zadunaisky J. A. (1984). The chloride cells: the active transport of chloride and paracellular pathways., In. Fish Physiology. (Eds), Hoar W.S. and Randall D.J., XB New York: Academic., p.103-172. 695.Zuchelkowski E. M. Lantz R C. and Hinton D.E. (1986). Skin mucous cells response to acid stress in male and female brown bullhead catfish, Ictalurus nehsiosus (LeSueur). Aquut. Toxicel., 8:139-148. 696.Zuchetkowski E. M., Lantz R. C. and Hinton U.E. (1981). Effects of acid-stress on epidetmal mucous cells of the brown bullhead Icruluns nebulurus (LeSueur): A rnorphomenic study. Ana[, flee., 20033-39. 697.Zuehelkowski E.M., Pinlutntf C.A. and Hinton D.E. (1985). Mucc,substance histochemistry in control and acid-stressed epidermis of brown bullhead catfish, Ictafurus nehulasur (LeSueur). Anat. Rec., 212:327-335. 698.Zydlewski J., and McCormick S. D. (7A01).Developmeuta] and Environmental Regulationof Chloride Cells in Young American Shad, Alosa sapidissirna. I. Exp. Zeal., 290:73-87. 178 Appendix -I (A) Immunohistochemistry Reagents: (a) Paraformaldehyde • Make 0.2 M phosphate buffer (pH 7.2) out of the stock solution given in (b) below solution A • 8% parafonnatdehyde in DDW — solution B • Mix these solution A and B in equal volume — solution C • Adjust the pH of solution Cat 7.2 by adding 2 N HCl slowly — solution D • Solution D is 4% paraformaldehyde in 0.1 M phosphate buffer which is used as fixative. (b) Phosphate Buffer Saline (PBS) 1M, pH 7.2: Stock: 20x (0.2 M pH 7.2) for 100 ml (a)Na2PO4 (Anhydrous) 2.18 got (b)NaHPO4.2H10 0.83 gat (c )NaC1 1.8.0 gm Dissolve in DDW and adjust the pH to 7.2 with NaOH solution DiLute 1:10 with DDW before use and adjust puI if necessary. (c) Triton X 100 (0.5%): store at 4°C Add 5O0µ1 of Triton X I00 and make up the volume to 100 ml of D.1 M PBS (pH 72) (d) Bloeldog Solution/ Bovine Serum Albumin: 100 ml PBS + 5ml Bovine serum (Sigma, B-4287) + 0.08% sodium Azide (NaN.) + D.5% Triton X100, mix on vortex. (e) ABC Peroxidase Take 1µI of streptavidin peroxidase diluted in proportion of 1:100 in PBS just prior to use. (f) Di-Amino Benzidine (DAB) Dissolve SDmg DAB powder in 2 m] DDW and the solution is aliquoted in 200pl tubes and stored at 4°C (stock). Working solution: 1 ml Iris buffer + 5glDAB+ 7g1 11z02 (1:10) (Mix ild H,02 in 10µI of Iris buffer (0.D5M)). (g) Mounting media 25 ml glycerol + 50 nil 0.5M PBS + 25 rut DDW VI[ (B) Fixatives: 2. Carney's fluid (100 ml) Absolute alcohol 60 ail Glacial acetic acid 20 ml Chloroform 20 ml b. Bouin's fluid(100 ml) Saturated Picric acid 75 ml Formaldehyde 20 ml Glacial acetic acid 5 not c. Formal calcium fixative: (For Acid hematein method) 40% Fomtalin 10 ml 10% CaCh 10 all Distilled water 80 ml d. Champy Maillet Fixative/ Osmium Zinc Iodide (OZf: (I) Zinc Iodine solution (5U ml), (to prepare right before) Zinc powder 250 gm Iodine 1.25 gm DDW 50 ml First, dissolve the Iodine-1.25 g in DDW add 2-3 drops HC1 (because iodine is not dissolve easily) and add 2.5 gm zinc powder, mix on magnetic stirrer, and filter through filter paper. Finally, yellow colour zinc iodine solution was obtained) (ii) 2% Osmium tetraoxide solution Mix 40 :nl zinc iodine solution with 10ru1 2°/ Osmium tetraoxide (C) Stains and Solutions: i. Dicromate calcium (100 ml) Potassium dicluvmate 5 gm CaCl2 (anhydrous) I gm DDW 100 ml ii. Acid Hematein Add 0.05 g hematoxylin in 48 ml DDW and exactly 1 ml 1% NaIO4 was mixed and heat until the water bail, cool it and add 1 ml glacial acetic acid. WIl iii. Borax- ferricyanide Potassium ferricyanide 0.25 gm Sodium tetraborate. 101120 0.25 gm DDW 100 ml This solution should be kept in dark iv. Harris heanrotoxvlin Haematoxylin 0.50 gm Aluminum ammonium sulphate 20.00 gm Mercuric oxide 0.50 gtn DDW 100 ml Boil the hematoxylin and aluminum ammonium sulphate in DDW and add the mercuric oxide. Prepare the stain at least one week before. v. Weigert's Iron heamotoxylin Solution A Ilematoxylin crystals I gm Alcohol 95% 100 ml Solution B Ferric chloride (29% aqueous) 4 ml DDW 95m1 Cone. I ICL I ml Mix the equal part of Solution A and Solution B vi. Gomori Solution KMn0y 0.30 gin Con. H2SO4 0.30 ml DDW 100 ml vii. Aldehyde fuchsin Basic Fuchsia 0.50 gar 70°%o Ethanol 100 ml Paraldehyde 175 ml Cone. HCI 1.25 ml Kept in 37°C in oven in early morning and will be ready by noon of succeeding day. viii. Biebrich scarlet-acid fuchsin solution (IOOnd) Biebrich scarlet (1% aquous) 90 HE Acid fuchsin (1% aquous) LO mL Glacial acetic acid I ml ix ix. Phosphomolybdic-phosphotungstic acid solution Phosphomolybdic acid 5g Phosphotungstic acid 5g DDW 200rnl x. Aniline blue solution Aniline blue 2.5g Glacial acetic acid 2m1 DDW 200m1 xi. Schiffs reagent Basic fuchsin 1 N-HCl 20ml Sodium metabisulphite 1 g Activated charcoal 2g DDW 200m1 Dissolve basic fuchsin in boil DDW, shake and cool to exactly 50°C, filter and add N-HCI, cool to 25°C and add Sodium metabisulphite. After standing the solution in dark for overnight, add activated charcoal, shake l min, filter and kept in dark at 4°C.