J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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THE STRUCTURE AND CHEMICAL COMPOSITION OF THE BODY WALL OF -PARASITIC

Jatinderpal Singh

Department of Zoology, Baring Union Christian College, Batala-143505 Punjab (India) Email: [email protected]

ABSTRACT The body is enclosed by a body wall comprising of three different layers. The outermost being a thick multilayered cuticle underlined by the middle hypodermal layer pronounced at four regions in the form of dorsal, ventral and a pair of lateral hypodermal cords. The innermost layer is constituted by a single layer of longitudinal muscle cells. Although the outer envelope of nematodes has been studied by a host of workers yet the controversy regarding certain aspects of its structure, chemical composition and function still prevail. The presence of proteins, carbohydrates, lipids, acid mucopolysaccharides and a variety of enzymes have been detected by various workers in different of larval and adult nematodes. Since the mere survival of a nematode parasite in the body of its host is mainly attributed to the uniqueness of its body covering, hence the study of its nature and chemical composition is of paramount importance in parasitic nematology. The present paper briefly reviews the histological and histochemical studies on the body wall of animal nematodes parasitic .

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J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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INTRODUCTION two-layered cuticle characterized the adult females of the family Heterodidae ( Nematodes are the most numerous and Rogers, 1965; Kampfle, 1966) and the multicellular animals present on the earth parasitic larvae of Trichinella spiralis (Maggenti, 1981). They parasitize man, (Beckett and Boothroyd, 1961). This two- livestock, crops and have a deleterious layered structure was believed to be due to effect on all. Not only is the economics of rapid periods of growth at some stages in nematode important but nema these animals. However, in pre-parasitic themselves are a fascinating subject for the larvae and males of Heterodidae and in the study of morphology, biology and host- adults of Trichuroidae the normal three- parasite relationship. Morphologically the layered cuticle prevailed (Sheffield, 1963 Nematode is an exceedingly variable and Wright, 1968). group and there hardly exists any statement that could be made regarding RESULTS AND DISCUSSION their histomorphology, which would apply to all forms. Different types of anatomical Structurally, the nematode cuticle is quite features are present in different species of diverse, not only in different genera and parasitic nematodes which are the result of families, but also within species. There adaptation influenced by their particular may even be marked differences in the type of habitat (Chitwood and Chitwood, cuticle of male and female worms and in 1950). the different body regions of the same individual. These differences in the cuticle Some recent studies on the body wall of can be more marked in parasitic animal parasitic nematodes have been nematodes. ( Cano-Martil et al, 2006). performed by Johal (1995), Johal and In Oxyuris equi, the cuticle Shivali (1996), Page (2001), Cano-Martil consisted of an outer and an inner cortical et al (2006), Rahemo and Hussain (2009) layers, a fibrillar layer consisting of two and Lalchhandama (2010). The distinct layers of fibres, a homogenous multilayered nature of the cuticle had long layer and two distinct layers of fine fibres, been established with the number of layers a homogenous layer and two fibre layers ( varying from two in the larva of Martini, 1912). Bird (1958) studied the Trichinella spiralis (Beckett and cuticle of Oxyuris equi and described an Boothroyd 1961) to eight in adult Oxyuris additional fibrillar layer under the cortical equi (Bird, 1958). Since then there has layer which was not mentioned by Martini been a general agreement on the basic (1912). The cuticle of O. equi, thus terminology for the nematode cuticle as a comprised eight layers, starting from the three-layered structure consisting of outermost layer inwards: (1) an external cortical, median and basal layers. The cortical layer; (2) an internal cortical layer; nematode cuticle has been (3) a fibrillar layer; (4) a part of comprehensively reviewed from time to homogenous layer; (5) and (6) two outer time by Maggenti (1981), Bird (1980 and fibrous layers; (7) and (8) two inner 1984), Wright (1987), Bird and Bird fibrous layers. There was no indication of (1991) and Page (2001). The nematode presence of any bounding layer or a basal cuticle nearly always consists of at least layer. two major layers, the cortical and the basal. However, an exception to this Lee(1965) reported that the cuticle of general rule was found in the insect adults of Nippostrongylus brasiliensis had parasitic nematode Bradynema spp. where three main layers, an outer cortex, a the normal cuticular structure has been middle matrix and an inner fibre layer. The replaced by microvilli (Riding, 1970). The cortex appeared to be a single layer which

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J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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varied in thickness, being thin at the apices epicuticle ; a narrow electron dense outer of the longitudinal ridges and much thicker cortical layer; an inner cortical layer a between the ridges. The cortex was matrix layer; three fibre layers and an separated from the fibre layer by a fluid inner fibrous basal lamella. The basal filled region, the middle layer. The fibre lamella possessed local areas associated layer was found to be composed of two with hemi-desmosomes on the hypodermal fibre layers and a basement membrane. membrane. The cuticle of the anterior half Using light and scanning microscopy, of the body of female was found to be Anya (1966) observed that the cuticle of similar to the male while the posterior half Aspiculuris tetraptera had three basic was enveloped by seven-layered structure layers namely, the cortex consisting of an consisting of an outer membrane like outer and inner cortical layer, middle layer, a narrow outer cortex, an inner matrix and inner fibre layer which in turn cortex, a thin matrix layer, two fibre layers is made of three layers. In addition, a thin and a fibrous basal lamella. osmiophillic superficial membrane on the surface of the cuticle, discernible only with Based on their ultrastructural and the electron microscope, was reported. cytochemical studies, some authors (Lee, 1965 and Bird, 1980) considered that the Tomita (1975) described the cuticle of outer covering of the cuticle was a “cell Thelazia callipaeda to be composed of two membrane” and referred to it as an layers, an external layer consisting of three epicuticle. Since then the use of terms such sub-layers forming the transverse body as “membrane”, “membrane-like”, striations and an internal layer composed “modified membrane” for different regions of six sub-layers. Franz (1980) made of the cuticle’s surface have created certain scanning and transmission electron confusions and implied various concepts microscopic studies on the cuticle of regarding its composition and function. Onchocerca volvulus and found it to be Zuckerman et al (1979) and Bird (1985) consisting of a cortical, a median and a used the term “glycocalyx” for the basal layer. The outer limitation of the carbohydrate moieties on the outermost cuticle was an irregularily folded surface surface of the cuticle which sometimes membrane, which showed a regular honey appeared as a fuzzy coating. The presence comb pattern and covered the entire worm of carbohydrate moieties and use of the with the exception of anterior and posterior term “glycocalyx” for the surface ends. Lee and Nicholas (1983) used membrane of the cuticle gave rise to the plasma etching to reveal the structure of impression of epicuticle being a cell the cuticle of adult Nippostrongylus membrane. This concept of the outside brasiliensis. The cuticle revealed an outer layer being a cell membrane had been membrane like layer, a single cortical discussed by Wright (1987) who pointed layer, a fluid filled layer, two fibre layers out that the surface structure was likely to and a basement lamella. vary considerably in different nematode species as a result of different functional Martin and Lee (1983) described the and anatomical requirements. To solve this structure of body wall of adult problem and minimize further confusion, Nematodirus battus. The cuticle of the he suggested that, it will be much male Nematodirus battus was divided into appropriate to use the term surface coat for three main layers namely, an outer cortex, the outer carbohydrate component of the a middle layer and an inner fibre layer. cuticle rather than the glycocalyx. He also These three major layers were further suggested that the epicuticle was not a cell divided into eight distinct layers, membrane, however, with studies it could namely,an outer membrane like layer or

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J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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be seen as a surface trilaminate The P-face of the epicuticle outer fracture differentiation containing lipids. plane presented a large number of densely- packed small particles and many Nevertheless, the term epicuticle now protuberances. appears to be accepted for the outside layer. Regarding remaining layers of the Neuhaus et al (1996) investigated the cuticle, there seems to be a consensus that ultrastructure, development and a generalized cuticle would consist of an morphogenesis of the body cuticle of adult epicuticle, a cortical layer, a median layer and juvenile Oesophagostomum dentatum and a basal layer (Bird and Bird, 1991). by light microscopy, scanning electron Fok et al (1992) described the cuticle of microscopy and transmission electron Nippostrongylus brasiliensis as a three microscopy. The cuticle of the first three layered structure and the outer part of the juvenile stages was consisted of a cuticle, the epicuticle, was found to be trilaminate epicuticle, an anormous layer covered with a polyanionic coat, the and a radially striated layer. In the last glycocalyx, which was stained by the juvenile stage and adult worm, the radially anionic dye ruthenium red. Lee et al striated layer was replaced by a fibrous (1993) studied the structure of the cuticle layer with three sublayers of giant fibres of adult Nippostrongylus brasiliensis by and a basal amorphous layer. freeze-fracture technique and transmission Consequently, the body cuticle of adult electron microscopy. The epicuticle Oesophagostomum dentatum revealad cleaved readily to expose E- and P- faces. three basic layers: an epicuticle, a three- The P- face of the cuticle possessed a zoned amorphous layer and a three-zoned small number of particles, similar to intra- basal fibrous layer. membranous particles, whilst the E-face contained a few widely scattered Martinez and Souza (1997) examined the depressions. Despite the presence of these cuticle of the infective third stage larvae of particles the epicuticle could not be Strongyloides venezulensis by transmission considered as a true membrane. Freeze- electron microscopy, ruthenium red fracturing of the remainder of the cuticle cytochemistry and deep-etch techniques. confirmed its structure as described by The cuticle was seen to be consisting of conventional transmission electron five layers: epicuticle, cortical, medial, microscopy. fibrous and basal. The epicuticle had a trilaminate appearance and the surface coat Since then a number of workers have stained with ruthenium red. At the level of authenticated the modern perspective cortical, median and basal layers, regarding the nature and structure of the interconnecting fibres and globular cuticle evidenced by their studies on larval structures were seen. The fibrous layer was stages of various nematodes. Ultra- formed by parallel bars of thick fibrous structural analysis of the cuticle of Brugia elements. Patel and Wright (1998) had malai micrifilarae indicated that it was homologized the different nomenclatures composed of two regions: the inner one of the cuticle given by earlier workers. with a homologous aspect and the outer They divided the cuticle into four layers: one designated as the epicuticle. Further, epicuticle, a combined cortical and median three laminae separated by electon- lucent layer, stratified layer and the fibrous mat regions were seen in the epicuticle (Araujo respectively, corresponding to the terms et al, 1995). Replicas of freeze-fractured surface sheet, cortex mat, striation band micrifilariae showed the presence of two and fibrous mat used earlier by Jackson fracture planes in the epicuticle and no and Bradbury (1970) and the terms fracture in the inner region of the cuticle. epicuticle, cortical, median and basal

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J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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layers respectively described by Kondo to 1:40. Exceptions to this were found in and Ishibashi (1989). Furthermore, Patel large ascarids (1:100) (Bird, 1971) and and Wright (1988) also followed the Ancylostoma (1:10) (Loss, 1905). The nomenclature for defining the different cuticle of Strongylus vulgaris being much layers of the cuticle as a modification of thicker than that of Oxyuris equi and also the commonly accepted three - layer plan. thicker than that of Ascaris lumbricoides The layer described as a basal layer by (Smyth, 1996). Bird and Bird (1991) was referred here as striated layer and fibrous mat. The layer Externally the cuticle revealed a variety of named as fibrous mat was found to be structures which were reviewed by analogous to the structural layer referred Chitwood and Chitwood (1950). These by Maggenti (1981) as the endocuticle. include much prevalent transverse The epicuticle appeared as a closely markings, longitudinal ridges and alae. applied electron dense layer but it is not Structure and pattern of the longitudinal possible to observe the trilaminar structure. ridges were discussed by a number of An indistinct external layer was observed workers: Lee (1965), Lee and Nicholas and this was assumed that this represented (1983), Martin and Lee (1983) and Nembo the surface coat. The cortical and median et al (1993). layers were observed as one homogenous zone. Lassaigne (1843) was the pioneer of performing chemical analysis on the Cano-Martil et al (2006) light cuticle of nematodes and from his microscopic and transmission electron observations, he established the chitinous microscopic studies on the cuticle of adult nature of the cuticle in Ascaris. Diplotriaena tridens and described three Subsequently, the presence of proteins in major zones namely, cortical, medial and the nematode cuticle was also reported. basal zones. This trilayered stratified Based on his extensive studies on the structure is externally covered by a chemical composition of the cuticle of trilaminar epicuticle. Ascaris lumbricoides, Chitwood (1938) come to the conclusion that the cuticle Rahemo and Hussain (2009) studied the consisted of at least five different body wall of galli. The cuticle substances namely, albumin, glucoprotein, was found to be consisting of three layers a mucoid, a fibroid, a collagen and a namely, outer cortex, middle matrix and keratin. inner fibre layer. The fibre layer in turn consists of several layers of dense tissue. The presence of proteins along with traces They also concluded the middle layer of of carbohydrates and lipids had been the cuticle as a homogenous layer without reported in the cuticle of various nematode any struts. The cuticle of Ascaridia galli species by a host of workers: Bird and was found to be a complex layer of Rogers (1956), Beams (1964) and Anya proteinaceous rings forming a hard and (1964 and 1966). In Toxascaris leonine, thick unit and is composed of several Johnson (1968) observed the presence of discrete concentric layers running proteins in all the layers except the fibrillar continuous around the body layer and the maximum concentration (Lalchhandama, 2010). being in the cortical layer. Kan and Davey (1968) while working on Phocanema Although the cuticular layers vary in decipiens indicated that the rich protein thickness but the average ratio of thickness content of the outer cortex was responsible of the cuticle to the diameter of the for the high resistance offered to the nematode was considered to be about 1:30 passage of host enzymes in parasite body.

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Jenkins (1970) observed the proteins with Carbohydrates were observed in the –SS- and –NH2 bound groups in different cortical as well as fibrillar layer of the cuticle layers of Trichuris suis, Singh and cuticle in Toxascaris leonina by Johnson Khera (1972) attributed a collagenous (1968). Gupta and Kalia (1978) reported nature to the cuticle of Dracunculus that the polysaccharides with 1:2 glycol medinensis. The presence of different group together with glycogen were present groups of proteins in all the seven layers of in high concentrations in the matrix as well the cuticle was observed by Lestan and as in fibrillar layers of Setaria cervi. The Dubinsky (1973) in Porocaecum. The presence of acid mucopolysaccharides on presence of –SS- group was depicted in the the surface of cuticle was observed in outer layer, collagen in the fibrillar and Diplotriaena tricuspis (Wajihullah and basal layers and elastin in the median Ansari, 1981), Oesophagostomum layer. The presence of –SS- and –SH columbianum (Johal, 1988) and Toxocara group bound proteins was also reported in canis (Page et al, 1992). Majumdar et al the inner cortical layers of Enterobius (1996) described the presence of hexose vermiculuris by Hulinska and Helinsky containing mucosubstances in the (1973).In Paranisakis kherai, the cuticle epicuticle- exocortex of Ascaridia galli. was found to be rich in proteins bound Concentration of lipid was reported with –SH, -SS- and -NH2 groups, which by Gupta and Garg (1976) in the matrix were distributed in moderate quantity in and fibrillar layers of the cuticle in the outer and inner cortical layers and in Paranisakis kherai. Prasad and Guraya high concentration in the matrix layer (1977) reported that the bound lipids were (Gupta and Garg, 1976). A substantial present in the outermost surface layer of amount of protein (collagen) was also cuticle and bound as well as free lipids in observed in the outer cortical and fibrillar the outer cortical layer in Ascaridia galli. layers of the cuticle of Pseudoproleptus Only a small amount of lipid was reported kherai by Sheikher and Garg (1979). in the cuticle of Tanqua anomala (Kankal, Ouazana (1982) stated that the collagen 1989). On the other hand, Majumdar et al formed the major constituent of the (1996) detected a substantial amount of nematode cuticle. Johal (1988) too, lipid in the epicuticle- exocortex and a described the presence of collagen like moderate amount of the same in the fibres in the basal layer of the cuticle of median zone of the cuticle of Ascaridia Oesophagostomum columbianum. galli.

By using immunostaining technique, The metabolically active nature of the Takahashi et al (1993) studied the cuticle was determined by the presence of ultrastructural localization of enzymes such as esterase in the cuticle of phosphorylcholine (a hapten) from the Ascaris by Lee (1962). Localization of inner layers of the body cuticle of esterase along with ATPase and RNA in Trichinella spiralis. Majumdar et al (1996) the nematode cuticle (Anya, 1966) further reported that the cuticle of Ascaridia galli authenticated that cuticle is a site for the was basically proteinaceous with a rich synthesis of cuticle enzymes. The presence quantity of elastin but poor quantity of – of a number of enzymes viz. esterase, acid NH2 bound proteins. Some tyrosin phosphatase and adenosine triphosphatase containing proteins were also located in was recorded in the cuticle of Trichuris the epicuticle region. The presence of suis and Haemonchus contortus by Jenkins chitin in the pharyngeal cuticle of (1970) and Sood and Kalra (1977). Oesophagostomum dentatum was detected by Neuhaus et al (1997). Anya (1966) was able to detect some RNA activity in the inner cortical of Aspiculuris

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J.Bio.Innov 1(6), pp: 250-263, 2012 ISSN 2277-8330 (online)

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tetraptera, Syphacia obvelata and Ascaris extensive accumulation of glycogen was lumbricoides. RNA in the matrix layer of observed in the hypodermis by Endo and the subcuticle of Diplotriaena tricuspis Trpis (1997). Presence of lipids in the and inner cortical layer of hypodermis of Trichuris ovis, Setaria Oesophagostomum columbianum was cervi, and Oesophagostomum reported by Wajihullah and Ansari (1981) columbianum was reported by Jenkins and Johal (1988) respectively. (1970), Gupta and Kalia (1978) and Johal (1988) respectively. The localization of The basic nature of nematode keratin, diffused phospholipids and hypodermis was described by Martini scattered lipids was recorded in (1903). A syncytial hypodermis was Dracunculus medinensis (Singh and reported by Loss (1905), Martin and Lee Khera, 1972) and Ascaris (Reznik, 1971). (1983), Neuhaus et al (1996), Rahemo and An appreciable amount of proteins was Hussain (2009) and Lalchhandama (2010) also observed by Johal (1988) in the lateral in Ancylostoma, Nematodirus, Ascaris and hypodermal cords of Oesophagostomum Ascaridia. Presence of three rows of nuclei columbianum. Proteins contained in the in the lateral and one to three in the haemoglobin were reported by Smith and median cords was reported by Hyman Lee (1963) and Okazaki et al (1967) in the (1951) and she also described that this hypodermal columns of Ascaris number is subjected to variation in lumbricoides. different species. However, Chitwood and Chitwood (1950) attributed a cellular Yanagisawa and Maki (1978) described nature to the hypodermis as they observed that certain species of nematodes revealed the presence of two sublateral rows of the presence of enzyme acid phosphatase unicellular hypodermal glands situated in in high concentration in the hypodermis. the lateral hypodermal cords opening by Acid phosphatase in the hypodermis was short ducts through pores in the cuticle in also detected by Sood and Kalra (1977). some species of nematodes. Johal (1994) The metabolic activity of hypodermis was reported the presence of large hypodermal established by the presence of RNA in gland cells, having prominent nuclei, Oesophagostomum columbianum (Johal, constituting the lateral hypodermal cords. 1988). These cells were found to communicate with the overlying cuticle by a large pore Schneider (1860) performed an exhaustive present in the centre of the cord. Neuhaus study on the somatic musculature of et al (1996) reported the attachment of nematodes and proposed descriptive hypodermis to the cuticle via nomenclature to each different category hemidesmosomes. Electron dense fine seen. He classified the muscles into three fibrils were reported to be extending from types based on the shape of the muscle the hemidesmosomes a short distance into cells, namely, Platymyarian (flat type of the basal amorphous layer of the cuticle. cells with fibres on the base), Von Kemnitz (1912) in Ascaris and coelomyarian (V- shaped type of cells with Martini (1916) in Oxyuris reported that fibres extending on lateral walls) and glycogen was stored in considerable circomyarian (cells in which fibres quantity in the hypodermal layer of the encircle the cytoplasm). Later, the same body wall. The presence of both glycogen author (1866) gave the classification of and fat in the lateral hypodermal cords of muscle cells according to the number of various nematode species have been rows present in each hypodermal area and reported by Fairbairn (1957), Anya (1964) introduced the terms, holomyarian (with and Singh and Khera (1972). In the no or two rows), meromyarian (two to five infective larvae of Onchocerca volvulus, rows) and polymyarian (large number of

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rows). Although this nomenclature has no third stage larva of Onchocerca volvulus. taxonomic significance and was simply Besides glycogen, Gupta and Kalia (1978) one of the convenience, but it is still not and Wajihullah and Ansari (1981) discredited. In Nippostrongylus described the presence of brasiliensis, Jamuar (1966) and mucopolysaccharides too, in the muscle Lalchhandama (2010) described that each cells of Setaria cervi and Diplotriaena muscle cell consisted of two regions, a tricuspis. Protein is regarded as main distal fibrillar zone and a sarcoplasmic constituent of the contractile part of zone containing mitochondria and nucleus. muscle cells in nematodes. In Toxascaris Orientation of muscle cells was described leonina, Johnson (1968) stated that the by Maggenti (1981), who concluded that contractile part of the body wall was found the muscles aligned anywhere from to be richest one as far as the amount of directly parallel to some 45 degree off the proteins was concerned. The presence of parallel. When parallel the cells were an appreciable amount of proteins was also staggered like bricks so that a spiral reported in the muscle cells by Jenkins around the pseudocoel was produced. (1970) in Trichuris suis and Johal (1988) Complex twisting, bending and rotational in Oesophagostomum columbianum. movements in nematodes is the outcome Kankal (1989) described that the protein of this orientation. The presence of formed the chief structural component of desmosomes for the attachment of the musculature of Tanqua anomala. coelomyarian muscles of Nematodirus Gupta and Garg (1976) and Johal and battus to the hypodermis was reported by Shivali (1996) detected the presence of Martin and Lee (1983). Large lipids from the fibrillar zone of muscle concentrations of glycogen granules in the cells. non-contractile part of muscle cells were REFERENCES reported by Hirsch and Bretschneider (1937). Lee (1960) stated that the amount Anya, A. O., The distribution of lipids and of glycogen in the muscle cells in the glycogen in some female oxyuroids. anterior region was less than that found in Parasitology, 54 : 555-566, 1964. the posterior region. Anya (1964) detected the presence of glycogen in non- contractile part as well as myofilaments of Anya, A. O., The structure and chemical the muscle cells in some female oxyurids. composition of nematode cuticle: Wajihullah and Ansari (1981) in Observation on some oxyuroids and Diplotriaena tricuspis, Johal (1988) in Ascaris. Parasitology, 56: 179-198, 1966. Oesophagostomum columbianum and Takahashi et al (1988) in Trichinella Araujo, C. G., Ana, Figueredo-Silvia, J., spiralis established that glycogen is concentrated mainly in the sarcoplasmic Souto-Padron, T., Dreyer, G., Noroes, J. zone of muscle cells. Kankal (1989) and De Suza, W., Scanning electron detected glycogen in both the contractile as microscopy of the adult Wuchereria well as non- contractile parts of muscle bancrofti (Nematoda: Filarioidea). Journal cells in Tanqua anomala. Storey and of Parasitology, 81 (3): 468-474, 1995. Ogbogu (1991) observed that though glycogen was the main constituent of the Beams, C. C., Phospholipids of Ascaris muscle cells of adult Litomosoides carinii but the same was found to be absent in the lumbricoides with special reference to larval muscle cells. Endo and Trips (1997) fatty acids and aldehydes. Experimental detected extensive accumulations of Parasitology, 15: 387-396, 1964. glycogen in the muscles of the infective

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