In Angelfish Pterophyllum Scalare

Home , Pterophyllum, Pterophyllum scalare

Ben-Gurion University of the Negev Jacob Blaustein Institutes for Desert Research Albert Katz International School for Desert studies

Swimbladder non-inflation (SBN) in angelfish Pterophyllum scalare:

Histological characterization and the role of environmental factors

Thesis submitted in partial fulfillment of the requirements for the degree of

"Master of Science"

By: Shira T. Perlberg

Under the Supervision of

Dr. Dina Zilberg1 and Dr. Arik Diamant2

1Albert Katz Department of Dry Land Biotechnologies

Ben- Gurion University of the Negev, Sde Boqer

2National Center for Mariculture Israel Oceanographic

Limnological Research, Eilat

Authors Signature…………………………………. Date…………..

Approved by the Supervisors……………………… Date…………..

Approved by the Director of the school………….. Date…………..

Ben-Gurion University of the Negev Jacob Blaustein Institutes for Desert Research Albert Katz International School for Desert studies

Swimbladder non-inflation (SBN) in angelfish Pterophyllum scalare:

Histological characterization and the role of environmental factors

Thesis submitted in partial fulfillment of the requirements for the degree of

"Master of Science"

By: Shira T. Perlberg

March 2006 I

Swimbladder non-inflation (SBN) in angelfish, Pterophyllum scalare: histological characterization and the role of environmental factors.

By: Shira T. Perlberg

This thesis is in partial fulfillment for the degree of Master of Science in Ben-Gurion

University of the Negev, The Jacob Blaustein Institutes for Desert Research, Albert

Katz international School for Desert Studies, 2006.

Failure to inflate the swim bladder has been regarded a major obstacle in the rearing of many fish species. Recently, a large-scale phenomenon of swim bladder non-inflation

(SBN) was reported in angelfish, Pterophyllum scalare, grown at a commercial ornamental fish farm in the Arava Valley, Israel (Zilberg et al. 2003). Failure to inflate the organ in the first two weeks post hatching resulted in swim bladder atrophy, dysfunctional buoyancy control, body deformities and decreased growth rates. A three- year survey was conducted in order to monitor SBN prevalence in the farm. The survey revealed no pattern for SBN occurrence in the farm and no relation to seasonality. To test the effect of environmental factors on SBN, eggs were hatched under different ambient conditions and larvae were examined under a light microscope for the presence of inflated swimbladders at 12 days post hatch (p.h.). Access to the air-water interface, feed concentration, delayed initiation of feeding (7th instead of 5th day p.h.), water conductivity (range of 110-1,015 μs) and tank coloration had no observable effect on

SBN. Methylene Blue (MB), a fungicide routinely prophylactically employed to treat eggs, significantly increased the prevalence of SBN at a concentration of 5 ppm (28% II vs. 9% in the control). Methylene blue is suggested to have a teratogenic effect on angelfish larvae at these concentrations. For histological characterization, larvae were sampled at 0 to 12 days p.h.. During normal development, the primordial swimbladder was first discernable at the end of the 1st day p.h as a primordium of epithelial cells with a central lumen surrounded by a sheath of connective tissue. Initial inflation occurred on the 4th day p.h. before initiation of feeding (day 5). Prior to inflation, the swimbladder epithelium consisted of an internal layer of columnar cells and an external layer of squamous cells. The columnar epithelial cells were filled with an amorphous material of unknown nature in their basal region and their nuclei were situated apically.

A pneumatic duct was not observed, suggesting that angelfish are physoclists.

A primordial rete mirabile was present on the ventral part of the swimbladder, extending towards its posterior end. Upon inflation, the swimbladder epithelium transformed into a cuboidal to squamous form, as the lumen swelled with gas and the amorphous material disappeared. A model for the role of the amorphous material in normal inflation is suggested. Abnormal swimbladders were apparent from the 4th day post hatch. Three types of abnormalities were identified: 1) swimbladders with little or no amorphous material in the epithelial cells; 2) swimbladders with folded epithelium; 3) swimbladders with disorganization of the epithelium and little or no amorphous material. In larvae that failed to inflate their swimbladders after the 4th day, the epithelial cells proliferated in a disorganized manner and the uniform monolayer lost its regular appearance. Histologically, non-inflated swimbladders in fish hatched in 5 ppm

MB were similar to abnormal swimbladders in fish raised under normal conditions (0.5 ppm Methylene blue). Understanding the way MB inhibits swimbladder inflation could shed light on SBN in angelfish.

III

Acknowledgement

I would like to thank my supervisors Dr. Dina Zilberg and Dr. Arik Diamant for their moral support and professional guidance along the way. I couldn’t have picked better companions for this journey.

I would also like to thank Dr. Rivka Ofir, for assisting with the more molecular point of view in the research.

I would like to thank Shaul and Monik Hareel, Nitsan and Janta from “Negev Angels” fish farm, for their great work on the survey and wonderful cooperation. I hope this work will be useful for you in the future.

I would like to thank Tamar Sinai, Shai Abutbul, Marcia Pimenta Leibovitz and Claudia

Sanabria for all the help with the experiments in the lab in Sde boker, and the administrative staff of the school: Dorit Levin, Ilana Saller and Dina Fiengold for making things so much easier.

A special thanks to all the people in the lab in Eilat: Barbara and Angelo Colorni,

Sharon Ram, Gilaad Hienish and Asaf lipshitz for hosting me for 4.5 month and making me feel at home.

Most of all, I would like to thank my amazing family, the Perlbergs:

My father and mother- for helping me look at things in the right proportions and giving me so many advices. I have learned so much from you both.

My brothers and sisters for always being there when I needed them.

And finally, my grandmother- for inspiring me to pursue my own dreams. IV

Table of contents

Page Abstract…………………………………………………………………………….. I Acknowlegement…………………………………………………………………… III Table of contents…………………………………………………………………… IV List of tables………………………………………………………………………... V List of figures………………………………………………………………………. V

1. Introduction…………………………………………………………………... 1 1.1. Architecture and function of the adult teleost swimbladder………………….... 1 1.2. Initial swimbladder inflation…………………………………………………… 3 1.2.1. Mechanism of initial inflation………………………………………………. 3 1.2.2. Timing of initial inflation…………………………………………………… 5 1.3. Swimbladder non-inflation in teleosts………………………………………….. 7 1.3.1. Factors that were reported to affect SBN...…………………………………. 8 1.4. Angelfish Pterophyllum scalare………………………………………………... 12 1.4.1. Swimbladder non-inflation in Angelfish……………………………………. 13 1.5. Research objectives……………………………………………………………... 14 2. Materials and methods………………………………………………………… 15 2.1. Fish hatching and rearing protocol in “Negev Angels” commercial fish farm…. 15 2.2. Field survey of SBN prevalence………………………………………………… 15 2.3. Fish experimental system……………………………………………………….. 16 2.4. Effect of environmental factors on the prevalence of SBN in angelfish………... 16 2.4.1. Access to the air-water interface…………………………………………….. 17 2.4.2. Effect of methylene blue (disinfectant) concentration on SBN……………… 18 2.4.3. Effect of food concentration and starvation on SBN………………………… 18 2.4.4. Effect of water conductivity (salinity) on SBN……………………………… 19 2.4.5. Effect of tank coloration on SBN……………………………………………. 19 2.5. Histological characterization of normal and abnormal swimbladder development in early larval stages…………………………………………………………….. 19 2.5.1. Normal swimbladder development………………………………………….. 19 2.5.2. Abnormal swimbladder development……………………………………….. 21 2.6. Statistical analysis………………………………………………………………. 22 3. Results………………………………………………………………………….. 24 3.1. Survey of SBN prevalence in a commercial angelfish farm during 2003-2005… 24 3.2. The effect of environmental factors on SBN in anglefish……………………… 25 3.3. Histological characterization of normal and abnormal swimbladder development in early larval stages…………………………………………………………….. 26 3.3.1. Normal swimbladder development in anglefish…………………………….. 26 3.3.2. Abnormal swimbladder developmet under routine conditions……………… 40 3.3.3. Abnormal swimbladder development in larvae hatched in 5 ppm methyelene blue…………………………………………………………………………... 44 4. Discussion………………………………………………………………………. 55 5. Further research……………………………………………………………….. 71 6. Reference……………………………………………………………………….. 72

V List of tables Page Table 1. Number of eggs per replicate used in each experiment…………………… 17 Table 2. The effect of environmental factors on SBN/live fish……………………. 25 Table 3. Summary of characteristics of different abnormal swimbladder types appearing on the 4th day p.h……………………………………………… 47

List of figures Page

Figure 1. A net box used to prevent the access to the air-water interface………… 18 Figure 2. Mean prevalence of SBN from live fish per month (%) in “Negev angels” commercial fish farm, during 2003-2005 ……………………………… 24 Figure 3. Larvae of angelfish Pterophyllum scalare at day 0 p.h…………………. 32 Figure 4. Larvae of angelfish Pterophyllum scalare at day 1 p.h…………………. 33 Figure 5. Larvae of angelfish Pterophyllum scalare at day 2 p.h…………………. 34 Figure 6. Larvae of angelfish Pterophyllum scalare at day 3 p.h…………………. 35 Figure 7. Larvae of angelfish Pterophyllum scalare at day 4 p.h…………………. 36 Figure 8. Larvae of angelfish Pterophyllum scalare at day 8 p.h…………………. 37 Figure 9. Electron-microscopic overview of the different layers of non-inflated swimbladders on the 4th day p.h…………………………………………. 38 Figure 10. Electron-microscopic view of different areas in an inflated swimbladder on the 4th day p.h………………………………………….. 39 Figure 11. Abnormal swimbladders in larvae of angelfish Pterophyllum scalare at day 4 p.h………………………………………………………………. 42 Figure 12. Abnormal swimbladders in larvae of angelfish Pterophyllum scalare at day 6 p.h and day 8 p.h……………………………………………….. 43 Figure 13. The frequency of swimbladder types from total non-inflated swimbladders in larvae exposed to 5 ppm methylene blue, on 4th day p.h ……………………………………………………………. 46 Figure 14. Schematic illustration of different abnormal swimbladder types from the 4th day p.h………………………………………………………………... 47 Figure 15. Larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue at day 1 p.h……………………………………………… 49 Figure 16. Larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue at day 2 p.h……………………………………………... 50 Figure 17. Larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue at day 3 p.h……………………………………………… 51 Figure 18. Non-inflated swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 4 p.h………………………….. 52 Figure 19. Non-inflated swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 7 p.h………………………….. 53 Figure 20. Electron-microscopic overview of non-inflated swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 7 p.h. and 9 p.h…………………………………………………… 54 Figure 21. Mechanism of methemoglobin reduction by MB……………………….. 59 Figure 22. The inhibiting effect of MB on vasodilation via the NO/c-GMP and PGI-2/c-AMP signaling pathways……………………………………… 60 Figure 23. A model of the changes in epithelial cell morphology appearing during normal swimbladder development in angelfish, Pterophyllum scalare… 65 1

1. Introduction

Except for typical bottom dwellers most freshwater teleosts posses a swimbladder. The swimbladder acts as a hydrostatic organ by replacing up to 8% of the fish’s body with gas (5% in sea water) and reducing the overall density of the fish’s body towards that of the water. The fish achieves neutral buoyancy and is able to maintain its position in the water with less effort (Steen, 1970). In most fishes, the swimbladder develops as a simple evagination of the alimentary canal (Boulhic and

Gabaudan, 1992; Govoni and Hoss, 2001; Johnston, 1953; McEwen, 1940; Steen, 1970;

Trotter et al., 2004). Fishes, which retain a connection between the swimbladder and the digestive tract (pneumatic duct) as adults, are called “physostomes”, while fishes that have no connection as adults are called “physoclists” (Steen, 1970). Some physoclists, such as Oreochromis mossambicus (Tilapia mossambica), have no patent pneumatic duct as larvae or as adults, and are termed “pure physoclists” (Doroshev and

Cornacchia, 1979; Doroshev et al., 1981). Most physoclists however, belong to an intermediate group termed “transient-physostomes”, which possesses a patent pneumatic duct as larvae and lose it as adults (Bailey and Doroshev, 1995; Trotter et al.,

2001).

1.1. Architecture and function of the adult teleost swimbladder

Typically, a “secretory section” (where gas is deposited into the swimbladder) and a “resorbent section” (where gas is re-absorbed back to the blood) of the swimbladder can be separated, but the actual anatomy of these sections varies from species to species (Evans, 1998). In the physostomous European eel, Anguilla anguilla, the pneumatic duct develops into the resorbent section of the swimbladder and is separated from the secretory section by a sphincter (Evans, 1998). In some physoclists 2 the resorbent area forms a posterior chamber separated from the secretory part by an adjustable membrane called a “diaphragm”. In others the resorbent area is limited to a small highly vascularized dorsal region, separated from the rest of the bladder by a muscular sheath called the “oval” (Steen, 1970). There are also physoclists, which have no separation at all (Steen, 1970). The wall of the secretory area in adult fish may consist of an outer layer of connective tissue termed “the tunica externa” rich in collagen fibers; a submucosa that may be impregnated with guanine crystals and may contain large amounts of membranous material; a “muscularis mucosa” containing smooth muscle cells; a “lamina propria” containing blood vesicles and connective tissue; and finally a layer of epithelial cells, lining the lumen (Evans, 1998; Steen, 1970;

Zwerger et al., 2002; Maina, 2000; Morris and Albright, 1975). In the European eel, the epithelium of the secretory part of the swimbladder is made up of gas gland cells whereas in the perch, Perca fluviatilis, the gas gland cells are concentrated in certain areas (Prem et al., 2000). The volume of the swimbladder in adult fish is regulated by two reflex mechanisms: inflation occurs by active secretion of gas from the circulatory system into the swimbladder via the highly vascularised rete mirabile and the metabolically active swimbladder gas gland cells in the secretory area; and deflation occurs by passive resorption of gases to the blood vesicles of the resorbent area (Steen,

1970). The resorption of gases is correlated to their transport capacity in the blood and their solubility. Most likely the quantitative extent of gas deposition and gas resorption in adult fish, is accomplished by regulation of the blood flow to the appropriate areas

(Steen, 1970). More details on the physiology of gas deposition and the function of the gas gland cells and rete mirabile, may be obtained in the review of Steen (1970).

3 1.2. Initial swimbladder inflation

Initial inflation of the swimbladder is considered one of the most critical stages of larval development (Blaxter, 1988). Most larvae inflate their swimbladder in early developmental stages, coinciding with the absorption of yolk reserves and the initiation of external feeding (Battaglene and Talbot, 1990; Doroshev and Cornacchia, 1979; Tait,

1960), but some species may inflate at a much later stage, such as the European eel in which swimbladder inflation occurs in 3 year old larvae, after migrating across the

Atlantic ocean (Zwerger et al., 2002).

1.2.1. Mechanism of initial inflation

The mechanism of initial inflation is poorly understood. Physostomous larvae are generally believed to inflate the swimbladder by gulping atmospheric air at the water surface and forcing it into the swimbladder via the alimentary tract and pneumatic duct (Steen, 1970; Tait, 1960). The physostomous Salmonoids: brown trout, Salmo trutta, rainbow trout, S. gairdneri, lake trout, Cristivomer namaycush, and whitefish,

Coregonus clupeaformis, failed to inflate their swimbladders while being denied access to the water surface (Tait, 1960). Detection of gas bubbles in the gut of these species prior to inflation (when access to air was granted) and in the swimbladder, pneumatic duct and gut of the physostome European eel during inflation supports this proposed mechanism (Tait, 1960; Zwerger et al., 2002). Another research conducted in European eel, revealed that during the initial filling of the swimbladder with gas, the gas gland cells could not accomplish their typical role in gas deposition because they lacked a developed baso-lateral labyrinth (that enlarges the surface area of the cells towards the blood vessels) and a close proximity to functional blood vessels as seen in the adults

(Zwerger et al., 2002), therefore concluding that the mechanism of gulping atmospheric air for initial inflation is most likely in this species. 4 Transient physosotomes, such as the striped bass, Morone saxatilis, striped trumpeter,

Latris lineata, Australian bass, Macquaria novemaculeata, walleye, Sander vitreus, spot croaker, Leiostomus xanthurus, and gilthead seabream, Sparus aurata, also displayed a need to reach the water surface for successful inflation (Battaglene and Talbot, 1990;

Chapman and Hubert, 1988; Chatain and Ounais-Guschemann, 1990; Govoni and Hoss,

2001; Marty et al., 1995; Trotter et al., 2005). Transient physosotomes are generally thought to initially inflate their swimbladder like physostomes, by gulping air at the surface and passing it through their transient pneumatic duct. Later the pneumatic duct regresses and the volume of the swimbladder is maintained by the vascular rete mirabile and gas gland cells (Steen, 1970). In striped trumpeter, Australian bass and spot croaker, a liquid-filled phase (swimbladder dilation) was reported prior to inflation with gas

(Battaglene and Talbot, 1990; Govoni and Hoss, 2001; Trotter et al., 2001). Recent studies in walleye suggest that a surfactant (surface active agent which reduces and modifies surface tension at the air-liquid interface) from the common bile duct together with gut peristalsis, aid in the fragmentation and passage of air bubbles through the pneumatic duct (Marty et al., 1995). In the European eel, surfactant has been observed in the swimbladder before the first filling (Zwerger, et al., 2002), but was produced in the gas gland cells rather than the bile duct (Prem et al., 2000). The surfactant may act also as an anti-glue to prevent sticking together of the swimbladder walls and may be important in this manner for initial inflation (Zwerger et al., 2002).

The mechanism of initial inflation in pure physoclists is the least understood amongst all swimbladder types. The larvae of these fish lack a pneumatic duct and therefore have no direct connection to atmospheric air for initial inflation. Doroshev et al. (1981) suggested that inflation in Oreochromis mossambicus might occur by active secretion of gas into the swimbladder from the circulatory system via the swimbladder epithelium.

This was supported by (a) a pronounced glandular swimbladder epithelium, that 5 appeared to become secretory at about the time of swimbladder inflation (b) the negative effect of hypoxic conditions caused by high stocking density on the rate of initial inflation, emphasizing the importance of sufficient dissolved oxygen in the ambient water as a source of gas for initial inflation (c) these fish were able to inflate their swimbladders when denied free access to water surface (Doroshev and Cornacchia,

1979). A different mechanism of inflation by internal gas secretion was also proposed in the transient physostome largemouth bass, Micropterus salmoides, and the pure physoclist jewelfish, Hemichromis bimaculata (Johnston, 1953; McEwen, 1940). In both species, swimbladder epithelial cells contained large vacuoles of unknown nature in their cytoplasm, close to the time of initial inflation. The authors suggested that the epithelial cells themselves were forming the gas used in the initial inflation, possibly by disintegration of organic material within the cells producing CO2. Though largemouth bass has a patent pneumatic duct, the authors did not believe it is used for transferring atmospheric air from the water surface and instead it is used for passing gas formed as a result of digestion, from the digestive tract (Johnston, 1953).

1.2.2. Timing of initial inflation

As mentioned before, the timing of initial inflation varies among species, and usually coincides with the final absorption of yolk and initiation of external feeding

(Battaglene and Talbot, 1990; Doroshev and Cornacchia, 1979; Tait, 1960). For larvae that commence exogenous feeding, the achievement of neutral buoyancy by inflating the swimbladder greatly reduces the energetic cost of swimming and improves predatory efficiency (Marty and Hinton, 1995). On the other hand, premature inflation could be a disadvantage in the wild, making the larvae more exposed to predation when they are buoyant, and also more vulnerable to visual predators, since inflated swimbladders are light refractive (Hall et al., 2004). Atlantic menhaden, Brevoortia 6 tyrannus and sand sillago, Sillago ciliata, for example, have a diel cycle of swimbladder inflation as larvae. They inflate their swimbladders during the night and deflate during the day (Battaglene et al., 1994; Govoni and Hoss, 2001). Inflation in the dark could be a behavioral adaptation in order to avoid predation in the photic surface water (Martin-

Robichaud and Peterson, 1998).

Several studies have noted a narrow “window of opportunity” for initial swimbladder inflation. In some fish species such as the transient physostomes Australian bass, striped bass, striped trumpeter and walleye, failure to inflate within a certain time interval

(which varied between the different species), was irreversible and resulted in abnormal swimbladder development (Battaglene and Talbot, 1990; Doroshev and Cornacchia,

1979; Marty et al., 1995; Trotter et al., 2004).

In other fish species, timing of inflation seems to be more flexible and a critical period does not exist. For example, in the physostomes brown trout, rainbow trout, lake trout, and whitefish, filling of the swimbladder was delayed artificially for weeks by the prevention of access to the water surface. As soon as access was granted, all individuals inflated their swimbladders, although it was much later than inflation would have occurred under normal conditions (Tait, 1960).

The physiological mechanism underlying the limited period for inflation is unknown.

In transient physostomes, it has been suggested that it is linked with the degeneration of the pneumatic duct (Bailey and Doroshev, 1995). In reality, however, the pneumatic duct stays open in many transient physostomes after the critical period for inflation is over (Marty et al., 1995; Trotter et al., 2004). Marty et al (1995), hypothesized that inflation in walleye could not occur later than 12 days post hatch because the development of the muscular pyloric sphincter produced a physical separation between the pneumatic duct (which opens to the stomach) and the bile duct (which opens to the intestine). As a result, surfactant from the common bile duct cannot reach and break up 7 ingested air bubbles to enable entry into the pneumatic duct. This did not explain the limited inflation interval in striped trumpeter, where the pneumatic and common bile duct ostia appeared to remain in close association during and after the initial inflation interval (Trotter et al., 2005). Lumen collapse and emptying of liquid following the dilation stage corresponded with the end of the ‘window of opportunity’ for swimbladder inflation in striped trumpeter (Trotter et al., 2005).

In striped bass and striped trumpeter, the timing of initial inflation and as a consequence the duration of the critical period for inflation, appears to be temperature dependent

(Bailey and Doroshev, 1995; Trotter et al., 2003).

1.3. Swimbladder non-inflation in teleosts

Failure to inflate the swimbladder is regarded a major obstacle in the rearing of many fish species, mainly transient physostomes. In Australian bass, gilthead seabream and striped trumpeter, up to 100% of swimbladder non-inflation may occur (Battaglene and Talbot, 1990; Chatain and Ounais-Guschemann, 1990; Trotter et al., 2001). Non- inflation in pure physoclists has been reported but it is rare (Doroshev et al., 1981).

Swimbladder non-inflation has been associated with decreased growth rates (Chapman and Hubert, 1988; Chatain and Ounais-Guschemann, 1990; Jacquemond, 2004; Martin-

Robichaud and Peterson, 1998); increased spinal deformities (Jacquemond, 2004;

Paperna, 1978); increased susceptibility to stress (Chapman and Hubert, 1988) and increased mortality rates (Battaglene and Talbot, 1990). Jacquemond (2004) hypothesized that the spinal deformities in fish that lack an inflated swimbladder are derived from the high pressure exerted on the vertebras by the incessant upward swimming, compensating the lack of natural buoyancy. Marty and Hinton (1995) showed that larvae of Japanese medaka, Oryzias latipes, with non-inflated 8 swimbladders had a higher oxygen consumption compared with normal larvae and explained their decreased growth rates by a higher energy demand for maintaining position in the water column and for other activities. This increased energy cost could also be a disadvantage in the case of competition on food between affected larvae and normal larvae bred in the same tank, discriminating the affected larvae and impairing their growth (Jacquemond, 2004). Histologicaly, failure to inflate the swimbladder has been associated with epithelial degeneration and necrosis (Bailey and Doroshev, 1995;

Doroshev and Cornacchia, 1979; Paperna, 1978); hypertrophy and hyperplasia of the epithelial cells (Paperna, 1978; Trotter et al., 2004); folding of the epithelium (Trotter et al. 2004); changes in epithelial cell nuclei to basophilic or intensified staining

(Doroshev and Cornacchia, 1979; Trotter et al. 2004); hyperplasia and atypical location of the rete mirabile (Padros et al., 1993) and increased dilation of the pneumatic duct

(Padros et al., 1993). Although swimbladder non-inflation does not always affect survival, its negative effect on growth and external appearance of the fish generally renders them unmarketable. This causes financial losses to the commercial fish farms.

1.3.1. Factors that were reported to affect SBN

Though SBN has been sighted in the wild (Egloff, 1996) it is mainly regarded as a problem of artificial rearing of larvae, where rearing protocols are under development and optimal, species-specific rearing conditions are to be finalized (Trotter et al., 2001).

Several factors have been reported to affect initial inflation: Access to the air-water interface appears to be essential for initial inflation in physostomes and transient physostomes (Chapman and Hubert, 1988; Chatain and Ounais-Guschemann, 1990;

Goolish and Okutake, 1999; Marty et al., 1995; Tait, 1960; Trotter et al., 2005).

Prevention of access to the water surface could be caused by a physical barrier like an oily surface film originated from the food (Chatain and Ounais-Guschemann, 1990), but 9 also by sub-optimal rearing conditions that impair the larvae’s ability to reach the surface. Examples for such rearing conditions were studied in several fish species:

Intense lighting, incorrectly applied in the case of the negatively phototactic Australian bass larvae, repelled them from the water surface and inhibited inflation (Battaglene and

Talbot, 1990); White colored tanks, caused disorientation of striped bass larvae, confusing the bright tank walls with the reflective water surface (Martin-Robichaud and

Peterson, 1998); and excessive water turbulence, due to vigorous aeration or surface skimmers, inhibited inflation in Australian bass larvae and gilthead seabream by making it more difficult for the fish to reach the water surface (Battaglene and Talbot, 1990;

1993; Chatain and Ounais-Guschemann, 1990). In striped bass on the other hand, increased turbulence and strong aeration seemed to improve inflation success rather than prevent it, possibly by disturbing the surface tension of the water (Chapman and

Hubert, 1988; Doroshev and Cornacchia, 1979). Battaglene and Talbot (1993) commented that “these apparent differences in optimal aeration between species need to be interpreted with caution because of the different rearing systems used and the possible confounding influence of surface films”. Ingestion of bacteria and organic debris from the surface film during inflation has also been thought to impede inflation of the swimbladder in physostomes and transient-physostomes (Marty et al., 1995;

Padros et al., 1993). The bacteria may enter the swimbladder lumen through the pneumatic duct and cause inflammation. Temperature has been shown to influence not only the timing of inflation (Bailey and Doroshev, 1995; Trotter et al., 2003) but also the success of initial inflation in striped trumpeter (Trotter et al., 2003). According to

Trotter et al. (2003), temperature influences initial inflation by affecting the mean size of the larvae at the onset of inflation, which is correlated with inflation success. Brood- stock nutrition was shown to affect initial inflation in gilthead seabream larvae (Tandler et al., 1995) where an addition of n-3 highly unsaturated fatty acids (HUFA) to the 10 brood-stock diet (through enriched artemia) caused a significant increase in initial swimbladder inflation. The requirement of n-3 HUFA is more critical for marine fish than for freshwater fish because the former have a limited ability to chain, elongate and de-saturate dietary linolenic acid (Harel and Place, 1998).

Salinity appears to affect inflation in estuarine larvae (Battaglene and Talbot, 1993). In the catadromous transient-physostome, Australian bass, in which spawning takes place in estuaries and larvae migrate to freshwater, salinities ranging between 15-30‰ were shown to increase inflation compared with 10‰, presumably by providing sufficient buoyancy to reach the water surface (Battaglene and Talbot, 1990; 1993). However, in striped bass, which often spawns in freshwater rivers and the larvae are carried downstream to more saline estuaries, a gradual increase in salinity from 0 to 6.5 or 10‰ did not affect inflation (Chapman and Hubert, 1988). Photoperiod was shown to influence inflation in striped bass, where a shorter photoperiod resulted in higher inflation rate (Martin-Robichaud and Peterson, 1998). Sand sillago required a regular photoperiod in the first 17 days post hatch to inflate the swimbladder and showed a diel pattern of nocturnal inflation (Battaglene et al., 1994). Stocking density in closed tanks had an effect on inflation in the pure physoclist Oreochromis mossambicus where a higher stocking density inhibited inflation irreversibly (Doroshev and Cornacchia,

1979). Doroshev and Cornacchia (1979) explained this decrease in inflation by the hypoxic conditions created in the closed tanks as the density increased, suggesting that initial inflation in this species is influenced by the amount of dissolved oxygen in the water. In the transient- physostome Australian bass, on the other hand, initial inflation increased in low to zero aeration (Battaglene and Talbot, 1993) and in walleye enrichment of the water with pure oxygen did not induce swimbladder inflation

(Summerfelt, 1991). 11 Water pH was shown to affect the rate of initial inflation in the physostomous common carp Cyprinus carpio L., where at a pH of 5-5.2 and 26 °C the majority of the larvae failed to inflate their swimbladders (Korwin-Kossakowski, 1988). The authors suggested that this is caused due to high mucus secretion associated with acid-induced stress that may lead to plugging of the air passage through the pneumatic duct.

The optimal rearing conditions for maximal swimbladder inflation appear to vary between fish species, depending on their swimbladder morphology (physostomous, transient-physostomous or physoclistous), mechanism of initial inflation (air gulping or other) and special behavioral and physiological adaptations that were part of the larvae’s survival strategies in the wild and were not lost in artificial rearing (inflation in the dark, photoperiod, etc.). Research on the subject seems to have focused on a limited number of species and cannot be generally applied. Learning more on the optimal conditions necessary for each species and their relationship with the species-specific swimbladder morphology and mechanism of initial inflation is crucial for reducing the obstacle of

SBN in artificial rearing.

The genetic basis for SBN has been poorly investigated. Harrell et al. (2002) found that in striped bass the heritability estimates for SBN were very low for half sibling families and moderate for full sibling families. They suggested that SBN might be predominantly influenced by dominance (the allele for normal inflation is dominant over the allele of non inflation), epistatis (the suppression of the phenotypic expression of one gene (SBN in this case) by another gene) or environmental factors. Their explanation to this was that swimbladder inflation is a fitness-related trait, and so SBN is most certainly lethal in the wild, and would have already been selected against in nature.

Failure of initial swimbladder inflation has been shown to be a sensitive indicator of toxicant exposure during early development of some fish species 12 (Hamm and Hinton, 2000; Henry et al., 1997; Kim and Cooper, 1999;

Sarnowski, 2004). In the common carp, exposure to heavy metals (Cu and Cd) resulted in non- inflation of the posterior and anterior chambers of the swimbladder in some larvae and delayed swimbladder inflation in others

(Sarnowski, 2004). The authors hypothesized that this effect could be a result of:

(a) metal-induced reduction of locmotory activity of the larvae making it difficult for them to reach the water surface (b) an effect on the metabolism of the larvae, inhibiting gas passage to each swimbladder chamber (c) a copper- induced reduction of air uptake ability and disturbance of the secretion and absorption of gas that fills the bladder or (d) changes in the length and entering point of the pneumatic duct to the posterior end instead of the cranial end. They also indicated that heavy metals might cause swelling and proliferation of epithelial cells, followed by necrosis, sloughing and heavy mucus secretion.

Excessive mucus secretion or hypertrophy of the epithelium may inhibit inflation by causing congestion of the pneumatic duct (Sarnowski, 2004).

However, in other researches, exposure to toxicants did not exclusively effect swimbladder inflation, and the development of other organs was affected as well

(Hamm and Hinton, 2000; Henry et al., 1997; Kim and Cooper, 1999).

1.4. Angelfish Pterophyllum scalare

Angelfish Pterophyllum Scalare (Lichtenstein 1823) (Class: Actinopterygii;

Order: Perciforms; Family: Cichlidae) is one of the most popular members of the family

Cichlidae in the global ornamental fish market. Numerous professional and amateur breeders worldwide culture it and a variety of strains have been developed over the years (Swann, 1994). The distribution of the angelfish is widespread. In South America: 13 the Amazon River basin, in Peru, Colombia and Brazil, along the Ucayali, Solimões and

Amazon rivers; Rivers of Amapá (Brazil), Rio Oyapock in French Guiana; Essequibo

River in Guyana (www.fishbase.org). In the wild, angelfish inhabit swamps or flooded grounds where the aquatic and riverine vegetation is dense (www.fishbase.org).

Angelfish occur naturally in the soft and slightly acidic water of the Amazon River

(Gobel and Mayland, 1998). They are typical substratum-spawners, and in the wild will typically choose a plant leaf as the spawning site. After hatching, both parents take care of the brood and the young (Gobel and Mayland, 1998).

1.4.1. Swimbladder non-inflation in angelfish

A large-scale phenomenon of SBN was recently reported in angelfish grown in a commercial ornamental fish farm in the Arava, Israel (Zilberg et al., 2004). The affected fish were incapable of maintaining their position in the water body and sank to the bottom. They were smaller in size than healthy fish from the same age group. Their head was pointed slightly upwards at an angle of 45°, towards the water surface and their swimming was laborious. Histologicaly, normal adult fish possessed a double- chambered inflated swimbladder, situated in the dorsal part of the abdominal cavity, with no evident connection to the alimentary tract. In fish that failed to inflate their swimbladders, the non-inflated swimbladder developed into a solid cluster of gas gland cells and a hyperplastic rete mirabile. In most cases a lumen could not be discerned but in others a narrow gap was observed. No bacteria were observed within the lumen of the swimbladder (Zilberg et al., 2004). A more detailed study of the process of normal swimbladder inflation in angelfish and possible causes for SBN was necessary.

14 1.5. Research objectives

In this study we aimed to investigate SBN in angelfish by:

1. Monitoring the occurrence of SBN in the commercial fish farm

2. Examining the effect of various environmental factors on the prevalence of SBN

3. Characterizing histologically the sequence of events leading to normal and

abnormal swimbladders development in angelfish larvae

2. Material and methods

2.1. Fish hatching and rearing protocol in “Negev Angels” commercial fish farm

Healthy appearing couples from different breeds are held in separate breeding aquaria and lay their eggs on a ceramic stick. The stick is removed from the parent’s aquarium within a few hours from laying time and placed in a hatching aquarium (20L) in a mixture of reverse osmosis water (Meitech, Hagor, Israel) and tap water, to provide conductivity of 400µs. Methylene blue (Sigma Aldrich, Steinheim, Germany) is added at a concentration of 0.5 ppm to prevent fungal contamination.

Hatching occurs within 3 days at a temperature of 26 ± 1 ºC. Tap water (pre-aerated over-night) is added at a rate of 10 % of the water volume on days 2 and 3-post fertilization. A 10% water exchange is applied from days 1-10 post hatch (p.h.). At the

5th day p.h., external feeding is commenced and biological box filters are placed in the aquaria. Fish receive newly hatched artemia brine shrimps (Brine shrimp eggs, Salt

Creek, Inc. Salt Lake City, Utah, U.S.A.) 3-4 times a day and excess artemia is siphoned once a day. At the 10th day p.h., every 3-5 cohorts are moved to a single aquarium (180L) where they will remain until reaching the age of one month when they are transferred to 1,000 L growing tanks. From day 21 p.h., fish receive a gradually growing amount of dry feed (TB, Coppens, Helmond, The Netherlands), and are weaned from the live feed by day 31 p.h..

2.2 Field survey of SBN prevalence

Field survey of SBN prevalence was conducted in “Negev Angels” commercial angelfish farm located at the Arava, Israel, during 2003-2005. At the age of one month, before moving to the large growing tanks, the fish are sorted manually and fish with non-inflated swim bladders are removed and documented. The process is based on 16 external appearance characterized by inability to maintain their position in the water, sinking to the tank bottom and arduous tail movements. The mean prevalence of SBN from live fish per month was determined based on farm data. Statistical significance was determined.

2.3. Fish experimental system

Eggs were obtained from “Negev Angels” angelfish farm, Arava, Israel. Two hatching sticks with 0-24 hr old eggs from parents with a history of producing healthy off spring

(high percentage of inflation in the larvae) were used in each experiment. Unless otherwise indicated, experiments were carried out in 10L aquaria and the farm-rearing protocol was followed. Temperature was kept at 26 ± 1 ºC and lighting regime was

12h:12h dark:light cycle. Fish were fed with artemia brine shrimps (Brine shrimp eggs,

Salt Creek, Inc. Salt Lake City, Utah, U.S.A.) 3 times a day from the 5th day p.h, at a density of 1 artemia per ml. Box biofilters were placed in each aquaria at the commencement of feeding. Ammonia, nitrite and oxygen levels were measured twice a week using Aquamerck commercial kits for ammonia and nitrite (Darmstadt, Germany) and YSI model 52 dissolved oxygen meter (YSI Incorporated, Yellow springs, Ohio). In case water quality deteriorated (i.e. ammonia and nitrite > 0.5 ppm), water exchange was increased. PH and conductivity were tested using a Cyberscan PC 300 pH and conductivity meter (Eutech Instruments, Singapore)

2.4. Effect of environmental factors on the prevalence of SBN in angelfish

Eggs were dispatched from the hatching sticks with a gentle siphon and equally divided between experimental aquaria, so that each aquarium contained the same number of eggs from both sticks. Number of eggs varied between experiments depending on the 17 amount of eggs on the two hatching sticks (table 1). Non-viable eggs (white color, non transparent) were excluded. Unless otherwise indicated, eggs were placed in open petri dishes glued to rubber corks (about two cm long) and sunk in 10L aquaria. Each treatment was conducted in 6 replicates. At 12 days p.h., fish larvae were examined under a light microscope for the presence or absence of an inflated swim bladder and

SBN prevalence in live fish was determined. To determine the effect of the treatment on growth, 5 normal larvae from each replicate were also measured for total length (TL).

The effect of the treatment on SBN prevalence, growth and survival was determined by statistical analysis.

Table1: number of eggs per replicate used in each experiment (n=6)

Factor tested Number of eggs

Access to the air-water interface 12 eggs per box

407 eggs in general tank

Methylene blue concentration 30 eggs per aquarium

Feed concentration 30 eggs per aquarium

Water conductivity 27 eggs per aquarium

Tank color 18 eggs per aquarium

2.4.1. Access to the air-water interface:

Eggs were hatched and grown in six 1L net boxes submerged in a single 100 L container. These conditions prevented access to atmospheric air but allowed water exchange through the box walls (fig. 1). Air stones were positioned as far as possible from the boxes, to prevent generation and trapping of air bubbles on the box’s nets.

Artemia was injected into the boxes, three times a day. For control, eggs were hatched 18 in the tank water, surrounding the boxes. Fish were examined for the presence of an inflated swim bladder at the age of 19 days p.h..

Fig.1. A net box used to prevent the access to the air-water interface.

2.4.2 Effect of methylene blue (disinfectant) concentration on SBN

Eggs were hatched in 3 different concentrations of methylene blue: 0.5, 2.5 and 5 ppm and a control treatment without methylene blue. The experimental concentration was applied at the beginning of the experiment and methylene blue was subsequently diluted with the water exchanges.

2.4.3. Effect of food concentration and starvation on SBN

Fish were fed with different concentrations of artemia brine shrimps: 0.25, 0.5 and 1 artemia per ml, 3 times a day. The effect of an initial two-day starvation

(commencement of feeding at 7 instead of 5 days p.h.) and then regular feeding (1 artemia/ml) was also examined. 19 2.4.4. Effect of water conductivity (salinity) on SBN

Eggs were hatched in either 140μs (distilled water) or 400μs (combination of tap and distilled water), followed by water exchange with 140μs and 1,100 μs (tap water) or 400

μs and 1,100 μs, respectively.

2.4.5. Effect of tank coloration on SBN

Eggs were hatched and grown in three different tank colors, black, white and transparent.

2.5. Histological characterization of normal and abnormal swimbladder development in early larval stages

2.5.1. Normal swimbladder development

Two hatching sticks with 24 hr old eggs, from parents with a history of producing healthy off-spring (low or no SBN) were used. Each hatching stick was placed in a 30L aquarium for the entire experiment. Larvae were reared under normal conditions and sampled from both sticks at different intervals p.h..

For external microscopic examination, larvae were sampled once a day, on days 0-6, 8,

10 and 12 p.h. They were placed in a petri dish, in a small water volume and examined under a light microscope at x25 magnification. Total length was measured with a millimeteric paper placed underneath the petri dish (5 larvae per sample), and larvae were photographed with an Olympus, C5050 Camedia digital camera (Tokyo, Japan).

For histological analysis, 10-20 larvae were sampled 2-4 times a day, from days 0-12 p.h. They were fixed in 10% Bouin's fixative for 24 hours and kept in 70% ethyl alcohol until processing. Samples were processed in a Leica TP 1020 automated tissue processor (Nussloch, Germany) in which they were dehydrated in an ascending ethanol 20 series (Ethanol 80% - 3hrs, Ethanol 96% x 3 – 3 hrs each, Ethanol 100% x 2 – 1 hr each), cleared (“Solvent for Histology”, Frutarom, Haifa, Israel, x 2 – 1 hr each) and infiltrated with “Histoplast” paraffin (Life science international, Cheshire, UK, LTD) (x

3 – 1 hr each). Samples were then sectioned (5-7µm) with a Leica RM 2135 microtome

(Nussloch, Germany). Sections were mounted on slides and left to dry overnight. They were then placed in 60°C oven for 30min – 1hr, to help paraffin melting and stained with hematoxylin & eosin (H&E) regressive staining method (Carson, 1997).

Hematoxylin & eosin regressive staining protocol: paraffin was removed with Bio Clear

Xylene substitute (Bio Optica, Milano, Italy) (5min), followed by hydration of the slides with absolute alcohol (10 dips), 95% alcohol (20 dips) and rinsing with distilled water.

Slides were immersed in Mayer’s hematoxylin solution for 10-15 minutes and then rinsed with distilled water. They were then dipped 5-10 times in 1% Hydrochloric acid in 70% alcohol (for differentiation), rinsed with tap water, and immersed for 30 seconds in Scott’s solution (bluing agent). After another rinse in tap water, slides were placed in

Putt’s Eosin for 10-15 minutes. They were dehydrated with 95% alcohol (5 min), absolute alcohol (5 min x 2) and cleared with Xylene substitute (5 min x 2). Finally, they were coversliped with DPX mountent for histology (Fluka/ Sigma Aldrich,

Steinheim, Germany). Slides were examined under a light microscope and photographed with a Nikon DN 100 digital camera (Japan). Normal swimbladder morphology and development was recorded (defined when present in >50% of the sample).

Sections from the 4th day p.h. were also stained with Lillie’s alochrome staining, PAS staining and Alcian blue (Sheehan and Hrapchak, 1980).

In order to determine percentage of inflated swimbladders, 50-200 larvae were examined at 5-7, 10 and 12 days p.h. under a light microscope.

21 For Transmitting Electron Microscopy (TEM), 2-5 Larvae were sampled 2-3 times a day from day 0-11 p.h. in one cohort and from day 0-3 p.h. in the other. They were fixed according to Lewis and Knights (1977) triple fixation, in 2.5% glutaraldehyde cacodylate buffer pH 7.2 for 48 hrs and washed in 2 changes of 0.2M cacodylate buffer pH 7.2. They were then fixed in osmium tetraoxide (2% in cacodylate buffer pH 7.2) for

1 hr at room temperature and rinsed in 0.2M cacodylate buffer pH 7.2 three times. They were then washed several times in sodium maleate buffer, pH 5.2, at 4°C for 30 min and fixed in 1% uranyl acetate in sodium hydrogen maleate buffer in the dark at 4°C for 2 hr. Then they were rapidly dehydrated according to the Standard Dehydration Schedule

(Glauert, 1975) in 70% alcohol (10 min), 95% alcohol (10 min), absolute alcohol (15 min x 2), 1:1 propylene oxide : absolute alcohol (10 min ) and propylene oxide (15 min x 2 ). For embedding, propylene oxide was removed and replaced with 1:1 propylene oxide : epoxy at room temperature for 30 min to 1 hr. The mixture was removed, replaced with epoxy and left at room temperature uncovered overnight. Specimens were transferred to dry capsules into which fresh epoxy was added and left overnight in 60°C to harden. Epoxy blocks were initially sectioned into semi thin sections of 0.1 μm stained with Toluidine blue and examined under a light microscope for orientation.

Blocks were subsequently sectioned with a diamond knife and thin sections were stained with lead citrate and examined under a JEM 100CX, Mark II, transmission electron microscope (Tokyo, Japan).

2.5.2. Abnormal development of the swim bladder

In the normal development experiment, swimbladders that had a different appearance were singled out and considered abnormal but these were scarce. In order to investigate abnormal development in a larger scale, methylene blue, which was shown to enhance the percentage of SBN (see results for the effect of methylene blue on SBN), was used. 22 24 hr old eggs from two pairs of parents were hatched in 5 ppm methyele blue. Rearing protocol was similar to the one used for determining normal development. On the 4th day post hatch, 100% of the water was exchanged to tap water with no disinfectant and box biofilters were applied on the 5th day post hatch. 10-20 larvae were sampled 1-4 times a day from days 0-9 p.h., for histology. They were separated from the 5th day p.h. to larvae with an apparent swim bladder and larvae without one, based on external observation. For Transmission Electron Microscopy (TEM), 2-4 larvae were sampled 1-

4 times a day from day's 3-9 p.h.. These were also separated from the 5th day to larvae with and without inflated swimbladders. Tissue processing, embedding and staining were done in the same manner as used for characterization of normal development.

Percentage of inflation was determined only on the 4th day and at 9:00 am on the 5th day, because sampling from this time on was done in a non-random manner due to separation of individuals with an inflated and non-inflated swimbladder. Larvae (40-

110) were examined under a light microscope for the presence of an inflated swimbladder.

2.6. Statistical analysis

(a) Analysis of the prevalence of SBN in the farm: Data of %SBN/live fish was

transformed using Arcsinus transformation and the difference between the mean

prevalence of SBN per month was determined using rank based One-Way

ANOVA analysis using SigmaStat (SPSS Inc, 1992-1997). Where significant

differences were found, means were analyzed with All Pairwise Multiple

Comparison Procedures (Dunn’s Test).

(b) Analysis of the effect of different environmental factors on the prevalence of

SBN, growth and survival of the larvae: Data of %SBN/ live fish and %live fish

/total eggs used, was transformed using Arcsinus transformation. The treatments 23 were compared with One-Way ANOVA analysis using SigmaStat (SPSS Inc,

1992-1997). Where significant differences were found, means were analyzed

with All Pairwise Multiple Comparison Procedures (Tukey’s test). Mean TL of

normal larvae from different treatments (except access to the air-water interface)

was analyzed using the same One-Way ANOVA and All Pairwise Multiple

Comparison Procedures without transformation.

test analysis using SigmaStat (SPSS Inc, 1992-1997).

3. Results

3.1. Survey of SBN prevalence in a commercial angelfish farm during 2003-2005

A three-year survey (2003-2005) of the prevalence of SBN in “Negev angels”, a

commercial angelfish farm located at the Arava, Israel, is presented. The total mean of

SBN/live fish calculated from the three years data was 14.65%, ranging from 0-100%

SBN/live fish per sample. Data is presented as mean prevalence of SBN from live fish

per month. The long-term survey of SBN during 2003-2005 in the farm revealed no

apparent pattern of occurrence. There seemed to be no relation to seasonality.

a (2003) b (2004) 30 a 30

25 25 ab a ab ab a 20 a 20 a ab a abc abc ab ab abc 15 15 bc abc ab c 10 10 abc bc bc 5 c 5 Mean SBN/ live fish (%) c fish (%) live / Mean SBN 0 0 123456789101112 123456789101112 Month Month

c (2005) Figure 2: Mean prevalence of SBN from 30 live fish per month (%) in “Negev angels”

commercial fish farm, Arava, Israel, during 25 2003-2005 (mean + S.E). Different letters a ab denote significant differences in mean 20 ab abc bcd SBN/live (%) between different months in abc abc abc abc cd the same year (P < 0.05). 15 bcd

d 10

5 Mean SBN / live fish (%) fish / live SBN Mean

0 123456789101112 Month 25

3.2. The effect of environmental factors on SBN in angelfish

The effect of selected environmental factors on SBN in anglefish is presented in table 2.

Prevention of access to the air-water interface, feed concentration, initial two-day starvation, water conductivity (140μs - 1100μs) and tank color did not significantly affect SBN. Methylene blue, a chemical routinely used to prevent fungal contamination of the eggs, caused a significant increase in SBN in a concentration of 5 ppm.

Table 2: The effect of environmental factors on SBN/live fish Factor tested Treatment %Live/eggs % SBN/live fish TL of normal (mean ± S.E.) (mean ± S.E.)1 larvae (mean ± S.E.)1 Access to the air- No access (boxes) 58.33 ± 10.76 0 13.21 ± 1.4 b water interface Access (general tank) 48.65 0 14.5 ± 0.43 a Methylene blue 0 ppm 58.9 ± 6.7 9.17 ± 3.76 a 10.97 ± 0.1202 a concentration 0.5 ppm 63.3 ± 6.72 12.63 ± 2.44 ab 10.67 ± 0.1909 a 2.5 ppm 62.22 ± 4.36 18.64 ± 3.38 ab 10.67 ± 0.0843 a 5 ppm 50.56 ± 7.57 27.67 ± 5.58 b 10.02 ± 0.1833 b Feed Two-day starvation 38 ± 8.73 4.29 ± 4.29 7.94 ± 0.1327 b concentration followed by feeding of 1 artemia/ml 2 0.25 artemia/ml 45.33 ± 1.2 2.22 ± 2.22 9.18 ± 0.2557 a 0.5 artemia/ml 45 ± 7.29 4.35 ± 1.40 9.233 ± 0.2765 a 1 artemia/ml 2 51.67 ± 8.46 4.31 ± 3.28 9.1583±0.1943 a Water 140μs - 140μs 59.26 ± 10.7 4.29 ± 1.44 9.05 ± 0.1586 conductivity 400μs - 400μs 56.8 ± 5.87 4.11 ± 3.08 8.733 ± 0.1820 140μs - 1100μs 56.17 ± 5.85 8.88 ± 3.45 8.4083 ± 0.1369 400μs - 1100μs 36.42 ± 6.72 3.04 ± 1.99 8.8 ± 0.1713 Tank color Black 71.1 ± 5.09 7.68 ± 3.44 6.4 ± 0.4 White 64.8 ± 7.4 0 6.917 ± 0.2386 Transparent 84.44 ± 4.08 6.76 ± 3.02 7.4 ± 0.4 1(a,b) different letters within each factor tested, denote significant differences between treatments (P < 0.05). 2 Ammonia levels in aquaria receiving this treatment reached 1ppm.

26 No significant differences in survival were apparent between any of the treatments in the factors tested. Larval growth was affected by: methylene blue concentration, access to air- water interface and starvation (table 2). In the methylene blue experiment, normal larvae that were hatched in 5 ppm were significantly smaller than those hatched in the lower concentrations. Postponing the initiation of feeding reduced growth compared to other treatments, even though they were fed 1 artemia per ml from the 7th day on.

Feeding with 0.25, 0.5 and 1 artemia per ml did not affect larval growth. Larvae that were grown in submerged net boxes were significantly smaller than larvae that were grown in the open tank water.

3.3. Histological characterization of normal and abnormal swimbladder development in early larval stages

3.3.1. Normal swimbladder development in angelfish

A histological analysis of swimbladder development in larvae reared under normal conditions is presented. The external observations refer to both cohorts, whereas the histological analysis represents only one cohort.

3.3.1.1. Hatching period

Eggs from both sticks hatched during the night between the second and third day after spawning. Since hatching was at night, the exact time was not sighted and therefore time is represented as hour and day of sampling (day 0 p.h. = the first day after hatching, third day after spawning). Larvae hatched with a considerably large yolk sack

(fig. 3 a, b) and stayed attached to the hatching stick for the next 24 hours. Larvae were equipped with cement glands emerging from the front of their head, which aided in attachment to the hatching stick (fig. 3b) and degenerated in the next few days (residues were still apparent in the 12th day p.h.). Several gill arches could be distinguished 27 around the pharynx (fig. 3b) and the mouth was covered with an orpharyngeal membrane. The gut appeared as a long straight tube, lined with columnar epithelial cells with a pale cytoplasm and centrally to basally located nuclei. The pronephric ducts ran dorsal to the gut at either side of the body and appeared unfolded. The eyes had no pigmentation. A primordial swimbladder was not seen at this stage.

3.3.1.2. Day 1 p.h.

At this stage, larvae dropped off the hatching stick and aggregated in clusters on the bottom of the aquaria. They stayed on the aquarium floor until the commencement of swimming (4 -5 days p.h.). The mouth opened during the day (not shown) and the cement glands were still apparent (fig. 4a). A pigmented layer started forming on the external side of the optic cup, and the lens was also seen (fig. 4b). The liver originated between the gut and the yolk sack as a cluster of cells with light colored cytoplasm (fig.

4c). An oval shaped gallbladder, containing traces of an eosinophilic material, also appeared between the liver and the gut, lined by a layer of squamous cells and surrounded by pigmentation (not shown). The intestine formed a small loop above the gallbladder, to which the bile duct connected. At the end of the first day (10:00 pm), a primordial swimbladder appeared for the first time in some of the larvae. It appeared between the oesophagus and the gut (figs. 4 b, c, d). A connection between the primordial swimbladder and the digestive tract could not be identified. The primordial swimbladder consisted of one layer of columnar epithelial cells with a central lumen.

The cells had a darkly stained cytoplasm with nuclei that were situated basally, centrally or apically towards the lumen (different location in individual cells) (fig. 4d). This appearance distinguished them from the gut’s epithelial cells, which had a faintly stained cytoplasm and basally located nuclei (fig. 4c) and from the epithelial cells of the pronephrous tubule that were cuboidal with a faintly stained cytoplasm and surrounded 28 a larger lumen (fig. 4c). The primordial swimbladder was surrounded by a connective tissue of presumably mesenchymal cells.

3.3.1.3. Day 2 p.h.

At 2 days p.h, primordial swimbladders were present in all larvae and had now further elongated on the antero-posterior axis (fig. 5c). At the beginning of the day (8:00 am), the cytoplasm of the epithelial cells surrounding the narrow lumen was still darkly stained and the nuclei position varied between cells (fig. 5c). As the day progressed, an amorphous material of unknown nature appeared at the basal end of the cells and the nuclei migrated to the opposite side, towards the lumen (fig. 5d). The connective tissue surrounding the primordial swimbladder became thicker as the dorsal area seemed to differentiate to a looser connective tissue and the ventral area became more condense.

The pronephric tubules started convoluting in the anterior part. Each gill arch developed a cartilaginous rod and the gill filaments started to form (fig. 5b). The heart contained a well-developed ventricle and bulbus arteriousus and a thin walled atrium (not shown).

Pigment deposition in the eye continued.

3.3.1.4. Day 3 p.h.

The primordial swimbladder reached its final form prior to inflation (figs. 6 c, d). It appeared as a flattened sack (longer on the antero-posterior axis than the dorso-ventral axis). It was comprised of a two layered epithelium surrounding a narrow lumen: an internal layer of columnar cells, and an external layer of squamous cells. The columnar cells appeared vacuolated, as the amorphous material seemed to have replaced most of the cytoplasm. Their nuclei were apically situated, creating a dense looking rim around the lumen. The amorphous material did not stain with any of the following stains: H&E,

PAS stain, Lillies alochrome stain and Alcian blue (sections not shown). At the 29 posterior end, a small cavity extending from the primordial swimbladder appeared (“the caudal extension”). Its small lumen was continuous to that of the primordial swimbladder’s. Similar to the primordial swimbladder, it was comprised of two layers of epithelium: a squamous outer layer surrounding a cuboidal inner layer (figs. 6 c, d).

The inner layer differed from that of the primordial swimbladder by the absence of amorphous material and basally situated nuclei. The caudal extension did not appear connected to anything other than the swimbladder. Capillaries formed a primordial rete mirabile in the ventral part of the swimbladder, extending towards its posterior end

(figs. 6 c, d). The first folds in the intestine could be observed, devoid of goblet cells at this stage. The junction between the oesophagus and the foregut was clearly seen underneath the primordial swimbladder. Towards the end of the day (8:00 pm), goblet cells started appearing in the oesophagus, and around the pronephros the hemopoietic tissue started developing (not shown).

3.3.1.5. Day 4 p.h.

At the beginning of the day (8:00 am) most larvae had non-inflated swimbladders resembling the form seen on the previous day. The primordial rete mirabile reached the posterior end of the swimbladder, and then turned ventrally towards the digestive tract, creating a loop next to the caudal extension (fig. 7d). It contained few erythrocytes. The first inflated swimbladders appeared at the beginning of the 4th day (8:00 am) but most larvae inflated their swimbladders during the course of the next 14 hours. The first inflated swimbladders had a columnar to cuboidal epithelium (fig. 7c). The cells seemed to have lost their vacuolated appearance as they flattened, containing much less amorphous material. In some of the bladders, an eosinophilic substance lined the lumen, adjacent to the epithelium. The caudal extension was not inflated at this stage (fig. 7c).

Swimbladder inflation continued through the day although most larvae had not started 30 swimming until the next day. Externally, inflated swimbladders appeared as a refractive bubble situated above the yolk sack (fig. 7a). The epithelium of the inflated bladders further transformed from cuboidal to squamous as the lumen grew in size (fig. 7e). The caudal extension also inflated and its epithelium turned squamous. A large amount of goblet cells were present in the oesphagus, the liver increased in size as the yolk sack shrunk. The gut started to convolute and folds could be seen in its mucosa. Gill lamellae started to form (not shown).

3.3.1.6. Day 5-10 p.h.

At the beginning of the 5th day (7:00 am) 98% of the larvae in one cohort and 100% in the other cohort had an inflated swimbladder (based on external appearance).

Exogenous feeding had commenced, and fish with inflated swimbladders had begun swimming. All inflated swimbladders had squamous epithelia and an inflated caudal extension. The rete mirabile was stretched along the bottom of the inflated bladders so it was harder to detect. During the course of the next few days the larvae changed greatly in size and appearance. Inner organs changed and developed but swimbladders remained unchanged, and only grew to occupy a larger body volume (fig. 8).

3.3.1.7. Ultrastucture of the swimbladder during normal inflation

The ultrastructure of the swimbladder cells on the 4th day p.h, during normal inflation, shows that prior to inflation (fig. 9), the inner columnar epithelial layer was filled with amorphous material, most of it concentrated in the basal region, but some also seen in the apical area. When examined closely, it appeared either in large masses or sometimes in non-continuous patches in the cell. In some areas, numerous crescent shaped vesicles at different sizes were surrounding it. The nuclei appeared at the apical region of the cell, containing densely stained nucleoli. The apical surface of the epithelium was 31 thrown into microvilli that extended into the primordial lumen. A layer of external squamous epithelial cells surrounded the inner columnar epithelial cells. An occasional erythrocyte could be observed lying within the epithelium. A labyrinth of finger like cytoplasmic protrusions could be seen in the baso-lateral region of some of columnar cells. After initial inflation (fig. 10), most of the amorphous material disappeared from the cells. The columnar epithelium then transformed to cuboidal and squamous. Some areas of the swimbladder appeared thicker, and multi-layered, whereas others appeared thinner, with only the two layered epithelium present. In the thicker areas, the epithelial cells seem to have the same finger like cytoplasmic protrusions in their baso-lateral region, seen prior to inflation. In some of the cells, small vacuoles could be seen.

32 a

Fig. 3. Larvae of angelfish Pterophyllum scalare at day 0 p.h., 9:00 am. a: External microscopic view. The large yolk sack (ys) and non-pigmented eyes (arrow) are apparent. Bar = 600 μm. b: Sagittal section of whole larvae. Cement glands (cg) can be seen, emerging from the front of the head. Several gill arches (g) are developing around the pharynx. The gut (gu) appears as a straight tube, lined with columnar epithelium and the pronephric duct (pd) is situated dorsally to the gut. The primordial swimbladder is not seen at this age. H&E Bouin’s fixation. Bar = 300μm

Fig. 4. (overleaf) Larvae of angelfish Pterophyllum scalare at day 1 p.h. a: External microscopic view at 5:00 pm. The yolk sack (ys) shrinks in size, pigmentation of the eyes (e) occurs and the cement glands (cg) appear on the forehead of the larvae. Bar = 700 μm. b, c, d: Sagittal section of larvae at 10:00 pm, H&E Bouin’s fixation. b: Further development of the gill arches (g) and a pigmented layer around the eye (e) is observed. The primordial swimbladder (psb) can be distinguished for the first time, dorsal to the yolk sack (ys), between the gut (gu) and the oesophagus (oe). The pronephric duct (pd) is situated above the gut. The liver (li) originates between the gut and the yolk sack. Bar = 300 μm. c: Differences in morphology of the gut, pronephric duct and primordial swimbladder can be seen: the cytoplasm of the gut epithelium is faintly stained with basally situated nuclei; The epithelium of the pronephric duct is cuboidal and surrounds a larger lumen; and the primordial swimbladder (in marked field) consists of darkly stained epithelial cells, surrounding a small lumen. Bar = 100 μm. d: (Enlargement of marked field in c) nuclei of primordial swimbladder are situated basally, centrally or apically towards the lumen. A sheath of connective tissue (ct) surrounds the swimbladder epithelium (ep). Bar = 30 μm. 33

a b

c d

a b

c d

Fig. 5. Larvae of angelfish Pterophyllum scalare at day 2 p.h. a: External microscopic view at 3:00 pm, showing further depletion of the yolk sack (ys), further deposition of pigment in the eyes (e), the cement glands (cg) and the heart (h). Bar = 700μm. b: Sagittal section of larvae at 8:00 pm. The elongated primordial swimbladder (psb) is located between the oesophagus (oe) and the gut (gu) in the dorsal part of the body. Each gill arch contains a cartilaginous rod (c) and newly formed gill filaments (gf). The gall bladder (gb) is situated between the yolk sack (ys) and the gut, adjacent to the liver (li) and appears with cuboidal epithelium. H&E Bouin’s fixation. Bar = 300μm. c: The primordial swimbladder at 8:00 am appears elongated in the antero-posterior axis. The epithelium is darkly stained (ep) and nuclei are located basally, centrally or apically. At 8:00 pm (d), an unknown amorphous material (am) appears in the basal region of the epithelial cells and nuclei (n) migrate to the opposite side towards the lumen (lu). The connective tissue (ct) seems denser in the ventral region as it differentiates. H&E Bouin’s fixation. Bar = 25 μm.

a b

c d

Fig. 6. Larvae of angelfish Pterophyllum scalare at day 3 p.h. a: External microscopic view at 2:00 pm. The gut (gu) is seen above the depleting yolk sack (ys). The primordial swimbladder cannot be distinguished externally. Cement glands (cg) are apparent on the forehead. Bar = 700 μm. b: Sagittal section of larvae at 8:00 am. The primordial swimbladder (psb) is located between the oesophagus (oe) and the gut (gu), caudal to the yolk sack (ys) and ventral to the pronephric duct (pd). An inflated gall bladder is located above the yolk sack adjacent to the liver (li). H&E Bouin’s fixation. Bar = 200 μm. c, d: The primordial swimbladder at 2:00 pm, in its final form prior to inflation, appears as a flattened sack with a narrow lumen (lu), with a two layered epithelium (ep), a basal squamous layer surrounding an apical columnar layer. The columnar epithelium has a vacuolated appearance as the amorphous material (am) replaces most of the cytoplasm. The caudal extension (ce) is present on the posterior end, its lumen contiguous with that of the bladder (d). A primordial rete mirabile (prm) starts to form in the ventral side of the swimbladder from the surrounding connective tissue. H&E Bouin’s fixation. Bar = 50 μm. 36

a b

c d e

Fig. 7. Larvae of angelfish Pterophyllum scalare at day 4 p.h, day of initial inflation. a: External microscopic view at 2:00 pm. The inflated swimbladder appeares as a refractive bubble above the almost depleted yolk sack (ys). Bar = 700 μm. b: Sagittal section of larvae with inflated swimbladder (sb) at 10:00 pm and surrounding organs: oesophagus (oe), gut (gu), yolk sack (ys) gall bladder (gb), heart (h) and liver (li). The gut started to convolute and folds can be seen in its mucosa. H&E Bouin’s fixation. Bar = 300 μm. c, d: The swimbladder at 2:00 pm, during initial inflation. The epithelium transforms from columnar to cuboidal and squamous as the lumen fills with gas. Most of the amorphous material disappears. The caudal extension (ce) does not appear inflated, and the rete mirabile forms a loop on the posterior part of the bladder (arrow). H&E Bouin’s fixation. Bar = 70, 90 μm respectively. e: At 10:00 pm, most swimbladders have a squamous epithelium and the caudal extension appears inflated. H&E Bouin’s fixation. Bar = 100 μm. 37

b c

Fig. 8. Larvae of angelfish Pterophyllum scalare at day 8 p.h. a: External microscopic view at 9:00 am. Larvae have grown in size. The inflated swimladder is still apparent (arrow). Bar = 1000 μm. b: Sagittal section of larvae at 9:00 am. There is no apparent change in the morphology of the swimbladder (sb). The yolk sack has disappeared completely, and the liver (li) is now adjacent to the gills. Prominent gill lamellae (gl) can be seen. Food particles can be seen in the digestive tract. H&E Bouin’s fixation. Bar = 250 μm. c: The inflated swimbladder has squamous epithelium (ep) and an inflated caudal extension (ce). The rete mirabile (rm) appears stretched along the ventral side of the swimbladder. H&E Bouin’s fixation. Bar = 120 μm. 38

a b

Fig. 9. Electron-microscopic overview of the different layers of non-inflated swimbladders on the 4th day p.h. at 8:00 am. a: The epithelium is composed of a layer of squamous cells (se) surrounding a layer of columnar cells. In the columnar cells, the amorphous material (am) fills most of the cell’s cytoplasm, situated mostly on the basal region, though some material is also observed in the apical side. The nuclei (n) are situated apically Numerous crescent shaped vesicles, are observed on the perimeter of the amorphous material. x 3,265. b: An erythrocyte (e) lies within the epithelium, and a labyrinth of cytoplasmic protrusions (cp) from the baso- lateral area of the adjacent columnar cell, appear in contact with it. The primordial lumen (lu) is lined with microvilli at the surface of the epithelial cells. x 11,755.

a b

Fig. 10. Electron-microscopic view of different areas in an inflated swimbladder on th the 4 day p.h. at 2:00 pm. a, b: Area of thicker epithelium. a: The epithelial cells have lost their columnar appearance and organized structure. Cytoplasmic protrusions can be seen between the cells in their baso-lateral region (white arrow). x 3,306. b: Some amorphous material (am) is still present in the cells, but most of it has disappeared. In some of the cells, small vacuoles can be identified (arrow). x 4,408 c: An area of thinner epithelium. The two layers of epithelium can be distinguished, an external layer (se) and an internal layer (ie) near the lumen (lu). The cells of the internal layer appear squamous and contain no amorphous material. x 3,416.

3.3.3. Abnormal swimbladder development under routine conditions

Swimbladders with abnormal appearance were apparent from the 4th day p.h., although they were rare. This could be a result of the choice of eggs used for this experiment, which were taken from parents with a history of low SBN in their progeny (as the primary aim was to define normal development). Type 1 of abnormal swimbladder was observed at 8:00 am on the 4th day, and is presented in figure 11a. Epithelial cells lacked the vacuolated appearance and contained little or no amorphous material. Cells appeared thinner and compressed to each other. Like in normal development, the internal cell layer of the epithelium was columnar and aligned around the lumen, with apically situated nuclei. The primordial rete mirabile appeared ventrally and a non-inflated caudal extension was present. Type 2 and 3 abnormal swimbladders were observed at

2:00 pm, and are presented in figures 11b and 11c respectively. In type 2 abnormal swimbladders, the epithelium folded on itself ventrally on the lateral left side, resulting in an abnormal crescent shaped swimbladder. On the lateral right side, the swimbladder was unfolded (fig. 11d). The columnar cells retained their vacuolated appearance and nuclei were situated apically toward the lumen. The caudal extension and the primordial rete mirabile were present and appeared normal. In type 3 abnormal swimbladders, the columnar cells appeared eosinophilic and contained little amounts of amorphous material, which concentrated in the basal region of the cell. In comparison with type 1 abnormality, the epithelium in the ventral wall of the swimbladder near the rete mirabile seemed to have lost its organized structure, and appeared thicker. In addition, eosonophilic material could be seen lining the lumen of the non-inflated bladder.

In all the non-inflated swimbladders isolated from day's 5-10 p.h. (fig. 12), the epithelium lost its organized, vacuolated appearance. Proliferation of the epithelial cells occurred, creating an increase in the number of cell layers. The internal layer of epithelial cells lost their columnar appearance and their nuclei were no longer situated 41 apically. The width of the hyperplastic epithelium was non-uniform. Cell’s cytoplasm appeared eosinophilic, due to paucity in amorphous material. In some bladders, abnormal folds of the epithelium occurred, possibly due to its continuous growth.

Quantity of the eosinophilic material lining the lumen increased. The caudal extension appeared inflated and its lumen continued to expand until the 10th day p.h. The rete mirabile grew in length and width.

On the 11-12 day post hatch, all larvae inspected in both cohorts had an inflated swimbladder.

a b

c d

Fig.11. Abnormal swimbladders in larvae of angelfish Pterophyllum scalare at day 4 p.h. a: Type 1 abnormal swimbladder, observed at 8:00 am. Epithelial cells (ep) contain little (arrow) or no amorphous material. Cells appear thinner and compressed. They retain their columnar appearance, organization around the lumen (lu) and apically situated nuclei. A primordial rete mirabile (prm) and an un-inflated caudal extension (ce) can be seen. H&E Bouin’s fixation. Bar = 70 μm. b, d: Type 2 abnormal swimbladder, observed at 2:00 pm. The epithelium folds on itself (arrow) on the lateral left side (b) but does not appear folded on the lateral right side (d). The cells retain their vacuolated appearance with an apically situated nuclei. H&E Bouin’s fixation. Bar = 70 μm. c: Type 3 abnormal swimbladder, observed at 2:00 pm. The epithelial cells (ep) contain little or no amorphous material. The epithelium appears disorganized on the ventral area of the swimbladder (arrow). Eosinophilic material lines the lumen (lu). H&E Bouin’s fixation. Bar = 70 μm. 43

a b

c d

Fig. 12. Abnormal swimbladders in larvae of angelfish Pterophyllum scalare. a: At day 6 p.h. 9:00 am. Proliferation of swimbladder epithelium (ep) in non-inflated swimbladders occurs, creating a lining epithelium with hyperplastic appearance. Cells cytoplasm appears uniformly eosinophilic due to paucity in amorphous material. Quantity of the eosinophilic material lining the swimbladder lumen (arrow) increases in some bladders. The caudal extension (ce) appears inflated. H&E Bouin’s fixation. Bar = 50 μm. b, c, d: Serial sections of an abnormal swimbladder from day 8 p.h. 9:00 am. b: The rete mirabile (rm) grows in length and width; c: The caudal extension’s (ce) lumen further expands; d: The swimbladder appears folded on itself on the lateral left side (arrow). H&E Bouin’s fixation. Bar = 70 μm. 44

3.3.5. Abnormal swimbladder development in larvae hatched in 5 ppm methylene blue

Histological analysis of fish hatched in 5 ppm methylene blue, the concentration shown to significantly increase SBN, was conducted in order to compare SBN morphology to that in larvae hatched in 0.5 ppm methylene blue (normal hatching conditions). External observations refer to two cohorts, whereas the histological analysis represents only one cohort.

3.3.5.1. Hatching till day 3 p.h.

Eggs from both sticks hatched during the night between the second and third day after spawning. Larvae seemed to develop normally. A primordial swimbladder appeared for the first time during the middle of the first day p.h. (12:00 pm) between the oesophagus and the gut. Its appearance was normal in all the fish observed and included a layer of columnar epithelium surrounding a small lumen. The cells had a darkly stained cytoplasm and nuclei that were situated basally, centrally or apically. A sheath of mesenchymal cells, which would differentiate eventually into the connective tissue, surrounded the primordial swimbladder. Some of the epithelial cells appeared to be undergoing mitosis (figs. 15 a, b).

At the beginning of the 2nd day p.h. (9:00 am) the primordial swimbladder appeared elongated on the antero-posterior axis (figs. 16 a, b). The connective tissue surrounding the primordial swimbladder differentiated into a loose connective tissue in the dorsal area, and a denser connective tissue in the ventral area. As in normal development, the amorphous material appeared in the basal area of the columnar epithelial cells, towards the end of the day (9:00 pm) and the nuclei migrated apically towards the lumen. In some of the larvae, the caudal extension appeared by the end of the second day (fig. 16 c, d). 45 On the 3rd day p.h., the swimbladder’s epithelia was comprised of an inner layer of columnar epithelial cells surrounding the narrow lumen, and an outer layer of squamous epithelial cells, appearing normal for this stage of development. The cells had the same vacuolated appearance as normally seen prior to inflation and nuclei were situated apically. The caudal extension was prominent by this stage and the primordial rete mirabile extended along the ventral part of the bladder towards the posterior end (figs.

17 a, b). One swimbladder seemed to have a slightly thicker epithelium (fig. 17c) but since there was only one row of nuclei around the lumen, cell proliferation hadn’t occurred. Other organs appeared similar to those in healthy fish hatched under normal conditions.

3.3.5.2. Day 4-5 p.h.

At the beginning of the 4th day p.h. (9:00 am), only few larvae from both cohorts had inflated their swimbladders (based on external observations). But as opposed to normal development, where normal inflation occurred during the day, by the end of the 4th day less than 10% of the larvae in both cohorts had inflated their swimbladders. Figure 13 shows the frequency of swimbladder types from total examined non-inflated swimbladders in a single cohort, per hour of sample, on the 4th day p.h.. The definition and schematic drawing of the different abnormal swimbladder types is summarized in table 3 and figure 14. Histologicaly, at the beginning of the 4th day p.h., most of the non-inflated swimbladders had a normal pre-inflated swimbladder appearance

(56.25%), similar to normal development in this stage. Non-inflated swim bladders, resembling type 1 and type 2 abnormalities (figs. 18 a, b), appeared during the beginning of the day (9:00 am) in very low percentages (6.25 %). A new type of non- inflated swimbladders, type 4, in which the epithelium hasn’t lost its vacuolated appearance, but seems to be thicker and less organized possibly due to cell proliferation 46 (fig. 18d), appeared in relatively higher percentages at the beginning of the day (31.25

%). Non- inflated swimbladders resembling type 3 abnormalities were not observed at this stage. As the day proceeded (2:00 pm), the percentage of normal pre-inflated swimbladders decreased to 20%. Non-inflated swimbladders resembling type 1 abnormality appeared in higher percentage (50%) and those resembling type 2 abnormality disappeared. Non-inflated swim bladders resembling type 3 abnormality

(fig. 18c) were apparent in small numbers (10 %), while non-inflated type 4 swimbladders decreased in percentage to 20%. By the end of the day (6:00 pm), normal pre-inflated swimbladders had disappeared, while 54.54% of the non-inflated swimbladders resembled type 1 abnormalities. Non-inflated swimbladders resembling type 3 abnormalities accounted for 36.36% while non-inflated type 4 swimbladders went down to 9.09%.

Normal (n=16) (n=10) (n=11) Type 4 60 Type 2 50 Type 3 Type 1 40

Precentage of total 10

0 9:00 14:00 18:00 Time of sampling

Fig. 13. The frequency of swimbladder types from total non-inflated swimbladders in a single th cohort, per hour of sampling, in larvae exposed to 5 ppm methylene blue, on 4 day p.h. “Normal” refers to normal looking pre-inflated swimbladders.

At the beginning of the 5th day p.h. (9:00 am) only 14.8% of one cohort and 23.4% of the second had inflated their swimbladder (based on external observations) as opposed to the very high percentage of inflation under normal conditions at this day. 47 Histological sections from the 5-6 day post hatch are not shown due to bad dehydration and therefore bad sectioning.

Table 3: Summary of characteristics of different abnormal swimbladder types appearing on the 4th day p.h.

Type Appeared in: Description 0.5 / 5 ppm methylene Inner epithelial cells contain little or no amorphous 1 blue material though the cells retain their columnar appearance and organized structure around the lumen. Nuclei are situated apically 0.5 / 5 ppm methylene The epithelium folds on itself. The inner epithelial 2 blue cells retain their vacuolated columnar appearance and the nuclei are situate apically 0.5 / 5 ppm methylene The inner epithelial cells contain little or no 3 blue amorphous material and lose their organized structure and columnar appearance. In some areas nuclei are no longer situated adjacent to the lumen 5 ppm methylene blue The inner epithelial cells have not lost their 4 vacuolated appearance, but they lose their organized structure and in some areas nuclei are no longer situated adjacent to the lumen

a b

c d

Fig. 14 : Schematic illustration of different abnormal swimbladder types from the 4th day p.h.. (a) Type 1 (b) Type 2 (c) Type 3 (d) Type 4. Grey colored cells= cells that contain little or no amorphous material; White colored cells= cell that are filled with amorphous material.

48 3.3.5.3. Day 7-9 p.h.

On the 7th day p.h., most non-inflated swimbladders had a multi-layered, disorganized epithelium due to cell proliferation with cells containing varying amounts of amorphous material. When compared with the abnormal swimbladder isolated on the 7th day p.h. in larvae reared under normal conditions, the epithelia seemed to be thicker, and the swimbladders had a more rounded rather than elongate form (fig. 19a). The caudal extension inflated despite the non-inflation of the swimbladder and the rete mirabile increased in size. The eosinophilic material, lining the lumen was present. This form of abnormal swimbladders persisted till the 9th day post hatch.

3.3.5.4. Ultrastucture of the swimbladder in fish hatched in 5 ppm methylene blue

The ultrastructure of non-inflated swimbladders from larvae hatched in 5 ppm methylene blue and sampled on the 7th and 9th day p.h. is presented (fig. 20). The swimbladder presented from the 7th day appears to belong to type 1 abnormality (fig.

20a). The internal columnar epithelial cells are organized around the lumen, and contain no amorphous material. The nuclei are situated apically. Compared with the cells of the internal epithelial layer in normal pre-inflated swimbladders, these cells appear to be depleted and thinner and their outline on the baso-lateral area appears to have lost its stretch. A complex labyrinth of cytoplasmic protrusions appears to have developed from the basal area of the cells, adjacent to a blood vessel. The swimbladder presented from the 9th day p.h., appears to contain a multi layered epithelium that had lost its organized pre-inflated appearance, due to cell proliferation (fig. 20b,c,d). The cells contain no amorphous material, and have lost their columnar appearance. Blood vessels appear submerged into the epithelium, and elaborate cytoplasmic protrusions appear near the blood vessels and between the cells. 49

a b

Fig. 15. Larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue at day 1 p.h 12:00 pm. a: First appearance of the primordial swimbladder (in marked field) dorsal to the yolk sack (ys), between the gut (gu) and the oesophagus (oe), similar to normal development. The pronephric duct (pd) is seen above the gut. H&E Bouin’s fixation. Bar = 100 μm. b: The primordial swimbladder consists of a layer of columnar epithelium surrounding a small lumen. The cells have a darkly stained cytoplasm and nuclei that are situated basally, centrally or apically. Some of the epithelial cells appear to be undergoing mitosis. A sheath of mesenchymal cells (ct) surrounds the primordial swimbladder. H&E Bouin’s fixation. Bar = 25 μm.

a b

Fig. 16. Larvae of angelfish Pterophyllum scalare hatched in 5 c ppm. methylene blue at day 2 p.h. a: Sagital section of a larva at

9:00 pm. General appearance seems normal. The primordial swimbladder (psb) is situated between the oesophagus and the gut, dorsal to the yolk sack (ys). The inflated gall bladder (gb) can be seen dorsal to the liver (li) and gill filaments (gf) are starting to form. H&E Bouin’s fixation. Bar = 300 μm. b: The elongated primordial swimbladder at 9:00 am seems normal to this stage, lacking amorphous material. H&E Bouin’s fixation.

Bar = 30 μm. c: Amorphous material (arrow) appears in the basal region of the columnar epithelium (ep) at 9:00 pm. A small caudal extension (ce) can be observed. H&E Bouin’s fixation.

Bar = 50 μm.

a b

Fig. 17. Larvae of angelfish Pterophyllum scalare hatched in 5 c ppm methylene blue at day 3 p.h. a: Sagital section of a larva at 9:00 am. The larvae appear to develop normally at this stage. A non-inflated primordial swimbladder (psb) is seen above the yolk sack (ys) and a dilated gall bladder (gb) is seen above the yolk sack. H&E Bouin’s fixation. Bar = 300 μm. b: The swimbladder at 9:00 am, reaches it final form prior to inflation and appears normal. The columnar epithelial cells (ep) are filled with amorphous material in their basal area, the caudal extension (ce) appears in the posterior end and the primordial rete mirabile (prm) forms ventraly. H&E Bouin’s fixation. Bar = 60 μm. c: A non-inflated swimbladder sampled at 3:00 pm, displays an abnormal thickened epithelium in the ventral area. H&E Bouin’s fixation. Bar = 60 μm

a b

c d

Fig. 18. Non-inflated swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 4 p.h a: Non inflated swimbladder resembling type 1 abnormality at 2:00 pm. The columnar epithelium is organized around the lumen but contains little(arrow) or no amorphous material. The caudal extension appears inflated. H&E Bouin’s fixation. Bar = 70 μm. b: Non inflated swimbladder resembling type 2 abnormality at 9:00 am. The epithelium appears folded on itself ventrally. The epithelial cells retain their vacuolated appearance. H&E Bouin’s fixation. Bar = 70 μm. c: Non inflated swimbladder resembling type 3 abnormality at 2:00 pm. Epithelial cells (ep) have lost their organized structure and columnar shape, possibly due to cell proliferation. The cells contain little or no amorphous material. H&E Bouin’s fixation. Bar = 80 μm. d: Non inflated swimbladder type 4 at 9:00 am. The epithelium retains its vacuolated appearance, but appear to have lost its organized structure (arrow), possibly due to cell proliferation. H&E Bouin’s fixation. Bar = 70 μm.

a b

c Fig. 19. Non-inflated swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 7 p.h. swimbladders who failed to inflate have a multi layered disorganized epithelium due to cell proliferation. a: The non- inflated swimbladder has a rounded appearance due to proliferation of the epithelium (arrow), and an eosinophilic material lines the lumen (lu). Cells contain little or no amorphous material. H&E Bouin’s fixation. Bar = 70 μm. b: Some non- inflated swimbladders retain a larger amount of the amorphous material in their cells (arrow). H&E Bouin’s fixation. Bar = 70 μm. c: Some of the non-inflated swimbladders have a more elongated appearance rather than rounded. The caudal extension (ce) appears inflated. H&E Bouin’s fixation. Bar = 80 μm.

Fig. 20. Electron-microscopic overview of non-inflated a b swimbladders in larvae of angelfish Pterophyllum scalare hatched in 5 ppm methylene blue on day 7 p.h. 2:00 pm (a) and day 9 p.h.10:00 am (b, c, d). a: Non-inflated swimbladder resembling type 1 abnormality. The columnar cells of the inner layer of the epithelium (ep) contain no amorphous material and appear depleted. The baso-lateral cell c membrane appears to have lost d its stretch. The nuclei (n) are situated apically. A complex labyrinth of cytoplasmic protrusions (white arrow) appears to have developed from the basal area of the cells, adjacent to a blood vessel, containing an erythrocyte (e). x 3,306. b, c, d: A Multi-layered non-inflated swimbladder. The epithelium (ep) has lost its organized structure and several cell layers can be be seen. The epithelial cells have lost their columnar appearance and nuclei (n) are no longer situated apically. Blood vessels (bv) appear submerged into the epithelium, and elaborate cytoplasmic protrusions (arrows) appear near the blood vessels and between the cells. x 5,061, x 5,714 and x 3,306 respectively.

4. Discussion

SBN is one of the major obstacles in larval- rearing of many fish species. In this research we attempted to study the causes and events leading to SBN in angelfish,

Pterophyllum scalare, using three approaches: (a) a study of the occurrence of the disease on the farm where it was first encountered (b) testing the effect of various environmental conditions that might induce SBN (c) histological characterization of

SBN in angelfish larvae.

The survey of SBN in the farm revealed no apparent seasonal pattern of occurrence for the disease. This is not entirely unexpected since most of the rearing conditions in the farm are monitored and constant throughout the year (e.g. temperature, lighting regime).

From observations made in the farm on individual cohorts, there was a large variation in

SBN prevalence between different cohorts, subjected to similar rearing conditions.

Some of the parent couples were thought to produce larvae with a higher percentage of

SBN than others, though an organized follow up was not done since they were replaced rapidly (personal communication with the farm). This could suggest a genetic basis for

SBN in angelfish causing a greater susceptibility in certain individuals, especially since the degree of inbreeding in the farm is very high. Previous research revealed differences in gene expression between healthy and affected 1-month old angelfish (Zilberg et al.,

2004). By the use of subtractive hybridization of transcripts, it was revealed that the genes for Hemoglobin beta-A chain, Angiotensin- converting enzyme and slc3a2 had an increased expression in affected individuals compared with healthy individuals.

Whereas the genes for Myosin light chain 2, Troponin T2 and HSP90beta had a decreased expression in affected individuals compared with healthy ones. Whether these differences are associated with SBN formation or result from it was unknown. Harrell et al. (2002) suggested that SBN in striped bass might be predominantly influenced by dominance or epistatis because of low heritability estimates for half sibling families vs. 56 moderate estimates for full sibling families. If indeed SBN is controlled by a recessive allele or by a gene that is suppressed by another gene, high degrees of inbreeding as seen in the farm could induce the expression of the disease.

In the present research we chose to test the effect of environmental and rearing conditions used in the farm on SBN in angelfish.

Angelfish were able to inflate their swimbladders while denied access to the water surface. This ability to inflate underwater was previously described in jewelfish

Hemichromis bimaculata and Tilapia mossambica, both of which are pure physoclistes from the Chilclid family (Doroshev and Cornacchia, 1979; McEwen, 1940). Though other Cichlids like Oreochromis grahami and Oreochromis niloticus possess a pneumatic duct prior to inflation, their need to reach the water surface hasn’t been investigated (Maina, 2000; Morrison et al., 2001). This ability was also reported in the transient-physostome zebrafish, Danio rerio (Goolish and Okutake, 1999; Pelster and

Burggren, 1996), and in the physostome European eel, Anguilla anguilla (Zwerger et al., 2002), contradicting the general assumption that both transient physostomes and physostomes initially inflate their bladders by gulping air at the water surface. In

European eel, inflation when denied access to the water surface was ascribed to artifacts in the experiment in which small-undetected bubbles accidentally entered the rearing tanks (Zwerger et al., 2002), but in zebrafish this was attributed to a different mechanism for inflation, involving hemoglobin transferred O2 (Pelster and Burggren,

1996). The ability of angelfish to inflate their swimbladders while being denied access to the water surface indicates that they use a mechanism for initial inflation, independent of gulping atmospheric air at the water surface. The significant difference in size between the larvae grown in boxes vs. the larvae grown in the surrounding tank did not have an effect on initial inflation and might be a result of the confinement of the boxes (Blaxter, 1988) as both treatments received the same amount of food. 57 The effect of different feed concentrations and an initial two-day starvation period was tested due to the proximity of initial exogenous feeding to initial swimbladder inflation.

We hypothesized that perhaps overfeeding of the larvae in the farm is creating an organic overload and by that impairing inflation. Ingestion of organic debris into the swimbladder via the pneumatic duct has been shown to impede inflation in the transient physostome walleye (Marty et al., 1995). Although we did achieve organic overload

(Ammonia levels reached 1 ppm in the higher feeding concentrations), this did not seem to have a significant effect on SBN in angelfish. Starvation of teleost larvae may cause: changes in growth and morphology, changes in the height of gut epithelia, shrinkage of the liver, changes in biochemical components of the larvae’s body, a decrease in the

RNA/DNA ratio, a decrease in sinking rate or an increase in buoyancy (due to increasing hypotonicity of body fluids and a decrease in body proteins), and a decrease in foraging efficiency (Blaxter, 1988). The initial two-day starvation had no effect on

SBN in angelfish although they were significantly smaller, as expected.

Tap water used in the farm originates from underground water wells in the area of

Hazeva, Israel. The water is mildly saline reaching a conductivity of 2.5 ms and is therefore pre-treated prior to drinking. In the farm, the tap water does not receive any treatment before use, except for overnight aeration to discard chlorine. Angelfish occur naturally in the soft and slightly acidic water of the Amazon River (Gobel and Mayland,

1998). Though larvae are hatched in the farm in a mixture of tap water and reverse osmosis that produces a conductivity of 400μs, the water is gradually replaced with tap water already from the 2nd day post fertilization. We tested the effect of different water types on the prevalence of SBN, and found no significant differences. A similar preliminary experiment was conducted in the farm using their own tap water (not presented), which showed no significant differences in SBN prevalence. The larvae are hatched and reared in the farm in transparent 20L aquaria. We tested the effect of 58 different tank colors on SBN in angelfish and found no significant effect. Bright tank colors affected swimbladder inflation in striped bass larvae, which confused the bright tank walls with the reflective water surface (Martin-Robichaud and Peterson, 1998).

This result in angelfish is consistent with the finding that angelfish do not require access to the water surface for initial inflation.

Methylene blue (MB) caused a significant increase in SBN when applied in high concentrations. Methylene blue (C16H18ClN3S ) is a synthetic cationic thiazine dye, widely applied in modern medicine (Schrimer et al., 2003). Some of its uses include: identification of anatomic and pathologic structures, diagnosis and targeted therapy for cancer (Cragan, 1999), treatment for methemoglobinemia (Bradberry et al., 2001), an antimalarial agent (Schirmer et al., 2003), and an experimental treatment of NO- mediated vasodilatory and septic shock (Faber et al., 2005; Haibo et al., 1995). It was also used in intra-amniotic injection, to detect premature rapture of the fetal membrane, and during genetic amniocentesis in di-amniotic twin pregnancies, to ensure sampling of both fetal sacks (Cragan, 1999). In aquaculture, MB is used to prevent bacterial and fungal contamination of eggs and as a treatment for ectoparasites of freshwater fish

(Noga, 2000). In the treatment of methemoglobinemia in humans, MB is used due to its ability to act as an electron donor in the non-enzymatic reduction of methemoglobin. An

NADPH methemoglobin reductase catalyses the reduction of MB to Leukomethylene blue (the colorless reduced form), which transfers electrons to methemoglobin non- enzymatically, restoring functional hemoglobin and becoming MB again (fig. 21)

(Bradberry et al., 2001). However, higher or repeated doses of MB have been shown to exacerbate methemoglobin formation, and cause formation of hydrogen peroxide as a by-product, causing oxidation of red-cell membranes, denaturation of hemoglobin, hemolytic anemia and Heinz body formation (Bradberry et al., 2001; Cragan, 1999;

Faber et al., 2005). 59

Fig. 21: Mechanism of methemoglobin reduction by MB (Bradberry et. al., 2001)

Methylene blue is experimented as a possible treatment for vasodilatory and septic shock in humans due to its activity as an NO-mediated vasodilatation inhibitor (Faber et al., 2005; Haibo et al., 1995). Methylene blue inhibits the production of NO from L- arginine in the vascular endothelium, by NO-synthases (NOS) (Faber et al., 2005).

Methylene blue also inhibits guanylil cyclase which catalyses the conversion of GTP to c-GMP (Faber et al., 2005). By this it prevents smooth muscle relaxation and vasodilatation signaled by the NO/c-GMP signaling pathway and the PGI-2/c-AMP signaling pathway (Cragan, 1999; Faber et al., 2005) as illustrated by figure 22.

From what is known of its biological activity, several plausible theories about the way

MB inhibits initial inflation in angelfish could be raised at this point. Despite that, it would be wiser to discuss first the histomorphological characteristics of normal vs. abnormal swimbladder development in angelfish.

Fig. 22. The inhibiting effect of MB on vasodilation via the NO/c-GMP and PGI-2/c-AMP signaling pathways (Faber et al., 2005)

During normal development, the primordial swimbladder in angelfish first appeared at the end of the 1st day p.h. (day 0 p.h – day of hatching). The simple primordial swimbladder morphology at this stage resembled the primordial swimbladders seen in striped trumpeter (Trotter et al., 2004), largemouth bass (Johnston, 1953), Dover sole

(Boulhic and Gabaudan, 1992) and jewelfish (McEwen, 1940). However, no connection between the swimbladder and the gut was observed. In most fish species, the swimbladder develops as a simple out-pocketing of the alimentary canal (Boulhic and

Gabaudan, 1992; Govoni and Hoss, 2001; Johnston, 1953; McEwen, 1940; Steen, 1970;

Trotter et al., 2004). In other species the swimbladder may develop as an unconnected mass of mesodermal cells that are later invaded by the pneumatic duct formed from an evagination of endodermal cells from the gut (Hoar, 1937). A third option is that the swimbladder originates as an independent mass of cells unidentified as to germ cell origin that later sends out a connection to the gut (Govoni and Hoss, 2001). In

Transient physostomes and physostomes, this primary connection develops into the pneumatic duct (Steen, 1970), whereas in the pure physoclist jewelfish, it eventually 61 disconnects prior to initial inflation (McEwen, 1940). We would therefore expect to see a primary connection between the gut and the primordial swimbladder in angelfish. The possible reasons for its absence could be: (a) the sagittal orientation of the sections making it harder to identify (b) it might have disappeared before hatching. The second explanation is unlikely because the primordial swimbladder appeared 2 days after hatching. Creating transverse sections of larvae in this day could give us further insight into this issue.

The general growth in size and shape of the primordial swimbladder in angelfish, prior to inflation, its location and the development of the surrounding connective tissue resembles that of other perciform species (Doroshev and Cornacchia, 1979; Johnston,

1953; McEwen, 1940; Morrison et al., 2001; Trotter et al., 2004). Epithelial vacuolization prior to inflation has also been reported in several fish species. In the transient physostomes, largemouth bass and striped bass, vacuoles appeared in the apical region of the epithelial cells of the ventral area of the swimbladder prior to inflation (Doroshev and Cornacchia, 1979; Johnston, 1953). However, vacuolization of the entire epithelium, and the basal location of the vacuoles in the epithelial cells observed prior to initial inflation in angelfish, were reported only from other members of the Cichlid family: jewelfish, Oreochromis mossambicus and Nile tilapia (Doroshev and Cornacchia, 1979; McEwen, 1940; Morrison et al., 2001), and therefore appear to be unique to Cichlids. Since jewelfish, Oreochromis mossambicus and angelfish were able to inflate their swimbladder without access to the water surface, and since epithelial vacuolization occurred near the time of initial inflation, it would appear that this vacuolization might have an important role in a unique mechanism of swimbladder inflation that does not involve gulping air from the water surface. The appearance of a primordial rete mirabile and caudal extension prior to inflation has also been seen in other fish species (Boulhic and Gabaudan, 1992; Doroshev and Cornacchia, 1979; 62 Marty et al., 1995; Morrison et al., 2001; Trotter et al., 2004), though in some species the rete mirabile develops in later stages (Govoni and Hoss, 2001; Johnston, 1953;

McEwen, 1940). This caudal extension resembles the “small cavity” reported from jewelfish, developing from the remnants of the primary connection between the primordial swimbladder and the gut (McEwen, 1940), and also to the “vesicle” reported from largemouth bass developing from the rudiments of the pneumatic duct (Johnston,

1953). It would seem reasonable that the caudal extension would develop into the posterior portion of the swimbladder seen in adult angelfish.

In angelfish, initial inflation occurs on the 4th day p.h., in contrast with what was previously reported in an earlier study that suggested that an inflated or dilated swimbladder with a pneumatic duct was present as early on as one day p.h in angelfish

(Zilberg et al., 2004). This was a misidentification, and the dilated organ observed on the 1st day p.h. was in fact the gall bladder, located between the yolk sack and the digestive tract, connected to the gut by the common bile duct. Timing of initial inflation on the 4th day p.h., coincides with commencement of swimming and exogenous feeding as reported in other fish species (Battaglene and Talbot, 1990; Boulhic and Gabaudan,

1992; Doroshev and Cornacchia, 1979; Morrison et al., 2001; Soares et al., 1994; Tait,

1960). The lag observed between the inflated appearance of the swimbladders in most of the larvae (externally and histologically) at the end of the 4th day and swimming which initiated sometime between the 4th and the 5th day, could be caused by the fact that yolk depletion was not completed on the 4th day, or that inflation had not reached a sufficient volume for neutral buoyancy. No substance was seen filling the bladder lumen in the histological sectioning, suggesting that a liquid dilation phase, prior to inflation, does not occur in angelfish. A liquid dilation phase was also not reported from any of the Cichlids showing similar development of the swimbladder. This matter requires further investigation, perhaps with the use of a simple experiment proposed by 63 McEwen (1940) which dissected jewelfish larvae underwater, upon initial inflation, and observed a gas bubble emerging from the inflated swimbladder, thus confirming the gas content of the swimbladder.

The mechanism of initial inflation is unclear. No apparent pneumatic duct was observed during inflation, suggesting that angelfish belong to the pure physoclist classification.

Since angelfish presented no need to reach the water surface for inflation, the question as to the source of gas for the initial filling of the swimbladder remains open. Previous researches suggested two theories about the source of gas for initial inflation in pure physoclistous Cichlids: (a) active secretion of gas into the swimbladder from the circulatory system via the swimbladder epithelium (Doroshev et al., 1981) (b) internal

CO2 production within the epithelial cells by disintegration of organic material

(McEwen, 1940). Doroshev et al. (1981) further observed a decrease in initial inflation in hypoxic conditions in Oreochromis mossambicus emphasizing the connection between the concentration of dissolved oxygen in the ambient water and initial inflation, and supporting the first theory. Both theories suggest that the vacuoles seen in the epithelial cells prior to inflation are gaseous and are subsequently secreted into the bladder upon inflation. To support this theory, attempts to stain these vacuoles in jewelfish with H&E, Mallory’s connective tissue stain (stains collagen fibers, reticular fibers, elastic fibers, nuclei and smooth muscle, red blood cells and myelin) and mucicarmine (stains epithelial mucin) had failed (McEwen, 1940).

In angelfish, the appearance of the amorphous material (creating the vacuolated appearance) in the epithelial cells near the time of inflation, and its disappearance upon inflation, could imply a connection to the inflation process. Our attempts to stain the amorphous material with H&E, PAS staining (stains carbohydrate-macromolecules such as glycogen, glycoprotein and proteoglycans), Lillies alochrome (stains nuclei, cytoplasm, muscle cells, collagen, reticular connective tissue and glomerular basement 64 membrane), alciane blue staining (stains polysaccharides and nuclei), uranyl acetate

(stains membranous structures and structures containing nucleic acids) and lead citrate

(stains RNA-containing structures and carbohydrates) had also failed. However, some findings are inconsistent with the previously suggested theories concerning its nature:

(a) if indeed the amorphous material is gas intended to be secreted into the swimbladder, why does it appear mainly on the basal region of the cell? The basal location of the majority of the material would imply secretion to the basal region instead. (b) From the electron microscopy images, the material has an amorphous morphology, rather than a vacuolated morphology, expected from a highly hydrophobic substance as gas. (c) The amorphous material started appearing on the 2nd day p.h while the capillary network of the rete mirabile seemed undeveloped yet. (d) The amorphous material seemed to fill all the epithelial cells and not only the ones adjacent to the primordial rete mirabile.

An alternative hypothesis on the role of the amorphous material in inflation in angelfish could be as an internal pressure producer (illustrated in fig. 23). The amorphous material increases inner cellular pressure prior to inflation, stretching the cell membrane and enlarging the overall cell surface. When inflation occurs, the material is disposed of

(from the basal region of the cell) causing a sudden increase in the cell's surface to volume ratio. This "extra surface membrane" minimizes the resistence of the cells to the stretching created by the first filling with gas, allowing them to easily assume their final squamous shape, with minimum risk of bursting. Gas for initial inflation, could originate from a different source, possibly from the circulatory system via the rete mirabile. In order for this to happen, the gas gland cells, producing and secreting acidic metabolites into the blood stream (in order to reduce the effective gas carrying capacity of the blood), have to be functional (Zwerger et al., 2002). We have no proof that indeed they are, but the existence prior to inflation of a primordial rete mirabile in close 65 proximity to the epithelium on one hand, and a labyrinth of cytoplasmic protrusions in

the baso-lateral area of the columnar epithelial cells (enlarging their cell surface) on the

other hand, could imply on exchange of substances between the epithelium and the

circulatory system. The cytoplasmic protrusions resemble the “basal labyrinth”

described in functional gas glands cells of adult teleosts, adjacent to the vascular supply

(Maina, 2000; Prem et al., 2000; Zwerger et al., 2002). On the other hand the

membranal proliferation could be a preparatory phase to initial inflation, ensuring ample

surface plasmalema during inflation.

ex ep mv lu in in plu ex am am bv

bv mv lu

in ex

bv am

Fig. 23. A model of the changes in epithelial cell morphology appearing during normal swimbladder development in angelfish, Pterophyllum scalare. Prior to inflation, the primordial swimbladder is composed of a two-layered epithelium (ep), an internal columnar layer (in) and an external squamous layer (ex) surrounding the primordial lumen (plu). The cells of the internal layer are filled with amorphous material (am) in their basal area, which is applying internal pressure, stretching the cell membrane and allowing it to grow a large surface area. They also have a labyrinth of cytoplasmic protrusions (cp) on their baso-lateral area, in contact with adjacent blood vessels (bv) and microvilli (mv) in their apical area. During inflation, gas from the circulatory system fills the lumen (lu) and the cells of the internal epithelial layer secrete their amorphous material towards the underlying connective tissue. This causes a sudden increase in their surface to volume ratio allowing them to easily assume their final squamous shape, with minimum resistance to the stretching and minimum risk of bursting. The labyrinth acts as a supply of the necessary surface plasmalema. 66 Seeing that the event of initial inflation requires a joint effort and coordination of so many different factors, it is not surprising that many things could go wrong. And indeed, abnormal swimbladders first appeared under routine conditions on the 4th day p.h, the day of initial inflation. The first type of abnormality had an organized epithelium that contained little amounts of amorphous material in the columnar epithelial cells. Since all the swimbladders contained amorphous material in their cells until the 3rd day p.h., this paucity in amorphous material on the 4th day, indicates its disposal rather than its total absence during development. The loss of vacuolated appearance has been described in other Cichlids upon inflation, when epithelial cells transformed from columnar to squamous (Doroshev et al., 1981; McEwen, 1940), but it hasn’t been associated with SBN. The effect of the disposal of amorphous material on

SBN is yet to be determined. The second type of abnormal swimbladders, consisted of swimbladders with folded epithelium. Folds in the epithelium of non-inflated swimbladders were reported before in gilthead seabream and striped trumpeter

(Paperna, 1978; Trotter et al., 2004), appearing as a result of inflation failure due to lumenal collapse. In angelfish, this swimbladder type appeared prior to inflation, although this does not necessarily mean that it will lead to SBN. The 3rd type of abnormal swimbladders, consisted of swimbladders that had lower amounts of amorphous material in their epithelial cells together with disorganizing of the epithelium in their ventral area. This disorganization could have resulted on the one hand from the onset of cell proliferation, which continued in abnormal swimbladders over the next few days, along with paucity in amorphous material in the cells, increased amount of eosinophilic material lining the lumen, proliferation of the rete mirabile and inflation of the caudal extension. However, the loss of the columnar appearance of the cells on the ventral area could also indicate a failed attempt of inflation, and collapse of the swimbladder. Proliferation of swimbladder epithelium and rete mirabile was 67 described in different species as a result of SBN (Padros et al., 1993; Paperna, 1978;

Trotter et al., 2004) and is consistent with the abnormal morphology of the swimbladder reported in adult affected angelfish (Zilberg et al., 2004). Eosinophilic material lining the lumen has been reported before in Oreochromis mossambicus, during and after inflation (Doroshev et al., 1981). This could be the surface-active surfactant, reported lining the gas-liquid interface of the epithelial border in the swimbladders of several teleosts (Daniels et al., 2004; Prem et al., 2000; Zwerger et al., 2002). Inflation of only one part of the swimbladder was reported in common carp exposed to copper and cadmium (Korwin-Kossakowski, 1988), and extreme dilation of the pneumatic duct associated with SBN was reported in turbot, Scophthalmus maximus (Padros, et al.,

1993). The inflation of the caudal extension in angelfish indicates that the mechanism of initial inflation is not entirely defected. Otherwise gas for inflation would not have reached the caudal extension. It would appear that inflation of the caudal extension would be easier than inflation of the swimbladder because it is smaller. If indeed the source of gas for inflation is the circulatory system, the close proximity of the rete mirabile to the caudal extension, along with its higher surface to volume ratio, could explain why its inflation is not impaired. Its inflation could also be a result of passage of gas from the anterior part of the swimbladder that failed to inflate.

Little information can be deducted from the small amount of abnormal swimbladders obtained in this experiment, as to the effect of the different abnormalities seen on SBN formation. Whether these abnormalities eventually lead to SBN or whether they are caused by it. A cohort with a higher percentage of SBN has to be used. Obtaining eggs with a high prevalence of SBN from the farm was not possible at this stage of the research, because cohorts were not bred separately, and so a follow up of SBN prevalence per couple was not done. Moreover, the heritability of SBN as a trait in anglefish has not been studied. We therefore decided to inspect SBN induced by 68 5 ppm MB, knowing that MB will increase our sample size and perhaps teach us more on the etiology of the disease. From the histological observations, SBN induced by 5 ppm MB appeared similar to SBN seen in routine conditions (0.5 ppm MB). In both cases development appeared normal until the 3rd day p.h and abnormal swimbladders appeared only on the 4th day p.h.. Similar types of abnormal swimbladders were observed on the 4th day in both conditions (types 1, 2 and 3), although type 4 appeared only in 5 ppm MB. Since a high percentage of the non-inflated swimbladders on the 4th day appeared abnormal as a result of exposure to 5 ppm MB, we could use the changes in frequencies of the different types to study the connection between them and the transformation from one type to another. During the 4th day, normal appearing pre- inflated swimbladders, type 2 and type 4 abnormalities (all of which had a high content of amorphous material in their epithelial cells) reduced in frequency while inflated swimbladders, types 1 and type 3 abnormalities (all of which had a low content of amorphous material in their cells) increased in frequency (Fig. 13). It would seem that the main event, taking place during the 4th day, is the disposal of the amorphous material, whether inflation occurs or not. Cell proliferation and epithelial folding does occur on the 4th day (types 2, 3 and 4), but it seems to be more prominent in the next few days. The morphology of non-inflated swimbladders after the 4th day p.h. was also similar between larvae exposed to 5 ppm MB and larvae reared under routine conditions. Most of the non-inflated swimbladders after the 4th day p.h. showed epithelial proliferation with varying amounts of amorphous material in the epithelial cells, larger amounts of eosinophilic material lining the lumen, inflation of the caudal extension and growth of the rete mirabile. However, the non-inflated swimbladders in 5 ppm MB did appear to have faster proliferation than those seen in routine conditions, which could imply on a separate effect caused by the exposure to 5 ppm MB. The ultrastructure of non-inflated swimbladders from the 7th and 9th day post hatch revealed 69 an abnormal development of the basal labyrinth. The membranal proliferation process appears to be a preparatory phase to normal initial inflation, serving to create the surface membrane needed for the flattening of the epithelial cells, and possibly allowing better exchange of metabolites between the epithelial cells and the blood vessels (fig. 23). It probably continues after inflation is successfully achieved, to allow growth of the swimbladder. In SBN fish the generation of extra membranes is ineffective and therefore results in the formation of dysfunctional “mega” labyrinths.

These similarities in morphology between SBN induced by 5 ppm MB and SBN created under routine conditions could imply that they are generated in similar ways. But does this mean that MB is the major cause for SBN in the farm, or are there other factors that haven’t been revealed yet?

Methylene blue is generally applied in the farm throughout the year, and all the larvae are exposed to it. In human medicine, evidence for MB toxicity and teratogenicity has been reported. The appearance of hyperbilirubinemia, hemolytic anemia, blue staining of the skin and methemoglobinemia among newborns, was associated with intra- amniotic injection of MB during obstetric procedures (Cragan, 1999). Methylene blue injected during mid-trimester amniocentesis was also associated with Jejuno-ileal atresia (small bowel atresia) in newborn babies (Cragan, 1999). Cragan (1999) suggested a few possible explanations for MB-induced atresia: (a) The vasoactive properties of MB in vitro, might induce arterial constriction in vivo, that would impair blood supply to the small bowel causing ischemic damage and atresia (b)

Methemoglobienemia and hemolytic anemia associated with intra-amniotic injection of

MB, might cause hypoxia that could enhance the effect of the arterial constriction and also lead to localized ischemia (c) There might be a direct toxic effect of MB on the bowel itself, causing vascular necrosis, mucosal ulceration, mural necrosis, and 70 extramural fat necrosis at the site of contact, since the amniotic fluid is swallowed by the fetus and thus the dye comes in direct contact with the bowel mucosa.

If indeed initial inflation in angelfish is associated with oxygen, or other gases transported in the circulatory system, biological activities that would damage the blood supply to the primordial swimbladder or oxygen transport capacity in the blood, like the ones demonstrated by MB in humans, could inhibit initial inflation. However, only a concentration of 5 ppm MB, ten times the concentration used in the farm, had a significant effect on SBN in our experiment. A low but existing degree of SBN was also observed in the control treatment, in larvae that were not at all exposed to MB. These findings disagree with the claim that MB is the primary cause for SBN in the farm, though it may act to enhance the effect of other factors, genetic or environmental.

Nevertheless, since similarities have been observed between SBN in routine conditions and SBN in fish exposed to 5 ppm MB, it might have a similar mechanism of effect on swimbladder development as the primary factor. Therefore, understanding the way MB in a concentration of 5 ppm inhibits initial inflation, could shed light on SBN in angelfish and bring the research a step forward. In this research we present strong evidence in vitro of MB teratogenicity to angelfish larvae in high concentrations, and therefore propose a more cautious use of the drug in the future.

The mechanism of initial inflation in teleosts in general, and in pure physoclist Cichlids specifically, is poorly understood. SBN in pure physoclistous fish has been reported only from one other fish species, and also has not been regarded as a phenomenon, rendering this phenomenon unique to angelfish amongst Cichlids. Since angelfish are very easy to breed, with high fecundities and small spatial requirements, using them as a research model of both initial inflation in pure physoclists and analysis of the factors inhibiting it, could promote larval rearing of Cichlids in the future, and possibly other teleosts as well. 71 5. Further suggested research

1. Further studying the mechanism of normal inflation in anglefish

a. Revealing the nature of the amorphous material - if the amorphous

material is a protein, an attempt to isolate the swimbladder prior to

inflation from live fish, and compare the protein content of the epithelial

cells to an inflated swimbladder (which would have disposed of the

amorphous material) might prove useful in identifying it. If it is of other

nature, perhaps different biochemical methods of identification could be

used.

b. Studying whether the swimbladder initially inflates with gas or dilates

with liquid on the 4th day p.h. using the experiment proposed by McEwen

(1940), which suggests dissecting the larvae and swimbladder under

water.

2. Studying the mechanism by which MB affects SBN in angelfish, by imitating its

biological effect in different means and comparing the result.

a. Generating methemoglobinemia with the use of high CO levels or

phenylhydrazine as proposed by Pelster and Burggren (1996).

b. Inhibiting the NO/cGMP pathway specifically by the use of specific

inhibitors such as Nωnitro-L-aginine, a non-selective inhibitor of nitric

oxide synthase (Schwerte et al., 1999).

3. Testing other environmental factors that have not been tested in this study such

as brood-stock nutrition, water quality parameters (pH, temperature, nutrients

and oxygen concentration).

4. Testing the heritability of SBN in angelfish, initially by comparing SBN

prevalence in different cohorts originating from the same parents (full siblings).

72 6. Reference

Bailey, H.C., Doroshev, S.I., 1995. The duration of the interval associated with

successful inflation of the swimbladder in larval striped bass (Morone saxitilis).

Aquaculture 131, 135-143.

Battaglene, S.C., Talbot, R.B., 1990. Initial swimbladder inflation in intensively reared

Australian bass larvae, Macquaria novemaculeata (Steindachner)

(Perciformes:Percichthyidae). Aquaculture 86, 431-442.

Battaglene, S.C., Talbot, R.B., 1993. Effects of salinity and aeration on survival and

initial swim bladder inflation in larval Australian bass. The progressive fish-

culturist 55, 35-39.

Battaglene, S.C., McBridge, S., Talbot, R.B., 1994. Swim bladder inflation in larvae of

cultured sand whiting, Sillago ciliata Cuvier (Sillaginidae). Aquaculture 128,

177-192.

Blaxter, J.H.S., 1988. Pattern and variety in development. In: Hoar, W.s., Randall, D.J.

(Eds.), Fish Physiology. Academic Press Inc., New York, pp. 1-58.

Boulhic, M., Gabaudan, J., 1992. Histological study of the organogenesis of the

digestive system and swim bladder of the Dover sole, Solea solea (Linnaeus

1758). Aquaculture 102, 373-396.

Bradberry, S.M., Aw, T., Williams, N.R., Vale, J.A., 2001. Occupational

methemoglobinemia. Occup. Environ. Med. 58, 611-616.

Carson, F.L., 1997. Histotechnology: A Self-Instructional Text. ASCP Press, American

Society of Clinical Pathologists, Chicago, 304 pp.

Chapman, D.C., Hubert, W.A., 1988. Influence of access to air and of salinity on gas

bladder inflation in striped bass. Progressive Fish Culturist 50, 23-27.

Chatain, B., Ounais-Guschemann, N., 1990. Improved rate of initial swimbladder

inflation in intensively reared Sparus auratus. Aquaculture 84, 345-353. 73 Cragan, J.D., 1999. Teratogen uptake: Methylene blue. Teratology 60, 42-48.

Daniels, C.B., Orgeig, S., Sullivan, L.C., Ling, N., Bennet, M.B., Schurch, S., Val,

A.L., Brauner, C.J., 2004. The origin and Evolution of the surfactant system in

fish: insights into the evolution of lungs and swim bladders. Physiological and

Biochemical Zoology 77, 732-749.

Doroshev, S.I., Cornacchia, J.W., 1979. Initial swim bladder inflation in the larvae of

Tilapia mossambica (Peters) and Morone saxatilis (Walbaum). Aquaculture 16,

57-66.

Doroshev, S.I., Cornacchia, J.W., Hogan, K., 1981. Initial swim bladder inflation in the

larvae of physoclistous fishes and its importance for larval culture. Rapp. P. -v.

Reun. Cons. int. Explor. Mer. 178, 495-500.

Egloff, M., 1996. Failure of swim bladder inflation of perch, Perca fluviatilis L. found

in natural populations. Aquatic Sciences 58, 15-23.

Evans, D.H., 1998. The Physiology of Fishes. CRC Press.

Faber, P., Ronald, A., Millar, B.W., 2005. Methylthioninium chloride: pharmacology

and clinical applications with special emphasis on nitric oxide mediated

vasodilatory shock during cardiopulmonary bypass. Anaesthesia 60, 575-587.

Glauert, A.M., 1975. Fixation, dehydration and embedding of biological specimens.

North-Holland Publishing Company, Amsterdam, 207 pp.

Gobel, M., Mayland, H.J., 1998. South American Cichlids. IV (Discus, scalare). Verlag

A.C.S. GmbH, Morfelden-Walldorf, Germany.

Goolish, E.M., Okutake, K., 1999. Lack of gas bladder inflation by the larvae of

zebrafish in the absence of an air-water interface. Journal of fish biology 55,

1054-1063.

74 Govoni, J.J., Hoss, D.E., 2001. Comparison of the development and function of the

swimbladder of Brevoortia tyrannus (Clupeidae) and Leistomus xanthurus

(Sciaenidae). Copea 2, 430-442.

Haibo, Z., Peter, R., Jean-Charles, P., Herbert, S., Panayotis, M., Gaetane, M., Jean-

Louis, V., 1995. Effects of methylene blue on oxygen availability and regional

blood flow during endotoxic shock. Critical care medicine 23, 1711-1721.

Hall, T.E., Smith, P., Jhonston, I.A., 2004. Stages of embryonic development in the

Atlantic cod Gadus morhua. Journal of morphology 259, 255-270.

Hamm, J.T., Hinton, D.E., 2000. The role of development and duration of exposure to

the embryotoxicity of diazinon. Aquatic Toxicology 48, 403-418.

Harel, M., Place, A.R., 1998. The nutritional quality of live feeds for larval fish.

Bulletin of the Aquaculture Association of Canada 98, 6-11.

Harrell, R.M., Van Heukelem, W., Jacobs, J.M., Schutz, J.R., Takacs, J.U., Jacobs, D.,

2002. Heritability of swimbladder inflation in striped Bass. North American

Journal of Aquaculture 64, 117-121.

Henry, T.R., Spitsbergen, J.M., Hornung, M.W., Abnet, C.C., Peterson, R.E., 1997.

Early life stages toxicity of 2,3,7,8- Tetrachlorodibenzo-p-dioxin in zebrafish

(Danio rerio). Toxicology and applied pharmacology 142, 56-68.

Hoar, W.S., 1937. Development of the swim bladder in the Atlantic salmon. Journal of

Morphology 61, 309-319.

Jacquemond, F., 2004. Separated breeding of perch fingerlings (Perca fluviatilis L.)

with and without initial inflated swim bladder: comparison of swim bladder

development, skeleton conformation and growth performances. Aquaculture

239, 261-273.

75 Johnston, P.M., 1953. The embryonic development of the swim bladder of the

largemouth black bass Micropterus salmoides salmoides (Lacepede). J.

Morphol. 93, 45-67.

Kim, Y., Cooper, K.R., 1999. Toxicity of 2,3,7,8-Tetrachlorobenzo-p-Dioxin (TCDD)

and Polychlorinated Biphenyls (PCBs) in the embryos and newly hatched larvae

of the Japanese medaka (Oryzias latipes). Chemosphere 39, 527-538.

Korwin-Kossakowski, M., 1988. Larval development of carp, Cyprinus carpio L., in

acidic water. J. Fish. Biol. 32, 17-26.

Lewis, P.R., Knight, D.P., 1977. Staining methods for sectioned material. North-

Holland Publishing Company, Amsterdam, 311 pp.

Maina, J.N., 2000. Functional morphology of the gas-gland cells of the air-bladder of

Oreochromis alcalicus grahami (Teleostei: Cichlidae): an ultrastructural study

on a fish adapted to a severe, highly alkaline environment. Tissue & Cell 32,

117-132.

Martin-Robichaud, D.J., Peterson, R.H., 1998. Effects of light intensity, tank colour and

photoperiod on swimbladder inflation success in larval striped bass, Morone

saxatilis (Walbaum). Aquaculture research 29, 539-547.

Marty, G.D., Hinton, D.E., 1995. Oxygen consumption by larval Japanese medaka with

inflated or uninflated swim bladders. Transactions of the American fisheries

society 124, 623-627.

Marty, G.D., Hinton, D.E., Summerfelt, R.C., 1995. Histopathology of swimbladder

noninflation in walleye (Stizostedion vitreum) larvae: role of development and

inflammation. Aquaculture 138, 35-48.

McEwen, R.S., 1940. The early development of the swim bladder and certain adjacent

parts in Hemichromis bimaculata. J. Morphol. 67, 1-57.

76 Morris, S. M., Albright, J.T., 1975. The ultrastructure of the swimbladder of the

Toadfish, Opsanus tau L. Cell and Tissue Research 164, 85-104.

Morrison, C.M., Miyake, T., Wright, J.R., 2001. Histological study of the development

of the embryo and early larvae of Oreochromis niloticus (Pisces: Cichlidae). J.

Morphol. 247, 172-195.

Noga, E.J., 2000. Fish disease - diagnosis and treatment. Iowa State University Press,

367 pp.

Padros, F., Minkoff, G., Sala, R., Crespo, S., 1993. Histopathological events throughout

the development of turbot (Scophthalmus maximus L.). J. Comp. Path. 109, 321-

334.

Paperna, I., 1978. Swimbladder and skeletal deformoities in hatchery bred Sparus

aurata. J. Fish. Biol. 12, 109-114.

Pelster, B., Burggren, W.W., 1996. Disruption of hemoglobin oxygen transport does not

impact oxygen-dependent physiological processes in developing embryos of

zebra fish (Danio rerio). Circulation research 79, 358-362.

Prem, C., Salvenmoser, W., Wurtz, J., Pelster, B., 2000. Swim bladder gas gland cells

produce surfactant: in vivo and in culture. Am J Physiol Regulatory Intergrative

Comp Physiol 279, R2336-R2343.

Sarnowski, P., 2004. The effect of metals on swimbladder inflation of common carp

(Cyprinus carpio L.) larvae. Electronic journal of polish agricultural universities

Schirmer, R.H., Coulibaly, B., Stich, A., Scheiwein, M., Merkle, H., Eubel, J., Becker,

K., Becher, H., Muller, O., Zich, T., Schiek, W., Kouyate, B., 2003. Methylene

blue as an antimalarial agent. Redox Report 8, 272-275.

77 Schwerte, T., Holmgren, S., Pelster, B., 1999. Vasodilation of swimbladder vessels in

the Europeal eel (Anguilla anguilla) induced by vasoactive intestinal

polypeptide, nitric oxide, adenosine and protons. The Journal of Experimental

Biology 202, 1005-1013.

Sheehan, D.C., Hrapchak, B.B., 1980. Theory and practice of histotechnology. CV

Mosby Co., St. Louis (MO), 481 pp.

Soares, F., Dinis, M.T., Pousao-Ferreira, P., 1994. Development of the swim bladder of

the cultured Sparus aurata L.: a histological study. Aquaculture and fisheries

managment 25, 849-854.

Steen, J.B., 1970. The swimbladder as a hydrostatic organ. In: Hoar, W.J., Randall, D.J.

(Eds.), Fish Physiology. Academic Press, New York, pp. 413-443.

Summerfelt, R.C., 1991. Non-inflation of the gas bladder of larval walleye

(Stizostedion vitreum): experimental evidence for alternative hypotheses of its

etiology. In: Lavens, P., Sorgellos, P., Jaspers, E., Ollevier, F. (Eds.), Larvi '91 –

Fish & Crustacean larviculture symposium. European Aquaculture society,

Gent. Belgium.

Swann, L., 1994. Reproduction of angelfish (Pterophyllum scalare). Illinois - Indiana

Sea Grant Program, Aquaculture extension, Purdue University. Fact sheet AS-4,

6pp.

Tait, J.S., 1960. The first filling of the swim bladder in Salmonoids. Can. J. Zool. 38,

179-187.

Tandler, A., Harel, M., Koven, W.M., Kolkovski, S., 1995. Broodstock and larvae

nutrition in the gilthead seabream Sparus aurata - new findings on its mode of

involvement in improving growth, survivel and swimbladder inflation. The

Israeli Journal of Aquaculture - Bamidgeh 47, 95-111.

78 Trotter, A.J., Pankhurst, P.M., Hart, P.R., 2001. Swim bladder malformation in

hatchery-reared striped trumpeter Latris lineata (Latridae). Aquaculture 198, 41-

54.

Trotter, A.J., Pankhurst, P.M., Battaglene, S.C., 2004. Morphological development of

the swim bladder in hatchery-reared striped trumpeter Lateris lineata. J. Appl.

Ichthyol. 20, 395-401.

Trotter, A.J., Pankhurst, P.M., Battaglene, S.C., 2005. A finite interval of initial

swimbladder inflation in Latris lineata revealed by sequential removal of water-

surface films. Journal of fish biology 67, 730-741.

Trotter, A.J., Pankhurst, P.M., Battaglene, S.C., Morehead, D.T., 2003. Effects of

temperature on initial swim bladder inflation and related development in

cultured striped trumpeter (Latris lineata) larvae. Aquaculture 221, 141-156.

Zilberg, D., Ofir, R., Rabinski, T., Diamant, A., 2004. Morphological and genetic

characterization of swimbladder non-inflation in angelfish Pterophyllum scalare

(Cichlidae). Aquaculture 230, 13-27.

Zwerger, P., Nimeth, K., Wurtz, J., Salvenmoser, W., Pelster, B., 2002. Development of

the swimbladder in the European eel (Anguilla anguilla). Cell and Tissue

Research 307, 155-164. www.fishbase.org