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Agata KORZELECKA-ORKISZ 1, Izabella SMARUJ 1, Dorota PAWLOS 3, Piotr ROBAKOWSKI 2, Adam TAŃSKI 1, Joanna SZULC 1, Krzysztof FORMICKI 1*

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Agata KORZELECKA-ORKISZ 1, Izabella SMARUJ 1, Dorota PAWLOS 3, Piotr ROBAKOWSKI 2, Adam TAŃSKI 1, Joanna SZULC 1, Krzysztof FORMICKI 1* ACTA ICHTHYOLOGICA ET PISCATORIA (2010) 40 (2): 187–197 DOI: 10.3750/AIP2010.40.2.12 EMBRYOGENESIS OF THE STINGING CATFISH, HETEROPNEUSTES FOSSILIS (ACTINOPTERYGII: SILURIFORMES: HETEROPNEUSTIDAE ) Agata KORZELECKA-ORKISZ 1, Izabella SMARUJ 1, Dorota PAWLOS 3, Piotr ROBAKOWSKI 2, Adam TAŃSKI 1, Joanna SZULC 1, Krzysztof FORMICKI 1* 1 Division of Fish Anatomy, Hydrobiology, and Biotechnology of Reproduction, 2 Division of Fisheries Management of Inland Waters, West Pomeranian University of Technology in Szczecin, Poland 3 West Pomeranian Research Centre in Szczecin IMUZ, Szczecin, Poland Korzelecka-Orkisz A., Smaruj I., Pawlos D., Robakowski P., Tański A., Szulc J., Formicki K. 2010. Embryogenesis of the stinging catfish, Heteropneustes fossilis (Actinopterygii: Siluriformes: Heteropneustidae). Acta Ichthyol. Piscat. 40 (2): 187–197. Background. The stinging catfish, Heteropneustes fossilis (Bloch, 1794), has recently raised interest among fish farmers, ornamental fish keepers, and pathologists. Its natural populations are threatened due to habitat loss and high fishing pressure. A number of factors may influence the reproductive success of this. The aim of this study was to assess the effect of one of such factors—the water hardness—on the course of the embryogenesis, the structure of the egg shell, the general morphology, and the behaviour of the hatched larvae. Materials and Methods. The fertilised eggs were incubated at a constant temperature of 23 ± 0.2°C in water of different hardness: 0ºGH (soft), 9ºGH (moderately soft), 18ºGH (moderately hard). Egg membranes of activated eggs were viewed under a scanning electron microscope. Also egg membranes strength and egg deformations were determined 1.5 h after fertilisation. Images of eggs and newly hatched larvae, recorded with the observa - tion sets described above, were measured and analysed. Results. The study showed the eggs were surrounded by thin, translucent, ~5 µm thick membranes equipped with numerous outgrowths on the external surface and porous on the inner side. The following observations were made within 828 h° (degree-hour) at 23 ± 0.2°C: the highest volume of eggs (1.16 ± 0.092 mm 3) was typical for eggs incubated in soft water (0°GH), while the lowest egg volume (0.99 ± 0.113 mm 3) was recorded in eggs incu - bated in moderately soft water (9°GH); yolks spheres were greenish in colour, the embryonic disc and the embryo itself being reddish; the embryo performed diverse movements (quasi-peristaltic, cardiac muscle contractions); after few hours of hatching, the pigments developed resulting in colour appearance; club-like primordial barbels were formed 24 h after hatching; the larvae commenced feeding on day 3 post hatching. Conclusion. Water hardness influences embryonic and larval development and the effects may be diverse. Low water hardness is recommended for egg incubation. However, the situation changed when the egg membrane pro - tection ceased to exist at hatching. Increasing water hardness a few hours prior to the expected hatching time pre - vents larval deformation. Keywords: stinging catfish, Heteropneustes fossilis , embryogenesis, eggs, embryonic motorics, embryonic morphometry, water hardness INTRODUCTION ing waters. The global interest in the species is under - The stinging catfish, Heteropneustes fossilis (Bloch, standable, because—from the Far to the Middle East— 1794), known also as the liver catfish or the fossil catfish, the species is a valued food product due to its tasty, high has increasingly attracted attention of fishermen and orna - quality meat rich in protein and low in fat (22.8% and mental fish keepers (Robins et al. 1991, Kahl et al. 1997). 0.6%, respectively); in addition, the meat is a good In contrast to Europe, the stinging catfish is of high com - source of dietary iron (226 mg per100 g of body weight). mercial value in Asia. However, in Europe, the species Those qualities make it a food recommended as a diet has recently gained interest of fish farmers, particularly component for patients recovering from illnesses those operating in discharge canals of power plant cool - (Pandey et al. 2001). * Correspondence: Prof. Dr hab. Krzysztof Formicki, Katedra Anatomii, Hydrobiologii i Biotechnologii Rozrodu, WNoŻiR, Zachodniopomorski Uniwersytet Technologiczny, Szczecin, Poland; phone: (+48) 91 449 6665, e-mail: [email protected] 188 Korzelecka-Orkisz et al. In addition to the species’ value for fish farmers, the trolled-temperature laboratory of the Division of Fish stinging catfish—due to some structural and physiological Anatomy, Hydrobiology, and Biotechnology of peculiarities—has become interesting also for the orna - Reproduction, West Pomeranian University of mental fish trade. Recently, the species has become a study Technology in Szczecin. object for pathologists focusing on transmittable zoonoses. Eggs and sperm were collected from three females The stinging catfish occurs naturally in tropical waters measuring 17–25 cm (TL) and four males (10–18 cm TL), of south Asia, general in bodies of water abundant in veg - respectively. Gametes were collected from spawners etation, oxygen-depleted due to decomposition of the plant which had been injected twice with carp pituitaries (a total material, and heated by the sun throughout the water col - amount of 5 mg). The second injection was applied 8 h umn (Munshi et al. 1976, Pandey 1978, Pethiyagoda 1991, after the first. Ovulation occurred 12 h after the second Kahl et al. 1997, Jayaram 1999). injection. The eggs were fertilised using the dry method. In its native habitat, the stinging catfish attains 70 cm The sperm, collected from the genital papilla with a of total length. The sexual dimorphism is distinct; the syringe equipped with elastic tubing, was diluted in the males are smaller and have a clearly pointed genital papil - immobilising fluid consisting of 200 mM NaCl (Linhart at la. The female fecundity is body size-dependent and varies al. 1987, Linhart at al. 2004) and was used to fertilise the from 3 thousand to 45 thousand eggs (Wheeler 1977, eggs. The fertilised eggs were transferred to rectangular Baensch and Riehl 1985, Rahman 1989). baskets constituting a part of a hydromechanical device, Under natural conditions, the stinging catfish repro - ensuring constant aeration and water temperature during duces from July to August. The reproductive activity is the incubation in 60 L aquaria. To prevent lumping and to temperature dependent, with 22°C being the reproductive provide enough oxygen, the eggs from three females were temperature optimum. The eggs are tended by both parents arranged separately in baskets in a single layer. who perform fanning movements with their fins to refresh The eggs were incubated at 23 ± 0.2°C in water of dif - –1 the water around the eggs (Sheel and Singh 1981, Saxena ferent hardness: soft (0ºGH = 0 mg CaCO 3 ·L ), moder - –1 and Sandhu 1994). Egg incubation was found to take from ately soft (9ºGH = 160.7 mg CaCO 3 ·L ), and moderate - –1 18 to 24 h, depending on the water temperature. The ly hard (18ºGH = 321.5 mg CaCO 3 ·L ) (°GH = Degrees knowledge on embryonic development of the stinging cat - of General Hardness). fish has been fragmentary, focusing on gamete morpholo - Egg membranes of activated eggs were fixed in gy and duration of the embryogenesis in relation to some buffered 4% formaldehyde, dehydrated in gradually more environmental factors, such as the water temperature and concentrated solutions of ethanol and acetone, and dried the oxygen content (Altman and Dittmer 1962, Thakur et in critical point liquid CO 2. They were subsequently dust - al. 1974). The newly hatched larvae, on average 2.72 mm ed and viewed under a scanning electron microscope total length, carry a fairly large yolk sac which becomes (SEM) (Fei Quanta 200). The observations were photo - completely resorbed on the fourth day after hatching. The graphically documented. larvae commence feeding on their third day after hatching Embryonic development was recorded live with a set (Singh et al. 1989, Roy and Pal 1986, Alok et al. 1993). consisting of a light microscope (Nikon ECLIPSE TE One of the factors having meaningful effect on the 2000S) and a dissecting microscope (Nikon SMZ 1500) embryonic development of this fish is the water hardness. coupled with a microprocessor control (Trol–8100/9100) Clarias gariepinus incubated in hard water (more than and a colour digital camera (Nikon DS Fi-1.), a screen –1 200 mg ·L of CaCO 3) had more malformed larvae (in (LG), a video recorder (JVC-HR-S7700), and a computer relation to control). Also soft water (0–10 mg ·L–1 of (PC). One set made it possible to view the egg from CaCO 3) has not been considered as a potential optimal above, the other providing a side view of the developing medium for developing embryos (Molokwu and embryo; the two observation sets, taken together, made it Okpokwasili 2002). possible to record details of changes in spatial distribution Taking into the consideration the results of the above- of various structures in the developing embryo. mentioned studies it would be interesting to learn about Duration of embryonic development was determined the morphogenesis of this fish. This could help to under - using thermal units—accumulated degree hours (hº), cal - stand the biological nature of the changes observed. culated as the sum of water temperature each hour during The presently reported study describes the structure of the development period. All types of motility in the devel - the egg membranes (from fertilization to hatching) and oping eggs were recorded and subsequently analysed. The the embryo morphology, as well as the behaviour of the number of somatic movements and the number of heart larvae from hatching until complete yolk sac resorption. contractions per minute were recorded and analysed and Particular attention was paid to the effects of water hard - measured with the specialised Nikon Imaging System- ness on the course of the embryogenesis. The acquired Elements BR. knowledge may translate into practical means affecting Egg membranes strength and egg deformations were cultivation procedures employed by fish growers.
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