UNIVERSITY OF PANNONIA GEORGIKON FACULTY KESZTHELY. Department of Animal Sciences and Animal Breeding. DOCTORAL (PhD) THESIS

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1 UNIVERSITY OF PANNONIA GEORGIKON FACULTY KESZTHELY Department of Animal Sciences and Animal Breeding DOCTORAL (PhD) THESIS Doctoral School in Animal and Agricultural Environment Sciences Head of School: Dr. Anda Angéla, DSc. (Legal Predecessor: Doctoral School in Animal Breeding Head of School: Dr. Szabó Ferenc DSc.) SEXUAL CHARACTERISATION OF THE BLACK SCORPIONFISH (Scorpaena porcus L., 1758) AND ITS ARTIFICIAL PROPAGATION Supervisor: Dr. Bercsényi Miklós, professor, PhD Németh Szabolcs Keszthely 2009

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3 1. INTRODUCTION AND OBJECTIVES Fish is of excellent composition from the dietetic point of view with high protein and low fat content. Presently, and most probably in the future as well, marine fisheries and fish culture play a much greater part in the production of fish than freshwater fisheries and culture. Marine fish catches reached 84.2 million tons and marine culture 18.9 million tons in 2006 whereas freshwater (and brackish water) catches and farm production were only 9.4 million and 28.9 million tons respectively (FAO, 2007). The way to the growth of fish production will definitely not be the increase of marine fisheries carrying the threat of overfishing but the increase of the proportion of marine fish farming. This supposal is based on the huge difference in the size of area covered by sea opposed to freshwater just as the low level of exploitation of the potentials of marine fish farming. Many authors have written about the achievements of artificial propagation and aquaculture breeding of marine fish still there are a number of economically valuable species with the breeding of which nobody has dealt so far. Though Hungary has no sea at the moment but Hungarian experience may contribute to the development of artificial breeding technologies for these marine species. This idea led me to attempt the artificial propagation of the delicious and economically valuable scorpionfish. Although, the largest and economically valuable species of scorpionfish of the Adriatic Sea (Globefish, 2007) is the largescaled scorpionfish (Scorpaena scrofa) (Picture 2.), I dealt with its close relative, - 2 -

4 the black scorpionfish (Scorpaena porcus) (Picture 1.) because of the limited volume of marine aquariua at my disposal. Picture 1. Black scorpionfish Picture 2. Largescaled scorpionfish (Photos: I suppose that based on the close relationship the propagation techniques developed for the black scorpionfish are adaptable to the other species of the Scorpaenidae family. The aims of the study were as follows: Assessing the ratio of sexes in the North-Adriatic population Description of the morphological differences between females and males or the establishment of the lack of sexual dimorphism The exploration of the significant features necessary for the artificial propagation on the basis of the structure of the reproductive organs The development of the injury-proof collection method of the sexually mature fish The development of artificial propagation at laboratory circumstances The establishment of the method of induced gametogenesis Development of techniques in connection with larva nursing - 3 -

5 2. MATERIAL AND METHODS 2.1. The establishment of the sex ratio in the North-Adriatic population of the black scorpionfish The identification of sex was done on the basis of microscopic slides of gonads. For sex determination 50 scorpionfish of different sizes was bought in the Pula fish market.. The gonads were conserved in 75 % ethanol. In the course of the preparation of the microscopic sections formaldehyde fixation and paraffin embedding was applied. The 2-3 micron slides were stained by haematoxilin and eosin By observing the slides the sexes could be unambiguously distinguished The induced gametogenesis of the black and largescaled scorpionfish. The artificial propagation of the black scorpionfish Catching, keeping and marking of the experimental animals I have caught 23 individuals of black and 5 of largescaled scorpionfish after their reproductive season (in September) at the shores of Pula and the Island of Krk with diving technique. I placed them into two 700 liter aquaria in the Tropicarium in Budapest. The temperature was regulated by two digitally controllable refrigerators. I marked the fish by fixing selfmade and numbered plastic tags to the dorsal fin

6 Induced gametogenesis of the black and the largescaled scorpionfish The fish was kept at 20 ± 0,5 C in sea water. Gametogenesis was induced by weekly injections of dried carp pituitary extract. Preceding the first injection the temperature was decreased to winter level (12 C) in 2 C per week steps. The fish was at this temperature for 4 weeks, then raised the temperature again to 20 ± 0,5 C. Induced gametogenesis took place at this temperature. The weekly dosage of CP was 6 mg/bwkg. dried carp hypophysis for the induced gametogenesis. The fish the sexes of which were unknown at the outset of the experiment had been treated with hypophysis for 12 weeks. Males had been treated only until the beginning of milt production when I could identify their sex. After this I injected only the males with 6mg/bwkg of hypophysis at the time of the females readiness to ovulate. The females had been treated with 6 mg/bwkg hypophysis until they reached a status close to ovulation ( significant swelling of the abdominal region and sudden weight gain), then I made them ovulate by a single dose of 12mg/bwkg of hypophysis. At each inoculation I weighed the fish with tenth of a gram accuracy. I started weighing the fish 4 weeks before starting the inoculations and continued until the tenth week after the last inoculation The artificial insemination of the black scorpionfish For I could only gain milt from the black scorpionfish I could only execute artificial insemination of this species. The milking of the gametes took place 24 hours after the inoculation of the determinative dose

7 Anaesthesia was not necessary for milking. Gaining the milk was carried out by extrusion; the milking of the spawn from the gelatinous hull was done with the combination of extrusion and drawing. I have tried both the wet and the dry fertilization technique. In the course of wet fertilization the milt flocculated and fertilization was not successful though the sperms showed movement under the microscope. Fertilization was successful when applying the dry fertilization technique Larva-nursing of the black scorpionfish I placed the fertilized, floating clumps of spawn into three 15 liter aquariua half-immersed into the big aquarium of the parent animals for ensuring the appropriate temperature. I constantly pumped some water from the greater waterspace onto the larvae. I examined the development of the larvae every hour. I have made microscopic records of the development of the larvae after they had hatched. Following the absorption of the yolk I tried to feed the larvae with living food (Artemia salina nauplius larva and Brachionus plicatilis, B. rotundiformis rotifier) and nutrients

8 3. RESULTS 3.1. The extreme sex ratio of the North-Adriatic population of the black scorpionfish. In case of the fish purchased in the fish market I managed to identify the sex the fish by the assessment of the sections. In the case of the fish kept in the aquarium the identification of sex was possible on the basis of gametes produced and milked under the effect of the series of inoculations. Table 1. shows the breakdown of experimental fish by sex. Table 1. The breakdown of experimental fish by sex. Females Males Total Fish purchased in the market pcs % Fish kept in the aquaria (injected) pcs % Total pcs % Table 1. shows that in the North-Adriatic population of the black scorpionfish (in the examined period) an extreme sex ratio evolved in which females dominate. The ratio of females and males is approximately 9:1. This rate significantly differs from the data collected at other sites according to scientific writings. The Turkish population is dominated by females. Total ratio is approximately 44:56. According to data from Tunisia - 7 -

9 the population there is dominated by females. The question arises: what may cause these differences? The selection by fishing and by size cannot be taken into account as a possible answer since it decreases the ratio of bigger fish and the data from Tunisia, the Black Sea and the North-Adriatic showed the dominance of females bigger in size. It is possible that apart from the mating season males and females separate so the data on sex ratio collected in different periods show significant differences. Since I collected fish only after the mating season (during a two-month period) the extreme sex ratio that I have found cannot be compared to the data concerning a whole reproductive period in scientific writings. By the assessment of an appropriate number of samples collected throughout a whole year the likelihood of separation of sexes in space can be decided. Due to the difficulties and costs of gathering a statistically significant number of samples this examination is attainable within the confines of another research. If there is no extreme sex ratio on the basis of statistically significant number of samples then the habitat and the behavior of sexes can be examined out of mating season. Attention has to be paid to the selection of the appropriate period for gathering female fish so that the number of males is not a limiting factor. In case the further examinations support the extreme sex ratio that I have found several other explanations may come up. For instance, the life span of females may be longer or (similarly to the silver carp Carassius gibelio- in Hungary) natural gynogenesis takes place. In the former case the sexes are identifiable by dating and by genetic examination in the latter

10 3.2. The microscopic examination of the sexual organs of the black scorpionfish Several conclusions could be drawn from the microscopic examination of the gonadsconcerning the reproductive features facilitating the induced gametogenesis and the artificial propagation. The slides of the ovaries showed that the black scorpionfish has the asynchronous type of ovary. Simultaneously several occytes at different stages of development are present in the ovary. Occytes at the same stage of maturity sit next to each other in a folded band-like arrangement. I assume on the basis of the asynchronicity of the ovary that the females are able to produce gametes several times within one spawning season, though probably not a big amount, therefore the number of larvae cannot be large either. The structure of the testicles of the scorpionfish is basically reminiscent of the testicles of the perch. It consists of tubules rolled densely next to each other The induced gametogenesis of the black and the largescaled scorpionfish The induced gametogenesis of the largescaled scorpionfish Due to the lack of space I had only three sexually mature and two immature samples of largescaled scorpionfish. I carried out injections on a mere experimental basis to find out if it was possible to execute induced gametogenesis in case of the largescaled scorpionfish. As a result of the injection I managed to retrieve spawn from two of the samples but - 9 -

11 unfortunately I was not able to retrieve any milt therefore I was not able to carry out artificial fertilization The induced gametogenesis of the black scorpionfish In the case of the black scorpionfish as a result of a series of 12 injections with carp hypophysis 17 samples resulted gametes, from 3 males and 14 females. Two females produced gametes twice during the series of injections. 34 and 15 days passed between the repeated spawnings. Since I gathered the fish in September and the temperature of the aquarium was lowered to 12 C and raised to 20 C during two weeks, the fish were surely out of reproductive season and the gamete maturing was generated by the hormone injections. pgsi values calculated from the proportion of body weight and the weight of the stripped eggs ( including the gelatinous hull) were between 16.4% and 61.3 %. The average pgsi value was 29.1 %. The predicted weight of spawn from the female fish of known weight can be estimated by the function: y = 0,1336x + 15,901, R 2 = 0,5415 (Figure 1.) The function was calculated by using linear regression on the data pairs

12 Scorpaena porcus pgsi Transmitted spawn (g) y = 0,1336x + 15, R 2 = 0,5415 p<0, Body weight (g) Figure 1. Stripped spawn plotted against body weight Following the 4 th week after the last series of injections some fish produced spawn again performing unexpected ovulation. The unexpected ovulationsoccurred for 5 weeks. The number of spawning fish is shown in Table 2. S.porcus hypophysis treatment Number of 4 spawning fish weeks Figure 2. The number of spawning fish per week as a result of hypophysis treatment

13 I estimated the quantity of occytes per spawnings between pieces. Compared to the species producing separate eggs (not coated in gelatinous matrix) the stripping wasrather the combination of pushing the belly and pulling the spawn then simple stripping The milked sperm was of opalescent colour and its quantity was only µl per fish. The spermia showed quick movement for 1.5 minutes following their activation by seawater. As a conclusion of the foregoing we can state that carp hypophysis treatment is an adequate method for the induced gametogenesis of the gametes in the case of both the black and the largescaled scorpionfish. (Table 2.) Table 2. The results of hypophysis injections Matured oocytes Matured sperm Black scorpionfish YES YES Largescaled scorpionfish YES NO 3.4. The artificial fertilization and incubation of the black scorpionfish eggs I carried out the successful artificial fertilization applying the dry method. I managed to obtain approximately 5000 pieces of mature spawns three times and milk some milt from the males as the same time. The rate of fertilization was about 30 % thus approximately 1500 pieces of larva hatched each of the three times. The embryos began to hatch at 20 o C 56 hours after fertilization

14 3.5. Experiences of the larva-nursing of the black scorpionfish The stages of larva development of the black scorpionfish My observations supported the scientific writings according to which the 2 mm-long, non-feeding larvae of the black scorpionfish have large yolk-sack at hatching which fully disappears on the 4 th day. They are vitrous and their eyes are not pigmented either. On the 4 th day their eyes begin to gain colour and small black spots appear at the sides of the pectoral fin. Their yolk sack absorbs. Their size is between mms. Their head begins to grow and their mouth is about 150 µm large. It is a new observation that the larvae move to the middle of the waterspace on the second day. Instead of gathering into small groups they scatter evenly not in the surface but close to it. On the fourth day they move to the bottom of the aquarium where they pick food from the bottom not in groups but scattered evenly. This observation is in contrast with the scientific writings about the larva development of the of the members of the Scorpaenidae family according to which the larvae change over to the benthic way of life from mm of length (Nagasawa and Domon, 1997; Terofal, 1996). According to my own observation larvae that consume the yolk sack and change over to individual food acquisition do not try to hunt for planktonic animals but undoubtedly all of them look for benthic sources of food. Their way of life does not change following the fourth day. I was able to nurse them until the age of 9 days then all of them perished most probably from famishment

15 Food tested in larvae-nursing The larvae of marine fish are usually small. The black scorpionfish is not an exception either with its non-feeding larvae of 2 mms. Small size renders breeding and obtainment of starting food more difficult. The nauplius larva of the Artemia salina definitely proved to be too big with their size of µm. In the Brachionus culture that I have bred myself most probably occurred some B. rotundiformis and a B. plicatilis species. Concerning size the smaller species (B. rotundiformis) was appropriate and the B. plicatilis seemed too big. The size of the B. rotundiformis seemed appropriate and a large number occurred in the larva breeding aquarium ( appr. 10 pcs/cm 3 ) still the larvae of the scorpionfish did not eat them since in contrast with the assumptions they did not take food from among the floating planktons but from the bottom. I haven t found any rotifiers in the stomach of the scorpionfish at any of the occasions of the microscopic observations. The larvae searched the detritus of the bottom for food. On the basis of the above described I have drawn the conclusion that it is worth trying sinking feed of 100 µm as starting food

16 4. CONCLUSIONS AND SUGGESTIONS Conclusions I have found that in the North Adriatic population of black scorpionfish there are much fewer males than females after the natural spawning season. The proportion of females and males is 9:1. I have found on the basis of microscopic slides of gonads that - similarly to the formerly examined species of Scorpaena notata - the females of the S. porcus have asynchronic type of ovary. This makes it possible to get spawnings several times within one reproductive season. I have found that 6mg/bwkg of carp hypophysis weekly injections were effective in promoting the game maturation of both the black and the largescaled scorpionfish and that 12 mg/bwkg was enough to induce ovulation. I have justified that the dry fertilization technique is effective at the black scorpionfish. I have found in the course of the incubation that 20 C º water temperature is adequate for the larval development. I have found in the course of the larva-nursing that the Brachionus plicatilis rotifer and the nauplius larvae of the Artemia salina brime shrimp usually applied in marine fish breeding is not adequate for the early feeding of the black scorpionfish. In the course of the larva-nursing I have disproved the assumption that larvae which consumed their yolk sack have planktonic way of

17 life. According to my own observations the larvae change over to benthic way of life on the fourth day and search for food not in the waterspace but in the bottom. Suggestions I suggest both at the black and largescaled scorpionfish a detailed study for the observation of the sex ratio in the North Adriatic, from round the year samples In case of the largescaled scorpionfish I suggest to continue the experiments on the hormone induced gamete maturing by an injecting the fish with 6 mg/bwkg of carp hypophysis and using double dosagefor the inducing the ovulation. I suggest fertilization applying the dry technique, but developing a better way of distributing the sperm on the egg mass. I suggest the testing of the sinking feed of the smallest possible size (100 µm) for feeding the black scorpionfish. I suggest the genetic (microsatellite or RAPD) identification of the three species of scorpionfish native to the Adriatic Sea (S.porcus, S. scrofa, S. notata) in order to make it possible to prove the presence or lack of hybridization between the species. This would help also to understand the role of extreme sex ratio

18 5. THE MAIN POINTS OF THE THESIS 1. I have found extreme sex ratio (right after the natural spawning period) in the North-Adriatic population of the black scorpionfish. The proportion of females and males is 9:1. 2. On the basis of the microscopic slides of the gonads I have found that they have asynchronic type ovary which makes repeated reproduction possible within one spawning season. 3. I managed to induce gametogenesis by carp hypophysis in case of two species of the scorpionfish. I could obtain mature ova from the largescaled scorpionfish and mature sperm from the black scorpionfish. 4. In case of the black scorpionfish I could carry out successful fertilization several times applying the dry technique. 5. I could nurse the larvae of the black scorpionfish until the age of 9 days. I have found that in contrast with the assertion of scientific publications that after absorption of the yolk the larvae do not choose the planktonic but the benthic way of life and search for food in the bottom

19 6. PUBLICATIONS IN THE SUBJECT OF THE THESIS 6.1. Articles in scientific periodicals Németh, Sz., Budaházi, A., Szűcs, R. and Bercsényi, M. (2009, Accepted), Out of the season artificial propagation of the black scorpionfish (Scorpaena porcus L.) in captivity. Mediterranean Aquaculture Journal Articles in scientific journals Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The artificial propagation of a scorpionfish species (Scorpaena porcus) of the Mediterranean Sea. Halászat, Vol. 101, pp Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2009), The extreme sex ratio of the black scorpionfish (Scorpaena porcus) and microscopic structure of the sexual organs. Állattenyésztés és Takarmányozás, Vol.58(1): pp Scientific presentations and posters Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), Extreme sex ratio of the black scorpionfish (Scorpaena porcus) and the microscopic structure of the sexual organs XIV. Ifjúsági Tudományos Fórum, Keszthely, CD, p. 6. Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), Experiments of the gametogenesis and propagation of the black scorpionfish (Scorpaena porcus) in captivity. XIV. Ifjúsági Tudományos Fórum, Keszthely, CD, p. 7. Németh, Sz. és Bercsényi, M. (2008), Observations in the course of the induced gametogenesis, spermiation and the ovulation of the black scorpionfish (Scorpaena porcus) I. Gödöllői Állattenyésztési Tudományos Napok, Gödöllő, CD, pp

20 Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The special sex ratio and the microscopic examination of the sexual organss of the black scorpionfish (Scorpaena porcus). XXXII. Halászati Tudományos Tanácskozás, Szarvas, Abstract book, p. 37. Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The induced gametogenesis and the attempts at the propagation of the black scorpionfish (Scorpaena porcus). XXXII. Halászati Tudományos Tanácskozás, Szarvas, Abstract book, p. 45. Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The establishment of the sex ratio of a scorpionfish species (Scorpaena porcus) of the Adriatic Sea and the microscopic structure of the sexual organs. XI. Tavaszi Szél Konferencia, Budapest, Abstract book, p Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The induced gametogenesis and the artificial propagation of the small- scaled scorpionfish (Scorpaena porcus). XI. Tavaszi Szél Konferencia, Budapest, Abstract book, p Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), The microscopic examination of the black scorpionfish (Scorpaena porcus) and the sex ratio within the species 50. Jubileumi Georgikon Napok, Keszthely, CD. Németh, Sz., Budaházi, A., Szűcs, R. és Bercsényi, M. (2008), Home achievements of the artificial propagation of the black scorpionfish of the Adriatic Sea (Scorpaena porcus) 50. Jubileumi Georgikon Napok, Keszthely, CD

21 7. PUBLICATIONS BESIDE THE SUBJECT OF THE THESIS 7.1. Publications in scientific journals Bódis, M., Ittzés, I., Németh, Sz. és Bercsényi, M. (2008), A new, Hungarian method of the artificial propagation of the pike perch closing of the genital aperture of the females previous to ovulation. Halászat, Vol. 101, pp Scientific presentations and posters Németh, Sz., Bódis, M., Ittzés, I. és Bercsényi, M. (2007), A new and saving method for closing the genital aperture of the females previous to ovulation. XXXI. Halászati Tudományos Tanácskozás, Szarvas, Abstract book, p Books Farkas, J. és Németh, Sz. (2009), The Living World of the Adriatic Sea. An introduction to Marine Biology. Pauz-Westermann Kiadó, Celldömölk, ISBN