1. Fish Reproduction: Special Genetic Issues Dr. Craig Kasper FAS 2263C

2. Genetics: What is it? Genetics: The science of heredity and variation. Genetics: The science of heredity and variation. Heredity: transmission from generation to generation (usually through the process of reproduction) factors which cause the offspring to resemble their parents (or not.)Heredity: transmission from generation to generation (usually through the process of reproduction) factors which cause the offspring to resemble their parents (or not.)

3. Genetics Terms Selective breeding-the choosing of individuals of a single strain and spp.Selective breeding-the choosing of individuals of a single strain and spp. Hybridization-the crossing of different spp.Hybridization-the crossing of different spp. Crossbreeding-the mating of unrelated strains of the same spp. to avoid inbreedingCrossbreeding-the mating of unrelated strains of the same spp. to avoid inbreeding

4. Selective Breeding “Artifical selection” as opposed to natural selection, results in reducing genetic variability in a population. Could be considered inbreeding if not closely monitored.“Artifical selection” as opposed to natural selection, results in reducing genetic variability in a population. Could be considered inbreeding if not closely monitored. If selective breeding involves excessive inbreeding physical abnormalities, metabolic deficiencies, and developmental abnormalities may occur.If selective breeding involves excessive inbreeding physical abnormalities, metabolic deficiencies, and developmental abnormalities may occur. Inbreeding depression-loss of fitness due to inbreedingInbreeding depression-loss of fitness due to inbreeding –more likely to observe occurence of recessive traits –Decrease in heterozygosity

5. Selective Breeding Although potential for deleterious results, selective breeding rationalized by:Although potential for deleterious results, selective breeding rationalized by: –size –color –shape –better FCR –reproductive capacity –disease resistance

7. Hybridization Already looked at hybrid striped bass.Already looked at hybrid striped bass. What other examples are out there?? Splake-brook trout/lake trout, Tiger Musky-northern pike/musky Sunfish hybrids (many combinations) Hopefully achieve “hybrid vigor,” at least in some trait.Hopefully achieve “hybrid vigor,” at least in some trait. Most true hybrids are sterile, but not all.Most true hybrids are sterile, but not all.

8. Gynogenesis Development of an ovum without fusion of gametesDevelopment of an ovum without fusion of gametes –usually haploid and die –sometimes diploid larva develop requires retention of 2 nd polar body during meiosisrequires retention of 2 nd polar body during meiosis Amazon mollies, cruzian carp do thisAmazon mollies, cruzian carp do this Induced by:Induced by: –radition, chemical, electrical currents, sperm from related species

9. Regulation of Ploidy Monosex, polyploidy, and beyond…

10. 1.Visual selection 2. Hybridization 3. Sex-reversal 4. Genetic manipulation Male Populations Mono-sex male tilapia populations can be produced by:

11. How DO you sex a tilapia?? Begin with 25 to 30 g fish (any smaller and you’ll go blind looking for genital openings.)Begin with 25 to 30 g fish (any smaller and you’ll go blind looking for genital openings.) Males and females can be separated by visual inspection of the genital papilla (next slide).Males and females can be separated by visual inspection of the genital papilla (next slide). The male genital papilla is pointed and contains only the small urogenital pore which is difficult to observe.The male genital papilla is pointed and contains only the small urogenital pore which is difficult to observe. The female papilla is rounded and contains a small opening to the urinary duct and a larger oviduct.The female papilla is rounded and contains a small opening to the urinary duct and a larger oviduct. Selection is usually based on the presence or absence of the oviduct.Selection is usually based on the presence or absence of the oviduct.

12. How DO you sex a tilapia?? Males are stocked for further growth while females can be used as brood stock or discarded.Males are stocked for further growth while females can be used as brood stock or discarded. Visual selection of male tilapia is costly because of the extra labor needed to separate males from females.Visual selection of male tilapia is costly because of the extra labor needed to separate males from females.

13. Male papilla Female papilla with oviduct anus Visual Selection of the Genital Papilla

14. Using Two Species Two species of tilapia can be crossed to yield all-male offspring.Two species of tilapia can be crossed to yield all-male offspring. Male Hornorum tilapia can be hybridized with the female Nile tilapia and offspring are all-male.Male Hornorum tilapia can be hybridized with the female Nile tilapia and offspring are all-male. However, the hybrid is fertile and can backcross with female Nile and Hornorum tilapia.However, the hybrid is fertile and can backcross with female Nile and Hornorum tilapia.

15. Using Two Species Only genetically pure Hornorum and Nile tilapia will result in 100% male offspring.Only genetically pure Hornorum and Nile tilapia will result in 100% male offspring. Maintaining pure lines of Nile and Hornorum tilapia over a long period of time is difficult.Maintaining pure lines of Nile and Hornorum tilapia over a long period of time is difficult. Additionally, hybrid fry yield per female is low compared with fry yield from pure species spawns.Additionally, hybrid fry yield per female is low compared with fry yield from pure species spawns.

16. Male Hornorum tilapia Female Nile tilapia All-male hybrid tilapia ZZ XX XZ Hybridization

17. Sex determination by hormones A popular way to produce all-male populations is with hormone sex reversal of fry.A popular way to produce all-male populations is with hormone sex reversal of fry. Tilapia fry (18 day) are fed a diet containing a male steroid (methyl testosterone) for 20 to 28 days.Tilapia fry (18 day) are fed a diet containing a male steroid (methyl testosterone) for 20 to 28 days. Females develop as “functional males” following hormone treatment.Females develop as “functional males” following hormone treatment. All-male populations are hard to produce!!All-male populations are hard to produce!! ~95 to 98% males are commonly produced.~95 to 98% males are commonly produced.

18. Sex Reversal of Tilapia Fry

19. 1) XX F + XY MXX + XY normal Genetic Manipulation

20. More genetic manipulation What if female Nile tilapia are homozygous (XX) and male Nile tilapia are heterozygous (XY) for sex.What if female Nile tilapia are homozygous (XX) and male Nile tilapia are heterozygous (XY) for sex. A pairing of genetically normal male and female Nile tilapia will result in 50% male and 50% female offspring.A pairing of genetically normal male and female Nile tilapia will result in 50% male and 50% female offspring. Now if we feed normal fry estrogen for 28 days, we get phenotypic all-female fry.Now if we feed normal fry estrogen for 28 days, we get phenotypic all-female fry.

21. 1) XX F + XY M estrogen XX F + XY ”F” Genetic Manipulation

22. However, 50% of these “feminized fry” are genetic females (XX) and 50% are genetic males (XY).However, 50% of these “feminized fry” are genetic females (XX) and 50% are genetic males (XY). These females are individually paired with normal males.These females are individually paired with normal males. A genetic female (XX) paired with a genetic male (XY) will have offspring that are 50% male and 50% female.A genetic female (XX) paired with a genetic male (XY) will have offspring that are 50% male and 50% female. Females from this crossing are discarded.Females from this crossing are discarded.

23. 1) XX F + XY M estrogen XX F + XY ”F” 2) XY ”F” x XY M XX F + 2 XY M + YY M Genetic Manipulation

24. When a genetically male female (XY) is crossed with a genetic male (XY) the offspring will be 25% female (XX) and 75% males (50% XY and 25 % YY).When a genetically male female (XY) is crossed with a genetic male (XY) the offspring will be 25% female (XX) and 75% males (50% XY and 25 % YY). Again, individual pairings with a genetic female (XX) are required to determine which male is YY.Again, individual pairings with a genetic female (XX) are required to determine which male is YY. The YY males or “supermales” will sire 100% male offspring (XY) when paired with genetic (XX) females.The YY males or “supermales” will sire 100% male offspring (XY) when paired with genetic (XX) females.

25. 1) XX F + XY M estrogen XX F + XY ”F” 2) XY ”F” x XY M XX F + 2 XY M + YY M 3) XX F x YY M 100% XY M ?? Genetic Manipulation

26. Transgenics: How do you get a transgenic fish? Transgenic or genetically engineered animals are developed by inserting single or multiple genes from one species into the DNA of another species.Transgenic or genetically engineered animals are developed by inserting single or multiple genes from one species into the DNA of another species. Three basic steps:Three basic steps: –Isolate and identify the genes that posses the desired trait. Genes may come from similar fish species, plants, bacteria, animals, and even humans. –Insert isolated material into the intended target fish’s eggs. –Grow your fish!!

27. Differences in terms? The terms genetically engineered, genetically modified, and transgenic are used synonymously.The terms genetically engineered, genetically modified, and transgenic are used synonymously. The term transgenic arises from the procedure of transgenesis, one method in which scientists have successfully inserted genes from one species to another.The term transgenic arises from the procedure of transgenesis, one method in which scientists have successfully inserted genes from one species to another.

29. What is a triploid fish? Triploid fish contain three sex chromosomes.Triploid fish contain three sex chromosomes. Heat and or pressure applied to females’ eggs to generate the extra chromosome.Heat and or pressure applied to females’ eggs to generate the extra chromosome. Proponents of G-EN suggest environmental risks associated with their release into open water would be rendered harmless.Proponents of G-EN suggest environmental risks associated with their release into open water would be rendered harmless. No guarantee of sterility!!No guarantee of sterility!! Competition is a reality for sterile fish too!Competition is a reality for sterile fish too!

30. Polyploidy Polyploidy has been well-studied in fish and shellfish.Polyploidy has been well-studied in fish and shellfish. Channel catfish triploids become larger than diploidsChannel catfish triploids become larger than diploids Triploids converted feed more efficiently than diploids (Wolters et al., 1982), had six percent greater carcass yield at three years of age.Triploids converted feed more efficiently than diploids (Wolters et al., 1982), had six percent greater carcass yield at three years of age.

31. Intersexed fish Hermaphrodites-contain ovotestes, can go either way...Hermaphrodites-contain ovotestes, can go either way... –MT treatment female after sex determination occurs (ovary has already started to develop) –intersexed fish must be killed to determine if the process work! Not very good for aquaculture?

32. Questions??