Production of Cloned Amago Salmon Oncorhynchus rhodurus* 3-5

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1 Fisheries Science 60(3), (1994) Production of Cloned Amago Salmon Oncorhynchus rhodurus* 3-5 Tooru Kobayashi,*1, õ Atsuhiko Ide,*2, õ Takayuki Hiasa,*2 Shozo Fushiki,*l and Koichi Ueno*2 *1Shiga Prefectural Samegai Trout Farm, Kaminyu, Maibara, Shiga 521, Japan *2 Department of Fisheries, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631, Japan (Received November 10, 1993) We induced homozygous clones of amago salmon by gynogenetic chromosome manipulation. Mitotic gynogenesis diploid (mitotic-g2n) as first generation was produced by suppression of the first cleavage of the eggs inseminated with UV-irradiated sperm of rainbow trout by hydrostatic pressure shock. The yield of apparently normal gynogenetic embryos was 2.26% when the eggs were shocked at 650 kg/cm2 of hydrostatic pressure for 6 min at 5 h 30 min (approximately 68 h Ž in cumulative temperature) after insemination. Homozygous evidence of gynogenetic juveniles was demonstrated by the segregation of Idh-3 and Pgm-1 loci. Five homozygous inbred strains were produced from the eggs of homozygous gyno genetic females by retaining the second polarbody, while outbred controls were produced for each strain by crossing with males of other strains. The clonal proof of these strains was demonstrated by the immunological acceptance of operculum allografts from inbred sisters. Similar survivals were shown among clones produced by mothers from identical strain. On the other hand, various survivals were seen between the clones using maternal parents from different strains. These results suggested that the line selection was a vital factor in clone production. Key words: clone, amago salmon, genetics, chromosome manipulation, gynogenesis, isozyme marker, tissue grafting, aquaculture Gynogenesis by chromosome manipulation should be a powerful tool with which to improve the genetic traits of rearing fish and to produce various useful races and strains in aquaculture. 1) Specially, completely homozygous diploids can be induced by suppressing the first cleavage of the egg developed gynogenetically. The gynogenetic dip loids produced by the suppression of the first cleavage (mitotic-g2n) become the foothold for induction of a clonal line in the next generation. Using this procedure, the thoroughbred clones can be established in only two generations.2) On the other hand, retaining the second polar body (meiotic-g2n) results in a high rate of gene centromere recombination. It is difficult to obtain complete homozygous diploids using this procedure.3) This phenome non has also been seen in some salmonid species4,5) and in others.6,7) Thus, meiotic-g2n methods are not effective for drastically increasing the fixation indices. The mitotic-g2n method is not influenced by gene-centromere recombination for induction of complete homozygous progeny. Therefore, the production of mitotic-g2n is indispensable for inducing the clonal lines. Clones are important for extensive biological studies. In addition, they seem to be useful for standardizing the size of seedlings in aquaculture. It had been considered that production of a clonal thoroughbred strain in fish requires reproduction for over 20 generations by brother-sister mating.8) The artificial mitotic-g2n procedure could easily induce clonal inbred lines in only two generations, and it is considered useful for rapidly improving the genetic characteristics of fish races.9,10) Gynogenetic trials have been conducted in some ex perimental species such as zebra fish Brachydanio rerio11) and medaka Oryzias latipes12) to provide a model for the induction of fish clones. Recently, these approaches were applied to species useful for fisheries such as ayu Plecoglossus altivelis9) and carp Cyprinus carpio.10) Amago salmon Oncorhynchus rhodurus is the salmonid species distributed on the western pacific side from the central region of Honsyu in Japan.13) This fish is an im portant species for inland fish cultures. Recently, it has been regarded as having potential for genetic breed improvement. Therefore, developing a procedure to produce clones in amago salmon should contribute greatly to salmon and trout cultures. In this study, we induced the completely homozygous diploid and clonal amago salmon. Evidence for the production of mitotic-g2n and clones was obtained using isozyme markers and by tissue grafting. We also examined the variance of growth in a colonal clony of amago salmon. Materials and Methods Hybridization Trials in Combination with Female Amago Salmon and Male Rainbow Trout, and Polyploidization of the Hybrids As sperm of the rainbow trout Oncorhynchus mykiss was employed as a trigger to start gynogenetic development of amago salmon eggs, we first examined the combination female amago salmon x male rainbow trout in order to know whether viable juveniles of diploid or triploid hybrid could be obtained. *3 Study of Biological Characters and the Genetics of Some Traits. of Triploid and Gynogenetic Diploid on Salmonid Fishes-III. *4 Contribution from the Shiga Prefectural Samegai Trout Farm. *5 Partly presented at the annual meeting of the Japanese Society of Scientific Fisheries, Tokyo, April, õ Present address: Shiga Prefectural Fisheries Experimental Station, Hassaka, Hikone, Shiga 522, Japan.

2 276 Kobayashi et al. Amago salmon were cultured at Samegai Trout Farm and were about the 7th generation of fish that originated from Gifu Prefecture. Eggs and sperm obtained from a single amago salmon and rainbow trout were used. The eggs were inseminated by the dry method, and the fertilized eggs were divided into two batches. One was exposed to heat shock (26 Ž for 20 min) 15 min after insemination, and the other was incubated at 12.0 Ž. Thereafter, the batches were incubated under fresh running water ( Ž) until hatching. Survival of embryos (eyed egg stage) and hatching were examined at 18 and 36 days after fertilization, respectively. Determination of the Optimum UV Dose to Genetically Inactivate Rainbow Trout Sperm The sperm of rainbow trout was diluted 100 times in artificial seminal plasma for salmonidae,*6 that consisted of 7.60 g NaCl, 2.98 g KCl, 0.37 g CaCl2 E2H2O, MgCl2.6H2O, 0.21 g NaHCO3 and 1000 ml distilled water, and exposed to ultraviolet (UV) rays at 40erg/mm2/s following the method of Onozato and Yamaha. 14) UV doses were tested between the range of erg/mm2 (0-100s). After insemination using bbalanced salt solution (BSS; 0.75 g NaCl, 0.02 g KCl, 0.02 g CaCl2.2H2O, g NaHCO3, and 100 ml distilled water), the eggs were incubated under fresh running water (12.0 Ž). The appearance of haploid embryos at the eyed stage was evaluated. These experiments were replicated three times. Determination of the Optimum Timing of the Hydrostatic Pressure Required to Supress the First Cleavage The eggs collected from 12 amago salmon were inseminated with UV-irradiated rainbow trout sperm ( erg/mm2) and were maintained at 12.4 Ž. To supress the first cleavage, hydrostatic pressure shocks of 650 kg/cm2 for 6min15) using a French Press (Ohtake Works Co., Tokyo) at 15 min intervals between 4 and 6 h after insemination were applied to the eggs. Thereafter, the eggs were incubated at 12.4 Ž, and the survival of the embryos was determined. The embryos were then divided into normal and haploid-syndrome (small eyes and deformed body). At 18 days after fertilization, the shapes of the hatched juveniles were exam ined. Production of Mitotic-G2N and Clones The eggs collected from two female amago salmon (S7 and S9) were inseminated with rainbow trout sperm irradiated with erg/mm2 of UV. Hydrostatic pressure of 650 kg/cm2 was applied for 6 min at 5 h 30 min (about 68 h Ž in cumulative temperature) after insemination to suppress the first cell cleavage. The eggs were then incubated in running fresh water, and the presumed mitotic-g2n progeny were raised in tanks (50, 500 1, and I t tanks, adjusted as they grew) for two years until maturation. The eggs obtained from 6 females of those mitotic-g2n (FO 1, F04, and F06 from S7, and F22, F23, and F51 from S9) were inseminated with the UV-irradiated sperm and given a high temperature shock (for 20 min at 26 Ž, 15 min after insemination) to retain the second polar body. 16) A the corium and subeutis, formed at the dorsal side of the host fish. Auto, alto and xeno-graft tissues were transplanted to the front, middle and rear pocket, respectively. After tissue graft manipulation, inflammation of the wounds was restrained with iodine. Thereafter the host fish were kept in water at about 12 Ž, and graft-tissue rejection or acceptance was checked every 10 days for two months. The donor and host fish used for the tissue grafting were about 15 cm in body length (9 months old). Results The Survival of the Diploid and Triploid Hybrids of the Combination Amago Samon?? x Rainbow Trout?? The survival of hybrid (female amago salmon x male rainbow trout) is indicated in Table 1. The survival rate (95.1 %) was high at the eyed egg and hatching stage in the normal cross. In this combination, polyploid eggs developed into normal embryos of the eyed egg stage (85.6%), and 85.1 % of the eggs hatched. Despite the high quality of the eggs, the hybrids (female amago salmon x male rainbow tout) completely failed to form normal embryos, irrespective Table 1. Survival of control and polypolidized embryos after heat shock in hybridized amago salmon Y x rainbow trout a and in normal fertilization between amago parents *l.2: AS, amago salmon; RT, rainbow trout; The maternal parent of each group was the same individual. *3 Heat shock (28 Ž for 10 min, at 15 min after fertilization). *4 Calculated as the rate to number of eggs used. portion of the eggs from each female was inseminated with the sperm of amago salmon (control-2n) as the outbred control to compare the survival at the eyed egg stage and the swim up stage of juveniles between 75 and 150 days after fertilization. The growth variance was examined in each group of juveniles reared in 500 l tanks for 18 months. Confirmation of Clones The induction of the mitotic-g2n was confirmed by isozyme marker analysis. The genotype of each locus was detected by means of starch-gel electrophoresis. The Mdh-1, Idh-3, and Pgm-l loci in amago salmon were studied, because they have polymorphic isozyme variation.*7 The technique of tissue grafting designed by Onozato and Nakanishi*s was applied to confirm clones of amago salmon. If the host and the donor possess identical genotypes, the grafted tissues will be accepted as selftissues of the donor. If they possess different genotypes, the graft tissues should be rejected. In the exchange among individuals of an intraclone, 100% of the graft tissues will be accepted and survive.9) Opercula were grafted in F01 and F06 following the method described by Han et al.9) Opercula from the donor fish were divided into small pieces (about 3 x 7 mm), and inserted into three small pockets, located between Fig. 1. The rates of eyed (open circles) and of normal appearing embryos (solid circles) inseminated with rainbow trout sperm irradiated by several doses of ultraviolet ray. Vertical lines represent mean+s.e.m. They were calculated at the eyed stage 24 days after insemination.

3 Clones of Amago Salmon 277 Table 2. Effect of the timing of the hydrostatic pressure (650 kg/cm2 for 6 min) on the survival of amago salmon eggs inseminated with UV-irradiated sperm of rainbow trout at the eyed egg stage. Eggs were incubated at 12.4 Ž of whether the eggs were polyploidized or not. The hybrid embryos were deformed, with stunted bodies, and did not survive to hatching. Optimum Dose of UV-ray Irradiation for Genetic Inactivation of Rainbow Trout Sperm The rate of eyed and normal looking embryos inseminated with rainbow trout sperm irradiated with several doses of UV is indicated in Fig. 1. In the intact control consisting of amago salmon eggs inseminated with the sperm of rainbow trout that was not UV-irradiated, no eyed embryos were observed. Irradiation with UV for only 20 s increased the rate of eyed eggs from 0 to about 80%. This rate peaked at 60 s, and thereafter gradually decreased until 100 s. Thus, we used a dose of s ( erg/mm2) of UV irradiation in the following experiments. Optimum Timing of Hydrostatic Pressure Required to Produce Mitotic-G2N by Suppression of First Cleavage Table 2 shows the relationship between the timing of hydrostatic pressure shock treatment and the survival of eggs 24 days after fertilization (300 Ž in cumulative temperature). Eyed eggs appeared when pressure shocks were applied to the eggs from 4 h 30 min to 6 h after insemination and the maximum rate of normal embryos (7.5%) was recorded at 5 h 30 min. No embryos developed from those shocked from 4 h to 4 h 15 min after insemination and the control. Survivors were observed among those shocked from 5 h to 6 h. Incidence of Mitotic-G2N and Confirmation of Its Induction In the trials to produce the mitotic-62n from the eggs of two individuals (S7 and S9) the incidence was 5.70 and 22.71% at the eyed stage, and 1.27 and 5.58% at the swim up stage. These fish were reared for two years until spawning. The female parents used in the induction of mitotic-uzln were heterozygous at Idh-3 in S7, and Idh-3 and Pgm-1 loci in S9 (Table 3). Genotype segregation and proportion at those loci in the mitotic-g2n progeny derived from heterozygous female S9 are given in Table 4. Although most of the genotypes of meiotic-g2n were heterozygous at those loci, all the genotypes of the mitotic-g2n progenies were Table 3. Genotype segregation of the isozymes from of parents, S7 and S9 Table 4. Genotype segregation of isozymes in meiotic-g2n and mitotic-g2n progeny of S9 Table 5. Genotype segregation of isozymes in mitotic-g2n progeny used to produce second generations homozygous. These mitotic-g2n progenies were all females and grew normally. In this experiment, the survival number of S7 and S9 at the swim up stage were 48 and 50. However, only 6 individuals (S7; 3 (6%) and S9; 3 (6%)) from the two strains survived until ovulating in the spawning season two years later. We could not examine the genotype segregation in S7, because no meiotic-g2n were alive long

4 278 Kobayashi et al. Table 6. Rates of normal embryos at the eyed egg stage and of swim up juveniles 24 and 75 days after fertilization; producing the gynogenetic second generation of mitotic-g2n, and their outbred controls *1: These data are represented in relation to the initial egg number. *2: Gynogenetic second generation of the mitotic G2N. Table 7. The number of graft-tissues accepted and rejected in the auto, allo, and xeno graft transplants in F01 and F06 of the S7 strain *1 Intra clone. *2: Outbred control. *3: Determined at the 10th day after implantation. *4. ND, no detectable. Fig. 2. The survival rate of clones and outbred controls from 75 to 150 days after fertilization. Clones and outbred controls are indicated as solid and open circles, respectively. A, F01; B, F06; C, F23. enough. Instead, the genotypes of the surviving mitotic-g2n are indicated in Table 5. All of the mitotic-g2n specimens surviving until spawning season except for F04 were homozygous. F04 was heterozygous on Pgm-1 locus, so it was excluded from clone production. Induction of Clones as the Second Generation of Mitotic- G2N Using five homozygous gynogenetic first generations of the two strains, the second mitotic-g2n generations were induced (Table 6). The rates of normal embryos at the eyed egg stage and of swim up juveniles were relatively high in both clones (CL; gynogenetic second generation produced from homozygous gynogenetic first generation) and in the outbred control (OC; crossing with precocious male of other strain) in strain S7. However, in strain S9, those values were very low except for the outbred control of F23; besides, viable juveniles could not be obtained from clones F22 and F51. After the swim up stage, the survival of two batches of strains S7 and S9-F23 was studied from 75 to 150 days after fertilization and it is shown in Fig. 2. The motility of the two batches was also very low in both CL and OC in strain S7. On the other hand, in S9-F23, the juveniles that survived until the swim up stage, gradually died from about 90 days after fertilization and the survival rate decreased to 4.8% in CL and 17.4% in OC at 150 days after fertilization. The Confirmation of Production of Clones by Tissue-Graftin The immune reactions to the graft-tissues at 60 days after implantation in strain F01 and F06 are shown in Table 7, and the reactive process is indicated in Fig. 3. The auto and allo-grafts were accepted by the dorsal regions of the host

5 Clones of Amago Salmon 279 Fig. 4. Body length and weight distribution in 18 month old offsprings of the clones (CL, N=31) and outbred controls (OC, N = 38) of the F0l strain g, respectively. In OC, the range of body length and weight were cm and g, respect ively. CL had the smallest variations of both measured values compared with the OC at 18 months old. The standard deviation of each trait in all the OC was bigger than in the CL (CL: OC, 1.52: 3.23 in length, 22.72: in weight). Discussion This investigation irradiated with amago salmon. showed UV can Normal that rainbow trout sperm produce gynogenetic diploids of hybridization of female amago salmon and male rainbow trout fails to produce viable offspring. 17,18) Hybrid embryos are deformed and die before hatching. Furthermore, polyploidization of these hybrid eggs by heat shock could not recover the survivability of the embryos. Therefore, the induction of gynogenetic progeny embryo offspring irradiated Fig. 3. An immune reaction test to confirm the clone using tissue grafts of amago salmon. The reaction of each graft tissue 10, 20, 30, 40, 50, ana 60 days after implantation on identical host individuals is shown in A-F. The tissues implanted in the pocket of the dorsal side were auto, allo and xeno graft from the left side, respectively. Auto, self-tissue of the host fish; Allo, tissue of an intraclonal sister of the host fish; Xeno, tissue of outbred control of the host fish. fish in a similar manner. All the xenografts of the outbred control were rejected. However, the rejection was slow, and xenografts gradually disappeared between 30 and 60 days after implantation (Fig. 3, C-F). All of the host fish used in this experiment survived during the observation periods of 60 days. Growth Variance The measured composition of body length and weight of CL and OC of FO 1 are indicated in Fig. 4. In CL., the range of body length and weight were cm and was easily confirmed developed. Using by whether or not the haploid this procedure, the viable of fish polyploidized as eggs fertilized sperm may be regarded as gynogenetic by UV diploids. Similar studies have been performed in various combina tions ) Further cytogenetic investigations, hybridiza tion between amago salmon egg and rainbow trout sperm, and early development of the embryo are required. Proof of a homozygous first generation arising from a gynogenetic line of fish has been demonstrated using isozyme markers.23) In this study, the meiotic-g2n induced from the heterozygous parent showed a very high frequency of heterozygote at the Pgm-1 (73.3%) and Idh-3 loci (40.0%), but the mitotic-g2n were all homozygous except for F04. Thus, heterozygotes were only one specimen (4.8%) in 21 mitotic-g2n examined in Table 4 and Table 5. F04 heterozygote was regarded as gynogenetic diploid induced by spontaneous retention of the second polar body (spontaneous meiotic-g2n).16,24,25) The rate of spontan eous meiotic-g2n contained in the mitotic-g2n were presumed to be 6.5% calculated from the frequency of heterozygotes at the Pgm-1 locus mentioned above. We therefore omitted F04 from being maternal parents used to produce a cloned generation, because clones seemed not to be inducible from those individuals. These phenomena

6 280 Kobayashi et al, suggested that some heterozygous individuals might have appeared in the first mitotic-g2n generation that was used to produce the first clonal generation, consequently the genetic uniformity among the progeny should be con firmed. That clones produced from F0l and F06, were confirmed by tissue grafting a piece of operculum into the pocket made on the lateral dorsal region.9) Auto and allografts were accepted, but the xenografts were rejected after 30 days at 12.5 Ž. This duration was similar to that seen in clonal ayu,9) and it may be necessary for the immune system to reject the graft-tissue. We concluded that the gynogenetic second generations of homozygous diploids produced in this experiment were intraclonal siblings of amago salmon. Furthermore, it seems to be necessary to make a detailed analysis of genetic uniformity of these cloned fish at the DNA level. In the production of a second generation from mitotic-g2n, the induction rate of clones varied extremely among different strains. Onozato*9 has reported that the induction of clones from salmonid fishes was not easy and the survival of the hatched larva were extremely low because of the bad quality of the eggs. Furthermore, Suzuki et al. r 9) reported that homozygous or near-homozygous individuals from artificial diploidy gynogenesis were not appropriate for fish farming due to considerable inbreedinnng depresssion. In this study, the eyed rates and survival rates after the swim up stage were extremely low in the first clone generation of strain S9, which may be due to the same reason. If the selected individual had several recessive malignant genes, their traits would be revealed as phenotypes. As a result of these, inferior viability or growth, and an increase in malformation would occur. It has been reported that the viability of fish induced by artificial diploid gynogenesis is actually much lower than that of normal development.19) However, the rapid rise of the inbred degree by one gynogenetic manipulation is also considered to remove such malignant genes. According to this view point, the individual in the mitotic-g2n that showed good growth, normal maturation and spawning, probably underwent no undesirable effects of gene homozygosity on development. Although, in the next cloned generation, there were only a few viable specimens. The eggs from which these viable gynogenetic fish hatched, might be of normal quality, because the maternal parent was normal. Onozato*10 described that if a recessive harmful gene is located on the locus related to development of the ovary or of oogenesis, the effects of the malignant genes would not appear in the homozygous first mitotic-g2n generation, but in the next generation. The results of this study suggest that a viable cloned gynogenetic second generation cannot be obtained, when fish having lethal genes and high mortality such as the S9 strain are used as the parent. In conclusion, the rate of production of a clonal line is considered to be affected by the selected strain. On the other hand, in the induced clonal colony, the growth traits of FO 1-CL were not completely the same, but the variances of growth traits of CL were extremely disproportionate from those of OC. The higher magnitude of trait variance in OC was partly due to the appearance of males in that group, but it could also be interpreted to reflect the magnitude of genetic variance. Variance in CL seemed to be induced by environmental effects, which must be sharply different from other environmental factors such as culture density, quantity or frequency of food admin istration, and other various culture conditions. Nakanishi and Onozato261 examined growth variability in isogenic crucian carp Carassius giberio langsdorfii, and found that even in an clonal population, there is growth vari ability. This suggests that trifling environmental factors affect each individual. These would include the superiority or inferiority in the struggle for food at the larval stage, variations even in a small aquarium, the environment for each egg in the ovary at oogenesis, and the locations where developing eggs settled. That is to say, it seems that a phenomenon such as growth is closely involved with causes other than genetics.") In other words, a link should not be sought between the induction of isogenic population and the production of a standard unified fish population. Although this study indicated a second magnification of growth variation by crossing with another strain, the report of Nakanishi and Onozato showed that the growth variation in a population of isogenetic crucian carp can be decreased. If the environmental factors could be standardized as much as possible, the variance in a cloned population could be minimized, and the introduction of the cloned strain to aquaculture should be greatly significant. However, raising techniques which fully enhance the characteristics of those clonal strains would need to be developed. In the present study, several homozygous clones of amago salmon were induced by means of gynogenetic manipula tion. Clonal fish should be very useful and effective in fish culture management, because they should have a unified seedling size, growth and other characteristics. These will relieve the labor required to select and standardize seedling uniformity. Furthermore, since the genetic backgrounds of each clone are completely identified, the effect of the environment will be measurable. Knowing the biological characteristics of clones should allow the relationship between inheritance and circumstance to be explained. Acknowledgements We are grateful to Dr. M. Matsuyama, Department of Bioresources, Mie University, and Dr. Y. Fujioka, Shiga prefectural Fisheries Experimental Station, for their useful advice and critical reading of the manuscript. This study was supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry, and Fisheries of Japan. References 1) G. H. Thorgaard: Chromosome set manipulation and sex control in fish, in "Fish Physiology" (ed. by W. S. Hoar, D. J. Randall, and E. M. Donaldson), Vol. 9, Academic Press, New York, 1983, pp ) C. E. Purdom, D. Thompson, and Y. D. Lou: Genetic engineering in rainbow trout, Salmo gairdnerii Richardson, by the suppression of meiotic and mitotic metaphase. J. Fish Biol., 27, (1985). 3) F. W. Allendorf and R. F. Leary: Heterozygosity in gynogenetic diploids and triploid estimated by gene-centromere recombination *9 H. Onozato: Abst. Meetg. Japan. Soc. Sci. Fish., April, 1990, p. 53 (in Japanese).

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