| The bluegill sunfish Lepomis macrochirus, a member of the sunfish family (family Centrarchidae) of order Perciformes, is currently recognized as one of the most valuable North American recreational fishes. It has long been commercially cultured to support recreational fishery stocking needs throughout the middle south, and southeastern United States. In the meantime, the bluegill sunfish has become increasingly important as a high-value species in aquaculture. In some states, like Ohio and Michigan in the United States, the bluegill sunfish has been listed as one of the top three culture species of fish. However, a common problem in small impoundments and farm ponds stocked with this species is overcrowded bluegill population due to their prolific reproductive nature and early maturation. An approach to the management of bluegill in small impoundments is to stock monosex populations. Monosex populations can eliminate the problem of prolific reproduction, precocious maturity and their consequences. A number of studies have shown that male bluegill sunfish appear to hold the greatest potential for the food market due to their more rapid growth capacity versus females. Therefore, monosex (all-male) culture will hold considerable potential as a method to increase the efficiency and profitability of bluegill sunfish aquaculture by improving growth rates. The objectives of our present study are to elucidate the sex determinination mechanism in bluegill and to use this information to develop mass production technology of genetically male populations. The main contents include:(1) Gonadal sex differentiation in the bluegill sunfish and its relation to fish size and ageA detailed understanding of the time of gonadal development and differentiation is critical to control sex and optimize culture. In the present study, we systematically studied gonadal sex differentiation of the bluegill sunfish and its relation to fish size and age from post hatching to 90 days post hatching (dph) using a slow-growing batch (SGB) and a fast-growing batch (FGB) of fish. The results indicated that the gonadal differentiation in bluegill was more related to body size than to age. In presumptive ovaries, germinal and somatic differentiation began between 13.2 and 16.0 mm (60 dph in SGB and 30 dph in FGB) in total length (TL). The outgrowths from the proximal and distal portions of the gonads and the fusion of the outgrowths to form the ovarian cavity occurred between 16.0 and 21.0 mm TL (80 dph in SGB and 50 dph in FGB) with germ cells undergoing meiosis. The gonads in the females larger than 25.5 mm TL always had peri-nucleolus oocytes. In presumptive testes, the efferent duct formed in the fish ranging from 25.4 to 28.0 mm TL (90 dph in SGB and 70 dph in FGB) with the onset of meiosis and testes contained spermatocytes exhibiting active meiosis in males larger than 33.0 mm TL. These findings indicate that bluegill is a differentiated gonochorist and sex differentiation occurs earlier in females than males. Based on our results, we suggest that the critical period of sex differentiation in bluegill occurs between 13.2 and 16.0 mm TL and histological sex differentiation is distinguishable in most fish larger than 21.0 mm TL.(2) Feminization effects of 17β-estradiol on bluegill sunfishWe systematically investigated the feminization of bluegill L. macrochirus by oral administration of various doses of 17β-estradiol (E2) and evaluated their effects on the growth performance, production and gonadal structure of sex-reversed female bluegill at both sex-ratio and histological levels. With positive control treatment, 30-day-old fry were fed E2 at 50 mg/kg,100 mg/kg,150 mg/kg and 200 mg/kg diet for 60 days. The survival of fish in the E2 treated and control groups were not significantly different (P> 0.05). The growth of the treated fish was significantly retarded during the period of treatment, while there was no side effect detected post-treatment and the retarded fish caught up during 120 days of culture after E2 treatment. All the treated groups produced 100% monosex female populations based on the macroscopic shape of gonads, and there were no significant differences detected between any E2 treatment and control group in the mean GSI of females during the spawning season from June to October (P> 0.05). Histologically,13.3% and 5.0% of the intersex fish were determined to come from the 50 mg/kg and 100 mg/kg E2 treatment groups, respectively, with 6.9% and 4.1% of the gonadal area containing testicular tissue. Most of genotypical male fish treated with exogenous E2 developed gonadal structures histologically indistinguishable from the gonads of females. This study suggests that 150 mg/kg E2 is the optimal dosage for feminization in bluegill, with 50 mg/kg and 100 mg/kg E2 being sub-optimal and 200 mg/kg E2 being over-optimal.(3) Masculinization effects of a nonsteroidal aromatase inhibitor on bluegill sunfishThe efficacy of Letrozole, a potent nonsteroidal aromatase inhibitor (AI), on gonadal sex differentiation and sex reversal was examined on bluegill sunfish. In Experiment 1 with AI diet treatments (50 mg/kg,150 mg/kg,250 mg/kg and 500 mg/kg) from 30 dph to 90 dph, AI interrupted ovarian cavity formation and the proportions of males in all treated groups were significantly higher than that in the control group. In Experiment 2 with AI immersion treatments (250μg/L,500μg/L, 1000μg/L) during 30 dph to 50 dph, the treated groups of 500μg/L and 1000μg/L produced significantly more males than control and 250μg/L groups. Histological examination revealed no differences in ovary or testis tissues between control and AI treated fish. There were no significant differences detected in body weight and length among the AI treated and control groups (P> 0.05) for both experiments. The results from these two experiments suggest that inhibition of aromatase activity by AI could influence sex differentiation in bluegill sunfish.(4) Temperature effects and genotype-temperature interactions on sex differentiation of bluegill sunfishThe effects of genotype by temperatures (17℃,23℃,29℃,34℃) on sex ratios of bluegill sunfish were tested on two batches of fry with different parents, each having two replicates. The fry were reared at each temperature from an initial mean size of 0.7 cm (4 days post hatch) to a final mean size of 6.3 cm. The sex of fish was then determined by macroscopic and histological examination of the gonads. The potential parents and fry were genotyped at 6 polymorphic microsatellite loci to perform parentage assignment and the result showed there were 11 and 8 full-sib families in the first batch and second batch, respectively. Chi-squared analysis comparing sex ratios of experimental groups for each treatment in both batches of bluegill sunfish with a theoretical 1:1 sex ratio indicated that there were significant deviations in 29℃and 34℃groups in first batch, and 34℃group in second batch bluegill (P< 0.05). The percents of males in 29℃and 34℃groups were significantly higher than that in 17℃and 23℃groups in the first batch (P< 0.05), while in the second batch, no significant differences were found between any treatments (P> 0.05). The pooled sex ratios were then compared to be each other and found that temperature had significant effects on sex ratios in the first batch fish (P< 0.001), while no significant effects on the second batch fish (P> 0.05). There were significant differences in 29℃and 34℃groups between these two batches fish (P< 0.05). Through histological examination, intersex fish were identified in 17℃and 34℃groups, where testicular and ovarian tissue occurred in the same gonad. These results indicated that high rearing temperature could alter the phenotypic sex ratios and increase the proportion of males in some families of bluegill, while not having the same effect in other families. It was concluded that genotype-temperature interactions were existed on bluegill sex determination and their existence suggests the interesting possibility of selecting thermosensitive genotypes in breeding programs for mostly male populations.(5) Production of all-male populations and discussion of sex determination mechanism in bluegill sunfishTwenty female from sex-reversed fish (150 mg/kg and 200 mg/kg E2) were mated with 20 normal males and two normal females were mated with 2 normal males as control groups. When fry reached the size of over 6 cm, the sex of the fry from each batch were identified. We successfully checked the sex ratios in 15 families from sex-reversed fish and 2 families from normal fish. The results showed the male to female ratios in the two normal batches were not significantly different from theoretical 1:1 ratio (P> 0.05), while male to female ratios in most sex-reversed batches were significantly different from theoretical 1:1 ratio (P< 0.05). Among the sex-reversed batches,61% of the males were found in one family, 70-80% in seven families,97% and 98% in two families, and 100% in five families. In the last seven families, the female to male ratios were not significantly different from theoretical 0:1 ratio (P> 0.05). Considering the principle of endocrine methods to produce all-male stocks and temperature effects on sex differentiation, we suggest the sex determination mechanism in bluegill sunfish is ZW/ZZ type and autosomal genes may be also involved in the process of sex determination.(6) Sex-specific tissue expression of estrogen receptor (ERal, ERa2 and ERβ) and aromatase (Cypl9a1a) genes in bluegill sunfishThe cDNA sequence of 3'-untranslated region of Cypl9a1a about 1498 bp was obtained for bluegill sunfish. Several tissues were analyzed from both male and female adult bluegill sunfish for sex-specific tissue expression. The genes ERal, ERa2 and ERβwere expressed in gill, stomach, spleen, brain, kidney, liver, heart, muscle and gonad. The expression of ERal and ERa2 in ovary was higher than that in testis. In males, ERa2 expression was highest in brain, while in females, ERa2 expression was highest in liver. ERβexpression in testis was higher than that in the ovary and stomach. Cypl9a1a expression was not detected in gill, stomach, spleen, heart and muscle. It was expressed in ovary, liver and brain of females, and in testis and liver of males, while expression in ovary was higher than that in testis. These results would be useful for sex identification in bluegill sunfish.(7) An AFLP-based approach to identify sex-specific marker in bluegill sunfishAFLP technique was used to identify sex-specific markers in bluegill. Four DNA pools including two females and two males (10 individuals in each pool) were screened by a total of 256 primer combinations. The results showed only 9 out of 12835 loci (0.73%o) exhibited presumed sex-associated amplifications. However, when individuals of each pool were involved to confirm these markers, the polymorphism among different individuals was responsible for the sex-associated amplifications. None was detected as sex-specific markers.Nine AFLP primer combinations were used to screen the genome DNA of bluegill individuals from three populations (Hocking, Wooster and Hebron). The results showed the mean percentages of polymorphic bands in the three populations were 28.7%,14.7% and 16.8%, and the Nei's gene diversity was 0.0866,0.0459 and 0.0617, respectively. The Hocking population had the highest genetic diversity, while Wooster population had the lowest value. The genetic distance between Hocking and Hebron was larger than any other two populations.This study systematically investigated the sex differentiation, sex control, sex determination mechanism and sex-related markers in bluegill sunfish. Sex determination mechanism in bluegill sunfish was suggested to be ZZ/ZW type, while autosomal genes are also involved in the process of sex determination. The present results indicated the feasibility of increasing the efficiency and profitability of bluegill aquaculture through monosex male culture. |
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