蓝鳃太阳鱼性控和性决定机制以及性别相关分子标记的研究
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摘要
蓝鳃太阳鱼(Lepomis macrochirus)隶属鲈形目,太阳鱼科,太阳鱼属。其耐低溶氧和肥水,肉质细嫩,无肌间刺,在美国密歇根和俄亥俄州,蓝鳃太阳鱼已经成为三大主要水产养殖种类之一。同时,由于其体色鲜艳,尤其在生殖季节,雄鱼的体色非常艳丽,其已作为一种观赏鱼类被很多人喜爱。但是蓝鳃太阳鱼具有性成熟早、繁殖快、年繁殖次数多等特性,使得在水库和池塘养殖过程中极易造成繁殖过剩、密度过大、生长速度较慢、个体过小且大小不均等不利情况,从而影响了蓝鳃太阳鱼养殖产业的进一步扩大。目前解决这一问题最为理想的措施是通过单性养殖有效的控制群体数量,消除大量繁殖、早熟以及其造成的影响。蓝鳃太阳鱼的生长表现出显著的性别差异,雄性比雌性体型大,生长速度快。因此,生产中培育全雄蓝鳃太阳鱼具有非常重要的应用和商业价值。本研究在蓝鳃太阳鱼性逆转基础上,对其性别决定机制进行初步的阐述,尝试采用分子标记方法寻找其性别特异性分子标记,对蓝鳃太阳鱼的单性育种进行研究。主要研究结果如下：
(1)蓝鳃太阳鱼性别分化及其与个体大小和年龄的关系
对鱼类性腺发育和性腺分化过程的了解有助于在其激素敏感时期采取外源激素的处理,从而在水产养殖实践中有效的调控其性别发育。本试验系统研究了蓝鳃太阳鱼从孵出到孵出后90天(90 dph)期间的性腺分化过程。通过设计慢速生长批次鱼(SGB)和快速生长批次鱼(FGB),研究了其性腺分化与个体年龄和生长的关系。在卵巢发育中,生殖细胞和体细胞的分化开始于鱼体体长为13.2-16.0 mm(SGB的60 dph,FGB的30 dph)之间。性腺基部和末梢组织的突起和延伸以及组织的融合发生在体长为16.0-21.0 mm(SGB的80 dph,FGB的50 dph)之间,生殖细胞在这个时期开始了减数分裂。体长大于25.5 mm的雌性卵巢已经含有了周边核仁期卵母细胞。在精巢发育方面,输出管形成于体长为25.4-28.0 mm(SGB的90 dph,FGB的70 dph)个体中,生殖细胞的减数分裂在这个阶段开始出现,体长大于33.0 mm的雄性精巢中精原细胞减数分裂活跃。这些结果表明蓝鳃太阳鱼属于雌雄异体分化型鱼类,雌性性腺的分化早于雄性,性腺分化与生长速度更相关,其性腺分化的关键时期发生在其体长为13.2-16.0mm之间。
(2)17β-雌二醇诱导蓝鳃太阳鱼雌性化研究
系统研究了口服不同剂量的天然雌二醇17p-estradiol(E2)对蓝鳃太阳鱼生长、性别比例、性腺发育的影响。从鱼苗孵出后30天开始投喂含不同剂量E2的饲料(50 mg/kg,100 mg/kg,150 mg/kg和200 mg/kg),设置一个空白组,每个组设置三个重复,投喂60天,结果发现E2的投喂对鱼存活率没有产生显著性影响(P>0.05)。E2投喂期间,鱼的生长速度受到了抑制,但在投喂E2完后的120天,投喂了E2的鱼生长速度出现了补偿效应。所有的E2处理组都诱导了100%的雌性,但在50mg/kg和100 mg/kg的处理组出现了兼性个体。待所有处理组鱼成熟后,比较了繁殖季节不同处理组鱼的性腺指数,没有显著差异(P>0.05)。E2处理组所得到的雌性的卵巢组织结构与对照组雌性的卵巢没有明显差别。综合比较雌性化效果、E2投喂期间和投喂后的生长速度以及个体性腺的发育,总结得出150 mg/kg的E2处理对蓝鳃太阳鱼的雌性化可以得到最佳的效果,50mg/kg和100 mg/kg的E2剂量不足,而200 mg/kg的E2过量。
(3)芳香化酶抑制剂诱导蓝鳃太阳鱼雄性化研究
使用芳香化酶抑制剂Letrozole对蓝鳃太阳鱼进行雄性化处理。实验1在鱼苗30-90 dph期间,投喂含不同AI剂量(50 mg/kg,150 mg/kg,250 mg/kg和500mg/kg)的饲料,结果发现500 mg/kg的AI处理影响了雌性卵巢腔的形成,在这个处理组发现了一个兼性性腺。所有处理组鱼的雌性比例都显著偏离理论值1：1,且雄性比例都显著高于对照组(P<0.05)。实验2,在鱼苗30-50 dph期间,每隔5天浸泡一次AI,设置250μg/L,50μg/L和1000μg/L AI三个处理组和一个对照组,每次浸泡8小时,结果发现500μg／L和1000μg/LAI处理组的雄性比例显著高于对照组和1000μg/L AI处理组(P<0.05)。各AI处理组和对照组鱼的体长和体重都不存在显著差异(P>0.05)。这些结果显示芳香化酶抑制剂AI可以诱导蓝鳃太阳鱼雌鱼雄性化,表明AI可以改变蓝鳃太阳鱼性腺的分化并通过抑制芳香化酶的活性促使性腺分化为雄性。
(4)温度以及温度-基因型相互作用对蓝鳃太阳鱼性别分化的影响
研究了不同的温度(17。C,23℃,29℃和34℃)处理对蓝鳃太阳鱼性别分化的影响,通过设计两批来自不同亲本的鱼,探讨了基因型×环境型相互作用对蓝鳃太阳鱼性别分化的影响。使用6对多态性高的微卫星标记对不同批次的鱼做了亲子鉴定,确定了其亲本来源,结果显示第一批鱼来自11个家系,第二批鱼来自8个家系。对不同温度处理组鱼的性别进行鉴定,结果显示第一批鱼的29℃和34℃处理组鱼,以及第二批鱼的34℃处理组鱼的雌雄比例显著偏离理论值1：1(P<0.05)。第一批鱼的29℃和34℃处理组的雄性比例显著高于17℃和23℃处理组(P<0.05),而第二批鱼的各温度处理组之间的雄性比例没有表现出显著差异(P>0.05)。比较两批鱼相同的温度处理组发现29℃和34℃两个处理组存在显著差异(P<0.05)。通过组织切片的观察发现17℃和34℃处理组存在兼性个体,在同一个性腺组织内同时存在精巢组织和卵巢组织。这些结果表明高温诱导了更多的雄性,低温诱导更多的雌性,但其诱导率受到亲本基因型的影响,不同家系个体对温度的敏感性不同。因此在蓝鳃太阳鱼性别决定中,存在温度-基因型的相互作用。
(5)全雄蓝鳃太阳鱼的获得及其性别决定机制的初步探讨
将通过E2处理的雌性化蓝鳃太阳鱼(150 mg/kg和200 mg/kg E2)与正常雄性交配,检测子代的性别比例,繁殖了20个家系,同时还繁殖了2个正常雌雄交配产生的家系,养殖过程中有5批性逆转雌鱼繁殖的家系全部死掉了,其余批次的鱼的存活率都达到了80%以上。性别鉴定结果显示2个对照组家系子代的性别比例都接近于理论值1：1,没有显著性差异(P>0.05)。而15个性逆转亲本的繁殖家系中,有1个家系的雄性所占百分比为61%；有7个家系的雄性比例在70%-80%之间；另外有2个家系的雄性比例为97%和98%,5个家系的雄性比例为100%,这7个家系的雌雄比例与预期值0：1没有显著性差异(P>0.05),相当于获得7个全雄蓝鳃太阳鱼家系。鉴于不同家系中雄性所占比例绝大部分在75%到100%之间,考虑到养殖期间温度的影响,根据三系配套法原理,可初步推测蓝鳃太阳鱼的性别决定机制为ZW/ZZ型,但常染色体上的一些基因或者是环境也会影响其性别决定。
(6)蓝鳃太阳鱼雌激素受体和芳香化酶基因在雌雄不同组织中的差异表达
雌激素受体ERal、ERa2和ERβ基因在蓝鳃太阳鱼雌性和雄性的性腺、鳃、胃、大脑、肾脏、肝脏、心脏、肌肉组织中都有表达,以β-actin基因做内参基因,比较表明ERal和ERa2基因在雌性卵巢的表达量高于雄性精巢,ERa2基因在雄性大脑组织中的表达高于雄性的其它组织,在雌性肝脏中的表达高于雌性其它组织,而ERβ基因在雄性精巢的表达量高于雌性卵巢,在雄性胃组织的表达量也高于雌性。通过3'-RACE得到了1498 bp的芳香化酶基因Cyp19a1a的cDNA序列,Cyp19a1a基因在蓝鳃太阳鱼雌雄鳃、胃、肾脏、心脏、肌肉组织中都没有表达,在雌性卵巢、肝脏和大脑中有表达,雄性精巢和肝脏中有表达,但在卵巢中的表达量高于精巢。这为通过基因表达情况鉴定蓝鳃太阳鱼的性别奠定了一定的基础。
(7)蓝鳃太阳鱼性别特异AFLP标记的筛选
采用AFLP分子标记方法,使用256对AFLP引物组合,在2个雌性DNA基因池和2个雄性DNA基因池中扩增,共扩增出了12835条清晰的条带,平均每个引物组合产生51条带,在四个基因池中共发现了531个多态位点,占总位点数的4.14%。其中9对引物组合在雌雄基因组DNA池中产生性别特异性扩增带,4个位点为雌性基因池特有,5个位点为雄性基因池特有。但将这9对引物组合在个体中评估没有发现基因池所具有的特异性扩增带在组成该基因池的所有个体中都存在,这表明在基因池中出现的差异位点都是由个体差异产生,而不是该性别所特有的标记,表明蓝鳃太阳鱼雌雄基因组信息差异不大。
利用9个AFLP引物组合,对蓝鳃太阳鱼Hocking, Wooster和Hebron三个群体进行了遗传结构分析。结果显示三个群体的多态位点百分率分别为28.7%、14.7%和16.8%,Nei's遗传多样性分别为0.0866、0.0459和0.0617,Hocking群体蓝鳃太阳鱼遗传多样性最丰富,Hebron群体其次,Wooster群体最低。Hocking和Hebron群体间的遗传距离最小,亲缘关系最近,Hocking和Wooster群体间的遗传分化最大。该结果为开展蓝鳃太阳鱼的遗传选育奠定了一定的基础。
本课题系统研究了蓝鳃太阳鱼性别分化、性别调控、性别相关基因表达等内容,初步鉴定蓝鳃太阳鱼的性别决定机制为ZW/ZZ型,但常染色体上的一些基因或者是环境也会影响其性别决定。本研究结果为其单性养殖提供了指导,有望通过繁殖性逆转鱼大规模得到全雄蓝鳃太阳鱼群体,用于其养殖业,这将大大提高蓝鳃太阳鱼养殖的经济效益。
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 age
A 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 sunfish
We 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 sunfish
The 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 sunfish
The 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 sunfish
Twenty 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 sunfish
The 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 sunfish
AFLP 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.
(1)蓝鳃太阳鱼性别分化及其与个体大小和年龄的关系
对鱼类性腺发育和性腺分化过程的了解有助于在其激素敏感时期采取外源激素的处理,从而在水产养殖实践中有效的调控其性别发育。本试验系统研究了蓝鳃太阳鱼从孵出到孵出后90天(90 dph)期间的性腺分化过程。通过设计慢速生长批次鱼(SGB)和快速生长批次鱼(FGB),研究了其性腺分化与个体年龄和生长的关系。在卵巢发育中,生殖细胞和体细胞的分化开始于鱼体体长为13.2-16.0 mm(SGB的60 dph,FGB的30 dph)之间。性腺基部和末梢组织的突起和延伸以及组织的融合发生在体长为16.0-21.0 mm(SGB的80 dph,FGB的50 dph)之间,生殖细胞在这个时期开始了减数分裂。体长大于25.5 mm的雌性卵巢已经含有了周边核仁期卵母细胞。在精巢发育方面,输出管形成于体长为25.4-28.0 mm(SGB的90 dph,FGB的70 dph)个体中,生殖细胞的减数分裂在这个阶段开始出现,体长大于33.0 mm的雄性精巢中精原细胞减数分裂活跃。这些结果表明蓝鳃太阳鱼属于雌雄异体分化型鱼类,雌性性腺的分化早于雄性,性腺分化与生长速度更相关,其性腺分化的关键时期发生在其体长为13.2-16.0mm之间。
(2)17β-雌二醇诱导蓝鳃太阳鱼雌性化研究
系统研究了口服不同剂量的天然雌二醇17p-estradiol(E2)对蓝鳃太阳鱼生长、性别比例、性腺发育的影响。从鱼苗孵出后30天开始投喂含不同剂量E2的饲料(50 mg/kg,100 mg/kg,150 mg/kg和200 mg/kg),设置一个空白组,每个组设置三个重复,投喂60天,结果发现E2的投喂对鱼存活率没有产生显著性影响(P>0.05)。E2投喂期间,鱼的生长速度受到了抑制,但在投喂E2完后的120天,投喂了E2的鱼生长速度出现了补偿效应。所有的E2处理组都诱导了100%的雌性,但在50mg/kg和100 mg/kg的处理组出现了兼性个体。待所有处理组鱼成熟后,比较了繁殖季节不同处理组鱼的性腺指数,没有显著差异(P>0.05)。E2处理组所得到的雌性的卵巢组织结构与对照组雌性的卵巢没有明显差别。综合比较雌性化效果、E2投喂期间和投喂后的生长速度以及个体性腺的发育,总结得出150 mg/kg的E2处理对蓝鳃太阳鱼的雌性化可以得到最佳的效果,50mg/kg和100 mg/kg的E2剂量不足,而200 mg/kg的E2过量。
(3)芳香化酶抑制剂诱导蓝鳃太阳鱼雄性化研究
使用芳香化酶抑制剂Letrozole对蓝鳃太阳鱼进行雄性化处理。实验1在鱼苗30-90 dph期间,投喂含不同AI剂量(50 mg/kg,150 mg/kg,250 mg/kg和500mg/kg)的饲料,结果发现500 mg/kg的AI处理影响了雌性卵巢腔的形成,在这个处理组发现了一个兼性性腺。所有处理组鱼的雌性比例都显著偏离理论值1：1,且雄性比例都显著高于对照组(P<0.05)。实验2,在鱼苗30-50 dph期间,每隔5天浸泡一次AI,设置250μg/L,50μg/L和1000μg/L AI三个处理组和一个对照组,每次浸泡8小时,结果发现500μg／L和1000μg/LAI处理组的雄性比例显著高于对照组和1000μg/L AI处理组(P<0.05)。各AI处理组和对照组鱼的体长和体重都不存在显著差异(P>0.05)。这些结果显示芳香化酶抑制剂AI可以诱导蓝鳃太阳鱼雌鱼雄性化,表明AI可以改变蓝鳃太阳鱼性腺的分化并通过抑制芳香化酶的活性促使性腺分化为雄性。
(4)温度以及温度-基因型相互作用对蓝鳃太阳鱼性别分化的影响
研究了不同的温度(17。C,23℃,29℃和34℃)处理对蓝鳃太阳鱼性别分化的影响,通过设计两批来自不同亲本的鱼,探讨了基因型×环境型相互作用对蓝鳃太阳鱼性别分化的影响。使用6对多态性高的微卫星标记对不同批次的鱼做了亲子鉴定,确定了其亲本来源,结果显示第一批鱼来自11个家系,第二批鱼来自8个家系。对不同温度处理组鱼的性别进行鉴定,结果显示第一批鱼的29℃和34℃处理组鱼,以及第二批鱼的34℃处理组鱼的雌雄比例显著偏离理论值1：1(P<0.05)。第一批鱼的29℃和34℃处理组的雄性比例显著高于17℃和23℃处理组(P<0.05),而第二批鱼的各温度处理组之间的雄性比例没有表现出显著差异(P>0.05)。比较两批鱼相同的温度处理组发现29℃和34℃两个处理组存在显著差异(P<0.05)。通过组织切片的观察发现17℃和34℃处理组存在兼性个体,在同一个性腺组织内同时存在精巢组织和卵巢组织。这些结果表明高温诱导了更多的雄性,低温诱导更多的雌性,但其诱导率受到亲本基因型的影响,不同家系个体对温度的敏感性不同。因此在蓝鳃太阳鱼性别决定中,存在温度-基因型的相互作用。
(5)全雄蓝鳃太阳鱼的获得及其性别决定机制的初步探讨
将通过E2处理的雌性化蓝鳃太阳鱼(150 mg/kg和200 mg/kg E2)与正常雄性交配,检测子代的性别比例,繁殖了20个家系,同时还繁殖了2个正常雌雄交配产生的家系,养殖过程中有5批性逆转雌鱼繁殖的家系全部死掉了,其余批次的鱼的存活率都达到了80%以上。性别鉴定结果显示2个对照组家系子代的性别比例都接近于理论值1：1,没有显著性差异(P>0.05)。而15个性逆转亲本的繁殖家系中,有1个家系的雄性所占百分比为61%；有7个家系的雄性比例在70%-80%之间；另外有2个家系的雄性比例为97%和98%,5个家系的雄性比例为100%,这7个家系的雌雄比例与预期值0：1没有显著性差异(P>0.05),相当于获得7个全雄蓝鳃太阳鱼家系。鉴于不同家系中雄性所占比例绝大部分在75%到100%之间,考虑到养殖期间温度的影响,根据三系配套法原理,可初步推测蓝鳃太阳鱼的性别决定机制为ZW/ZZ型,但常染色体上的一些基因或者是环境也会影响其性别决定。
(6)蓝鳃太阳鱼雌激素受体和芳香化酶基因在雌雄不同组织中的差异表达
雌激素受体ERal、ERa2和ERβ基因在蓝鳃太阳鱼雌性和雄性的性腺、鳃、胃、大脑、肾脏、肝脏、心脏、肌肉组织中都有表达,以β-actin基因做内参基因,比较表明ERal和ERa2基因在雌性卵巢的表达量高于雄性精巢,ERa2基因在雄性大脑组织中的表达高于雄性的其它组织,在雌性肝脏中的表达高于雌性其它组织,而ERβ基因在雄性精巢的表达量高于雌性卵巢,在雄性胃组织的表达量也高于雌性。通过3'-RACE得到了1498 bp的芳香化酶基因Cyp19a1a的cDNA序列,Cyp19a1a基因在蓝鳃太阳鱼雌雄鳃、胃、肾脏、心脏、肌肉组织中都没有表达,在雌性卵巢、肝脏和大脑中有表达,雄性精巢和肝脏中有表达,但在卵巢中的表达量高于精巢。这为通过基因表达情况鉴定蓝鳃太阳鱼的性别奠定了一定的基础。
(7)蓝鳃太阳鱼性别特异AFLP标记的筛选
采用AFLP分子标记方法,使用256对AFLP引物组合,在2个雌性DNA基因池和2个雄性DNA基因池中扩增,共扩增出了12835条清晰的条带,平均每个引物组合产生51条带,在四个基因池中共发现了531个多态位点,占总位点数的4.14%。其中9对引物组合在雌雄基因组DNA池中产生性别特异性扩增带,4个位点为雌性基因池特有,5个位点为雄性基因池特有。但将这9对引物组合在个体中评估没有发现基因池所具有的特异性扩增带在组成该基因池的所有个体中都存在,这表明在基因池中出现的差异位点都是由个体差异产生,而不是该性别所特有的标记,表明蓝鳃太阳鱼雌雄基因组信息差异不大。
利用9个AFLP引物组合,对蓝鳃太阳鱼Hocking, Wooster和Hebron三个群体进行了遗传结构分析。结果显示三个群体的多态位点百分率分别为28.7%、14.7%和16.8%,Nei's遗传多样性分别为0.0866、0.0459和0.0617,Hocking群体蓝鳃太阳鱼遗传多样性最丰富,Hebron群体其次,Wooster群体最低。Hocking和Hebron群体间的遗传距离最小,亲缘关系最近,Hocking和Wooster群体间的遗传分化最大。该结果为开展蓝鳃太阳鱼的遗传选育奠定了一定的基础。
本课题系统研究了蓝鳃太阳鱼性别分化、性别调控、性别相关基因表达等内容,初步鉴定蓝鳃太阳鱼的性别决定机制为ZW/ZZ型,但常染色体上的一些基因或者是环境也会影响其性别决定。本研究结果为其单性养殖提供了指导,有望通过繁殖性逆转鱼大规模得到全雄蓝鳃太阳鱼群体,用于其养殖业,这将大大提高蓝鳃太阳鱼养殖的经济效益。
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 age
A 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 sunfish
We 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 sunfish
The 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 sunfish
The 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 sunfish
Twenty 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 sunfish
The 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 sunfish
AFLP 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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