Temperature-induced Feminization of Channel Catfish
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摘要:
目的 在性别分化前利用高温诱导斑点叉尾鮰性别逆转，探究雄性斑点叉尾鮰雌性化的合适水体温度。 方法 设置（30±0.5） ℃ （T-30，CK）、（33±0.5） ℃ （T-33）和（36±0.5） ℃ （T-36） 3个温度组对1dah试验鱼进行为期30 d的温度诱导。对卵巢已分化成型的60日龄试验鱼测量并统计生长数据、存活率、卵巢形成比例，同时结合遗传性别鉴定结果计算各组的性逆转率。分别在解剖学和组织学水平观察各组试验鱼的性腺结构和卵母细胞发育水平。进一步采用qRT-PCR方法分别检测雄性性别标志基因dmrt1和雌性性别标志基因foxl2在150日龄XX雌鱼、XY伪雌鱼卵巢中的表达水平。 结果 T-30组、T-33组和T-36组试验鱼的存活率分别为95.33%、91.33%、82.67%；随着温度的升高，各组试验鱼的体长、体重先增加后减小，T-30组、T-33组和T-36组试验鱼的体长分别为9.13 cm、10.14 cm、8.80 cm；T-30组、T-33组和T-36组试验鱼的体重分别为6.31 g、9.76 g、6.11 g；T-30组、T-33组和T-36组试验鱼的卵巢形成比例分别为51.00%、66.67%、77.67%。性逆转效果评估结果显示：T-30组XX雌鱼和T-33组、T-36组伪XY雌鱼卵母细胞以Ⅱ期卵母细胞为主，T-30组中XX雌鱼和T-36组XY伪雌鱼出现Ⅲ期卵母细胞；T-33组中XY伪雌鱼出现卵巢发育缓慢、轮廓不清、与体腔黏膜相连的现象。对斑点叉尾鮰性别相关基因检测显示：foxl2基因在XY雌鱼中被激活，dmrt1基因在XY雌鱼中的表达被抑制。 结论 对性别分化前的斑点叉尾鮰持续进行高温诱导，可以使斑点叉尾鮰的生理性别向雌性化方向分化。 Abstract:Objective Water temperature required to induce feminization on male channel catfish fries before sex differentiation was determined. Method One-dah channel catfish fries were treated for 30 d in water at the temperature of (30±0.5) ℃ (T-30, CK), (33±0.5) ℃ (T-33) or (36±0.5) ℃ (T-36). Growth and rates of survival, ovarian formation, and sex reversal of individual fish that had ovaries differentiated and formed in 60 d with positive genetic sex identification were measured, calculated, and recorded. In each group, the ovarian development of XX and XY females as determined by the anatomy and H&E staining sections was compared and analyzed. Subsequently, qRT-PCR was used to detect the expressions of foxl2 and dmrt1 in XX and XY female ovaries as well as XY male testis at 150 dahs. Result The survival rates of the fries under T-30, T-33, and T-36 were 95.33%, 91.33%, and 82.67%, respectively. The body length of the fish under T-30 measured at 9.13 cm, under T-33 at 10.14 cm, and under T-36 at 8.80 cm, while the body weighed at 6.31 g for CK, 9.76 g for those under T-33, and at 6.11 g for those under T-36. The ovarian formation percentages were 51.00% under CK, 66.67% under T-33, and 77.67% under T-36. On sex reversal, the majority of oocytes in the XX females were at stage Ⅱ under T-30, but under T-33 and T-36 in the XY females. The stage Ⅲ oocytes were found in the XX females under T-30 as well as in the XY females under T-36. Under T-33, the XY female channel catfish showed slow oval development with unclear outline and coelomic mucosa. The expression of foxl2 was upregulated and that of dmrt1 downregulated in the XY females. Conclusion The physiological sex before sexual differentiation of channel catfish could be converted to female by continuous high-temperature induction. -
Key words:
- Channel catfish /
- sexual reversal /
- temperature /
- feminization
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图 1 60日龄斑点叉尾鮰卵巢组织
图A、C、G是T-30、T-33、T-36组中XX雌鱼的卵巢组织切片图，图E、I是T-33、T-36组中XY伪雌鱼的卵巢组织切片图；图B、D、H是T-30、T-33、T-36组中XX雌鱼的卵巢组织解剖图，图F、J是T-33、T-36组XY伪雌鱼的卵巢组织解剖图。
Figure 1. Histological sections and anatomy of ovaries of 60 dah channel catfish
A, C, and G: histological ovarian sections of channel catfish (XX) under T-30, T-33, and T-36, respectively; E and I: histological ovarian sections of sexual reversal channel catfish (XY) under T-33 and T-36, respectively; B, D, and H: ovarian anatomy of channel catfish (XX) under T-30, T-33, and T-36, respectively; F and J: ovarian anatomy of sex-reversed channel catfish (XY) under T-33 and T-36, respectively.
图 2 斑点叉尾鮰性别分化基因表达
A为斑点叉尾鮰雄性性别分化基因dmrt1表达图，B为斑点叉尾鮰雌性性别决定基因foxl2表达图。图中小写字母表示显著性差异（ P ＜0.05）。
Figure 2. mRNA expressions of sex differentiation genes in channel catfish
A: mRNA expression of dmrt1; B: mRNA expression of foxl2. Means with different lowercase letters indicate significant difference at P＜0.05.
表 1 开口饵料及微粒饲料调整方案
Table 1. Particulate feed adjustment for channel catfish
饲料
Feed试验鱼体长
Body length/mm投喂天数
Feeding days/d卤虫 1～6 3 微粒饲料S1 6～10 6 微粒饲料S2 9～14 6 微粒饲料S3 13～18 6 微粒饲料S4 18～28 6 
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表 2 qRT-PCR反应引物表
Table 2. Primers for q-PCR
基因
Gene引物
Primers序列 (5′-3′)
Sequence (5′-3′)长度
Length/bp退火温度
Tm/℃dmrt1 dmrt1-F
dmrt1-RGTGATTACGGCTTTGCGGTG
TAGCGGGAAGGCTGACAAAA20
2057
56foxl2 foxl2-F
foxl2-RTGATGCACTGCCCTTACTGG
CACAAGTCTCGGGTAGTGGG20
2057
58α-tubulin α-tubulinF
α-tubulinRAGCCATACAATTCCATCCTGACC
GCGGCAGATGTCGTAGATGG23
2056
58
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表 3 试验鱼生长特性和雌鱼比例
Table 3. Growth and survival of channel catfish and proportion of females in each experimental group
组别
Group存活率
Survival rate/%体长
Body length/cm体重
Body weight/g卵巢形成比例
Ovarian ratio/%性逆转率
Sex reversal rate/%T-30 95.33±0.94 a 9.13±0.75 6.310±1.58 51.00±1.03 a 0 a T-33 91.33±1.25 a 10.14±1.39 9.761±2.80 66.67±1.22 b 31.98±1.34 b T-36 82.67±1.25 b 8.80±0.81 6.113±1.62 77.67±1.35 c 54.43±1.96 c 同列数据后不同小写字母表示差异显著 （P＜0.05）。
Means with different lowercase letters on the same column indicate significant difference (P＜0.05).
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