凡纳对虾高低繁殖力群体卵巢组织学观察及相关候选基因的表达分析

Ovarian histology and expression of related candidate genes in high- and low- fecundity populations of Penaeus vannamei

中文关键词: 凡纳对虾  繁殖  卵巢  组织学  基因  表达模式

英文关键词: Penaeus vannamei  Reproduction  Ovary  Histology  Genes  Expression pattern

中文摘要:

      在生产中，凡纳对虾(Penaeus vannamei)雌虾的繁殖性能表现出巨大差异。本研究以一个生产周期中雌虾产卵频次为繁殖力指标性状，以高繁殖力和低繁殖力雌虾为研究对象，对雌虾不同卵巢发育阶段(增殖期、小生长期、大生长期和成熟期)进行组织学观察。针对在前期选择清除分析中筛选到的繁殖候选基因TRX2、PARD3、PLCβ4和RERE，在高、低繁殖力雌虾不同卵巢发育阶段，采用实时荧光定量PCR方法，首次分析比较这些基因在卵巢、眼柄组织中的表达规律。组织观察结果显示，低繁殖力雌虾卵巢在整个生产周期中无法发育至成熟期，高繁殖力雌虾卵巢在卵黄颗粒的形成和积累、皮质棒形成等关键过程的速度要快于低繁殖力雌虾。基因表达检测结果显示，在卵巢小生长期和大生长期，TRX2、PLCβ4和RERE基因在高繁殖力组卵巢的表达量均显著高于低繁殖力组卵巢(P<0.05)，4个基因在高繁殖力组成熟期卵巢中均维持较高的表达水平；在不同时期的眼柄组织中，低繁殖力组中TRX2、PARD3、PLCβ4和RERE基因的表达量均高于高繁殖力组。研究表明，上述4种基因可能在凡纳对虾卵巢发育过程中发挥重要作用。以上结果为深入研究凡纳对虾雌虾繁殖力产生差异的分子机制以及分子辅助育种提供了重要参考。

英文摘要:

      The Pacific white shrimp Penaeus vannamei is one of the most productive shrimp species in China and the world. Domestic demand for P. vannamei has increased annually because of its fast growth, easy breeding, strong resistance to stress and disease, and high meat yield. The primary challenge in the development of the shrimp industry is seed rearing. The demand for broodstocks of P. vannamei in China is as high as 1,000,000 pairs per year, and the total weight of shrimp seedlings exceeds 1.3 trillion. China needs to import 300,000 pairs of P. vannamei every year. The cost of each pair of P. vannamei is approximately 200 US$, thus equating to hundreds of millions of US$ in total. During the production of P. vannamei, the spawning frequency varies greatly among female shrimp. Cultivating high-fecundity P. vannamei through genetic improvement is an effective way to reduce the cost of seedling farms and improve economic benefits. To further study the molecular mechanisms of fecundity differences in female P. vannamei, the ovarian histology and expression of four candidate genes in populations with high- and low-fecundity were analyzed in this study. Before the experiment, the 7-month-old parent shrimp of P. vannamei were fed a commercial parent shrimp fortified diet, a maturation promoting diet, and squid for nutritional enhancement. After one month of promoting maturity, the unilateral eyestalk of female shrimp was cut using forceps, and next-generation family construction began after 15–20 days of recovery. The spawning frequency of 439 female shrimp in the 30-day production cycle was calculated and used to indicate fecundity. Those with a spawning frequency of 0 were included in the low-fecundity group, while those with a spawning frequency of >3 were included in the high-fecundity group. The ovarian development of P. vannamei was divided into four stages (proliferation, small growth, large growth, and maturation) according to the color, size, and shape of the ovaries. In the high-fecundity group, the ovaries exhibited development up to stageⅣ, indicating their attainment of maturity. In the low-fecundity group, ovarian development was limited to stageⅢ, with progression to stageⅣ proving difficult. Ovarian tissues from the same part of female shrimp of stageⅠ–Ⅳ in the high-fecundity group and stageⅠ–Ⅲ in the low-fecundity group were taken and immediately immersed in tissue fixative for paraffin sectioning. The research team previously identified candidate genes related to fecundity in genomic regions undergoing selective sweep in isolated populations. These included thioredoxin 2 (TRX2), partitioning-defective 3 (PARD3), phospholipase Cβ4 (PLCβ4), and arginine-glutamate acid dipeptide repeats (RERE), which play important roles in oocyte maturation, cell proliferation, and early development in other species. qRT-PCR was used to analyze and compare their expression in the ovary and eyestalk tissues of high- and low-fecundity females at different stages of ovarian development. The results of paraffin section showed that the ovarian development of the low-fecundity group was slower than that of the high-fecundity group. In the high-fecundity group, stageⅠ ovaries primarily consisted of oogonia and oocytes during the early phase of yolk formation, with the emergence of small spherical yolk granules. StageⅠ ovaries in the low-fecundity group primarily consisted of oogonia, with no observable yolk granule formation. StageⅡ ovaries in the high-fecundity group were mainly composed of oocytes with vitelline formation, accompanied by a notable augmentation in both the size and number of yolk granules. StageⅡ ovaries in the low-fecundity group primarily comprised oocytes in the early phase of vitelline formation with yolk granules beginning to form. StageⅢ ovaries in the high-fecundity group consisted mainly of oocytes during the late phase of yolk formation. Many spherical yolk granules were formed in the cytoplasm, and cortical rods began to appear, although the lengths of the cortical rods were short. StageⅢ ovaries in the low-fecundity group were composed of oocytes during yolk formation. The cytoplasm was full of spherical yolk granules in the absence of cortical rods. In the high-fecundity group, stage Ⅳ ovaries were filled with mature oocytes, large yolk granules filled the entire cytoplasm, and thick cortical rods were formed in the oocytes. qRT-PCR showed that the four genes maintained high expression levels in the mature ovaries of the high-fecundity group. Their expression levels in the ovaries of the high-fecundity group were higher than those in the ovaries of the low-fecundity group in the small and large growth stages. TRX2, PLCβ4, and RERE expression levels were significantly different between the groups (P<0.05). In eyestalk tissues at different stages, TRX2, PARD3, PLCβ4, and RERE expression levels in the low-fecundity group were higher than those in the high-fecundity group. The results suggest that these four genes may play important roles in the ovarian development of P. vannamei. The expression levels of all four genes in the high-fecundity group were lower than those in the low-fecundity group in eyestalk tissues. However, in ovarian tissues, except for the PARD3 gene in stageⅠ, which was expressed at lower levels in the high-fecundity group than in the low-fecundity group, the expression levels of all four genes in the other stages were higher in the high-fecundity group than those in the low-fecundity group, which is opposite to the expression trend in eyestalk tissues. Therefore, we speculate that these genes also promote cell proliferation, development, and target gene expression in the eyestalks of the low-fecundity group. Their high expression in the eyestalks of the low-fecundity group may enhance the secretion of ovarian inhibitory factors, thereby inhibiting ovarian development. Further research is needed to reveal their functional mechanisms. The above results provide an important reference for the in-depth study of the molecular mechanisms of differences in the fecundity of female P. vannamei.