Insights into Eyestalk Ablation Mechanism to Induce Ovarian Maturation in the Black Tiger Shrimp

Eyestalk ablation is commonly practiced in crustacean to induce ovarian maturation in captivity. The molecular mechanism of the ablation has not been well understood, preventing a search for alternative measures to induce ovarian maturation in aquaculture. This is the first study to employ cDNA microarray to examine effects of eyestalk ablation at the transcriptomic level and pathway mapping analysis to identify potentially affected biological pathways in the black tiger shrimp (Penaeus monodon). Microarray analysis comparing between gene expression levels of ovaries from eyestalk-intact and eyestalk-ablated brooders revealed 682 differentially expressed transcripts. Based on Hierarchical clustering of gene expression patterns, Gene Ontology annotation, and relevant functions of these differentially expressed genes, several gene groups were further examined by pathway mapping analysis. Reverse-transcriptase quantitative PCR analysis for some representative transcripts confirmed microarray data. Known reproductive genes involved in vitellogenesis were dramatically increased during the ablation. Besides these transcripts expected to be induced by the ablation, transcripts whose functions involved in electron transfer mechanism, immune responses and calcium signal transduction were significantly altered following the ablation. Pathway mapping analysis revealed that the activation of gonadotropin-releasing hormone signaling, calcium signaling, and progesterone-mediated oocyte maturation pathways were putatively crucial to ovarian maturation induced by the ablation. These findings shed light on several possible molecular mechanisms of the eyestalk ablation effect and allow more focused investigation for an ultimate goal of finding alternative methods to replace the undesirable practice of the eyestalk ablation in the future

Relative expression levels in term of fold change (ΔΔCt) of a known ovary-relevant marker, <i>Vitellogenin</i> (<i>Vg</i>), to those of the housekeeping genes in three ovary sample groups:

<p><b>(A) Wild broodstock (WB) from four different ovarian maturation stages compared to those of the housekeeping genes in WB stage 1, (B) Domesticated shrimp at 18-month-, 14-month-, and 10-month-old compared to those of the housekeeping genes in domesticated shrimp at 4-month-old, (C) Domesticated broodstock after the ablation for 1 (D1), 4 (D4), and 7 (D7) days compared to those of the housekeeping genes in before the ablation (D0).</b> Different letters above the bars of each graph signify statistical differences in gene expression levels within the sample group.</p

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

<div><p>Gene expression of reproductive system of the black tiger shrimp (<em>Peneaus monodon</em>) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for <em>P. monodon</em> reproductive organs is lacking. In this study, the stability of four potential reference genes (<em>18s rRNA</em>, <em>GAPDH</em>, <em>β-actin</em>, and <em>EF1-α</em>) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, <em>EF1-α</em> and <em>GAPDH</em> ranked first and second as the most stable genes in testis groups whereas <em>GAPDH</em> and <em>EF1-α</em> were for ovaries from wild-caught broodstock and domesticated groups. <em>EF1-α</em> and <em>β-actin</em> ranked first and second for the eyestalk ablated ovaries. For geNorm, <em>EF1-α</em> and <em>GAPDH</em> had the best stability in all testis and ovaries from domesticated groups whereas <em>EF1-α</em> and <em>β-actin</em> were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, <em>Dmc1</em> and <em>Vitellogenin</em>, were used to validate these reference genes. When normalized to <em>EF1-α,</em> the expected expression patterns were obtained in all cases. Therefore, this work suggests that <em>EF1-α</em> is more versatile as reference genes in qPCR analysis for reproductive system in <em>P. monodon</em>.</p> </div

Testis and ovary samples from <i>P. monodon</i> used in this study.

*<p>GSI is gonadosomatic index calculate as a percentage of testis weight by total body weight.</p

Primer pairs for quantitative real-time PCR (qPCR).

<p>Primer pairs for quantitative real-time PCR (qPCR).</p

Stability values and ranking order (in parentheses) of the candidate reference genes measured by the NormFinder software.

<p>The genes with the highest stability values were hightlighted in each case. TT = testis samples, OV-WB = ovary samples from different ovarian maturation stages, OV-DS = ovary samples in different growth stages, and OV-EA = ovary samples before and after eyestalk ablation.</p

Average expression stability values (M), which is the mean pair-wise variation between an individual gene and all other tested genes, determined by geNorm software.

<p>(A) Average M value of 46 testis samples in <i>P. monodon</i> (TT), (B) Average M value of ovary samples from wild broodstock with different ovarian maturation stages (OV-WB), (C) Average M value of ovary samples from domesticated shrimp with different growth stages (OV-DS) and (D) Average M value of ovary samples from domesticated broodstock before and after eyestalk-ablation (OV-EA) in <i>P. monodon</i>.</p

Threshold cycle values (Ct) of four housekeeping genes (<i>18s rRNA</i>, <i>GAPDH</i>, <i>β-actin</i>, and <i>EF1-α</i>) determined by qPCR from (A) testis samples of wild broodstock from different locations (Andaman sea and Gulf of Thailand) and domesticated shrimp with different growth stages (18-, 14-, 10-, and 4-month-old domesticated shrimp, DS), (B) ovary samples from wild broodstock with different ovarian maturation stages (Stages I–IV), (C) ovary samples of domesticated shrimp from different growth stages (18-, 14-, 10-, and 4-month-old domesticated shrimp, DS), and (D) ovary samples from 14-month-old domesticated broodstock before and after eyestalk ablation for 1, 4 and 7 days.

<p>Different letters above the bars signify statistical differences.</p