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

Distribution of number of genes assigned to predicted pathways from the KEGG database.

<p>A total of 117 pathways were retrieved from the mapping analysis of the 682 differentially expressed transcripts.</p

Putative pathways affected by the eyestalk ablation.

<p>The gonadotropin-releasing hormone (GnRH) signaling pathway, calcium signaling pathway, and progesterone-mediated oocyte maturation were identified as putative affected pathways containing early induced genes after the eyestalk ablation. The lighter red boxes represent the genes whose expression levels were induced more than 2 fold, whereas the dark red boxes represent the genes whose expression levels were induced less than 2 fold at Day 1 after the ablation. Abbreviation: GnRHR: gonadotropin-releasing hormone receptor; G q/11: guanine nucleotide-binding protein G(q) subunit alpha; PLC beta: phospholipase C beta; InsP3: inositol-1,4,5-triphosphate; IP3R: inositol-1,4,5-triphosphate receptor; CALR: calreticulin; ER: endoplasmic reticulum; Ca2+: Calcium ion; CaM: calmodulin; CaMK: calmodulin kinase; CaN: calcineurinB; PMCA: Ca2+ transporting ATPase; GPCR?: G protein-coupled receptor; G: guanine nucleotide-binding protein G(i) subunit alpha; AC: adenylate cyclase; PKA: cAMP-dependent protein kinase; Cdc25: cell division cycle25; Cdc2: cell division cycle2; CycB: cyclinB; MAPK: mitogen-activated protein kinase; Rsk1/2: p90 ribosomal S6 kinase; Myt1: membrane-associated tryrosine and threonine-specific cdc2 inhibitor kinase.</p

Diagram of the eyestalk ablation experiment and physiology of shrimp and ovary.

<p>(A) Eyestalk ablation experiment was conducted over 7 days. Ovary samples were collected before eyestalk ablation (D0) and after eyestalk ablation at Days 1, 4 and 7 (D1, D4, and D7, respectively). (B) Examples of representative shrimp (upper panel) and their ovaries (lower panel) from each time point.</p

The pathways with the highest numbers of participated genes.

<p>*only genes with differential expression level.</p

Summary of microarray experiment.

<p>*Gonadosomatic Index (GSI) = % gonad weight/body weight.</p

List of genes participated in the putative pathways analyzed by KASS.

<p>List of genes participated in the putative pathways analyzed by KASS.</p

Summary of shrimp samples used in this study.

<p>*Gonadosomatic Index (GSI) is calculated as a percentage of ovary weight by total body weight.</p><p>Different superscript letters indicate significant differences between groups (<i>p-</i>value<0.05, <i>Tukey</i> test).</p

Primers used in Reverse-transcriptase Quantitative PCR (RT-qPCR) analysis.

<p>Primers used in Reverse-transcriptase Quantitative PCR (RT-qPCR) analysis.</p