Reverse-transcriptase quantitative PCR (RT-qPCR) analysis.

<p>The relative expression levels of the selected transcripts in ovaries before (D0; <i>n</i> = 5) and after eyestalk ablation of <i>P. monodon</i> broodstock at Days 1, 4, and 7 (D1, D4, and D7, respectively; <i>n</i> = 5 each) were measured using RT-qPCR to confirm microarray results and identify other genes potentially involved in ovarian maturation via eyestalk ablation. A total of 12 transcripts were selected to represent three different expression patterns: (A) Late induced, (B) Repressed, and (C) Early induced groups. Different letters above each bar indicate significant difference (<i>P<0.05</i>; Tukey test).</p

Gene expression analysis by cDNA microarray.

<p>Transcript levels in ovaries after eyestalk ablation at Days 1, 4 and 7 (D1, 4 and 7, respectively; Cy5 Red) were compared to those from non-ablated broodstock (D0, pooled from n = 7; Cy3 Green) using cDNA microarray. (A) A total of 2,743 transcripts present in at least 7 of the 8 microarrays with expression differences greater than median values ± SD (∼1.9-fold) in at least one arrays were selected. (B) Hierarchical clustering analysis of the 682 transcripts with expression difference greater than 2-fold change in at least 4 of the 8 microarrays. (C) Clusters I–III exhibit various differentially expressed patterns: Late induced (I), Repressed (II), and Early induced (III) groups. Transcripts in blue letters were further characterized by reverse- transcriptase quantitative PCR (RT-qPCR).</p

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

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

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

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

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

Gene Ontology (GO) annotation.

<p>Pie diagrams of the percentage distribution of sequences among the three principal GO categories were shown for (A) all 5,568 features on UniShrimpChip and (B) the 682 differentially expressed transcripts. Bar graph showed comparison between all features on UniShrimpChip (darker colored bar) and the differentially expressed transcripts (lighter colored bar) in each GO sub-category: (C) biological processes, (D) cellular components and (E) molecular functions.</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