Relative expression levels in term of fold change (ΔΔCt) of a known testis-relevant marker, <i>Dmc1</i>, to those of housekeeping genes in 5 testis sample groups; wild broodstock from Andaman sea (black), wild broodstock from Gulf of Thailand (diagonal lines), 18-month-old domesticated shrimp (DS) (gray), 14-month-old DS (horizontal lines), and 10-month-old DS (diamond), were compared with four housekeeping genes (<i>18s rRNA</i>, <i>GAPDH</i>, <i>β-actin</i>, and <i>EF1-α</i>) in 4-month-old domesticated shrimp.

<p>Different letters above the bars of each graph signify statistical differences in gene expression levels within the sample group.</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

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

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

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

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

The proposed scheme to induce ovarian maturation by eyestalk ablation in <i>P. monodon</i>.

<p>Eyestalk ablation releases gonad- or vitellogenesis- inhibiting hormones (GIH or VIH) allowing a synthesis of yolk protein. GnRH activates the calcium signaling pathway resulting in increased levels of intracellular calcium ion through the GnRH signaling and calcium signaling pathways, which subsequently activates folliculogenesis, estradiol and progesterone production in ovary. Progesterone may then induce progesterone-mediated oocyte maturation process from oocyte stage 1 to 4; Oc1–4. “XX” indicates that this type of cell is not known in shrimp but it is pituitary gonadotrope cell in other organisms. Abbreviation: GnRH: gonadotropin-releasing hormone receptor; GIH: gonad inhibiting hormones; VIH: vitellogenesis inhibiting hormones; GV: germinal vesicle; GVBD: germinal vesicle breakdown.</p

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