EXPRESSION LEVELS OF VITELLOGENESIS-INHIBITING HORMONE IN THE WHITELEG SHRIMP Litopenaeus vannamei IN RELATION TO MOLTING

Vitellogenesis-inhibiting hormone (VIH) synthesized by and secreted from the X-organ/sinus gland complex in the eyestalk, is known to negatively regulate the process of vitellogenesis, which is an essential component of female reproduction in decapod crustaceans. However, information on how VIH fluctuates in accordance with the maturation and molt cycles has been lacking. This study was conducted to determine expression levels of the VIH gene in eyestalks in relation to molting in the whiteleg shrimp, Litopenaeus vannamei, one of the most significant species in commercial aquaculture world-wide.

VIH expression levels were determined in eyestalks using quantitative real-time RT-PCR. Total RNA was extracted using an RNeasy Mini Kit and then purified using an RNeasy MinElute Cleanup Kit (Qiagen) to remove pigments. Quantitative real-time RT-PCR was performed as a two-step procedure using a High Capacity RNA-to-cDNA kit and TaqMan® Fast Universal PCR Master Mix (2x, No AmpErase UNG), based on real-time monitoring of the fluorescence signal generated during PCR on a 7500 Fast Real-Time PCR system (Applied Biosystems). Expression levels were quantified in relative terms using standard VIH mRNA normalized to beta actin mRNA. In order to plot standard curves, an eyestalk cDNA library and testis cDNA library were serially diluted, and respectively used for VIH and beta actin expression. The standard curves were linear to six orders of magnitude (assigned arbitrary values of 3.7 x 10⁷ - 1.2 x 10⁴ for VIH, and 1.0 x 10⁷ - 3.2 x 10³ for beta actin).  

In this study, we therefore established a quantitative real-time RT-PCR system for VIH mRNA in L. vannamei. The calculated regression coefficient of the standard curves was always greater than 0.98. Using this system, VIH expression levels according to molt stage were preliminarily assessed in adults (body weight, 38.2 ± 7.0 g, mean ± SD) and subadults (body weight, 15.6 ± 3.2 g, mean ± SD). In both adults and subadults, VIH mRNA expression fluctuated in relation to molting and showed a peak at molt stage D0, but there was no significant difference.