cDNA cloning, expression, and immunolocalization of gonad-inhibiting hormone (GIH) in Litopenaeus vannamei

Abstract

In this study, the full-length GIH cDNA sequence from Litopenaeus vannamei was cloned from the eyestalk by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The fulllength GIH cDNA was 865 bp with a 288 bp open-reading frame, which encoded a 96 amino acid prepro-GIH with 17 amino acid signal peptide. L. vannamei GIH (LvGIH) can be classified as a member of type-II crustacean hyperglycemic hormone polypeptide family. LvGIH shares 93.8 and 66.7 % amino acid sequence identity with GIH from Penaeus monodon, and the molt-inhibiting hormone from Marsupenaeus japonicas, respectively. By quantitative real-time PCR (qPCR), LvGIH mRNA transcripts were detected in fertilized eggs, nauplius, zoea, mysis and juveniles of 25, 35 and 40 days old. LvGIH transcript levels increased significantly with the development from fertilized eggs to juveniles. LvGIH transcript levels were highest in juveniles at 35 days old. By RT-PCR, LvGIH mRNA transcripts were detected only in the eyestalks and brains but not in the muscles, intestines, gills, heart, hepatopancreas, ovaries and testes of adults, and there was no difference in the expression level of LvGIH between males and females. Using the P. monodon anti-GIH antibody, we showed that LvGIH was located mainly in the XO-SG and slightly in axon, with similar fluorescence intensity found in XO and SG. To summarize, we have cloned and characterized the GIH of the shrimp L. vannamei. In addition to the GIH properties described in other crustaceans, a peak of LvGIH expression was identified at the time of sexual differentiation (i.e., day 35 larvae) suggesting that LvGIH may also be involved in the control of this process.

Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades

“…According to phylogenetic analysis, VIH is considered a member of the crustacean hyperglycemic hormone (CHH) family [ 11 , 12 ]. In crustaceans, VIH is predominantly synthesized in the X-organ/sinus gland complex in eyestalks [ 9 , 10 , 13 ], with the exceptions that in some species, the brain is another major source of VIH [ 14 – 16 ]. Therefore, ablation of the eyestalk has been extensively practiced in commercial shrimp culture as a technique to accelerate ovarian maturation, based on the assumption of diminishing VIH production in the eyestalk [ 17 – 19 ].…”

“…After that, our group identified another candidate for the L . vannamei VIH from the shrimp eyestalk, which belonging to the type-II CHH subfamily and possessing vitellogenesis-inhibiting activity [ 15 , 16 ]. Given that the first and second VIH candidates in L .…”

“…As opposed to the type-I VIH (SGP-G), the newly cloned VIH-2 in L . vannamei is predominately generated in both the brain and the eyestalk [ 15 , 16 ]. VIH -2 transcript levels have been found to increase significantly as fertilized eggs develop into juveniles, peak at sexual differentiation [ 16 ], under a negative estrogen regulation [ 30 ], and decrease in the brain after unilateral eyestalk ablation [ 15 ].…”

“…vannamei is predominately generated in both the brain and the eyestalk [ 15 , 16 ]. VIH -2 transcript levels have been found to increase significantly as fertilized eggs develop into juveniles, peak at sexual differentiation [ 16 ], under a negative estrogen regulation [ 30 ], and decrease in the brain after unilateral eyestalk ablation [ 15 ]. The recombinant VIH-2 protein is effective in inhibiting the hepatopancreatic Vg mRNA expression in female shrimp by both in vitro primary cell culture and in vivo injection approaches, and in reversing ovarian growth induced by eyestalk ablation [ 15 ].…”