In the present study, double strand RNA technology (dsRNA) was used to inhibit transcripts of vitellogenesis-inhibiting hormone (VIH) that mainly synthesized and secreted from the central nervous system in Scylla olivacea females. The results presented in this study clearly demonstrate the potential dsRNA-VIH was highly effective to inhibit VIH in the eyestalks of females injected with dsRNA-VIH on the 3^rd, 7^th and 14^th day, respectively. The dsRNA-VIH injections were performed at 14-day intervals, a single dsRNA dose of 0.6 µg/gram body weight was enough to suppress VIH expression until 14^th day after injection. The dsRNA-VIH injection significantly increased gonad-somatic index (GSI) and hemolymph vitellin level at day 14 and 28 when compared with control groups. The histological observation found that the number of oocyte step 4 in dsRNA-VIH group was significantly higher than that of the control group. Also, dsRNA-VIH has stimulatory function on other reproduction-related genes such as the Scyol-PGES and Scyol-ESULT that both genes gradually increased their expressions in brain and ventral nerve cord. In conclusion, the silence of VIH gene could reduce the production of VIH from eyestalk and brain that affected other downstream genes related to ovarian maturation in the mud crab.

The mud crab Scylla olivacea is widely cultured for its economic value, but reproduction issues limit its production. Vitellogenesis-inhibiting hormone (VIH), serotonin (5-HT), and gonadotropin-releasing hormone (GnRH) are important neurohormones that control reproduction in crustaceans. Mimicking crab hormone stimulation during reproduction has scarcely been reported. Comparison of the single hormone and multiple hormone approaches to crab hormonal control in S. olivacea is limited. In situ hybridization showed that injection of dsRNA-VIH could abolish its gene expression in neuronal clusters of female S. olivacea eyestalks, potentially reducing its inhibitory effects on ovarian maturation. This was confirmed by assessing the ovarian gonadosomatic index (GSI), hemolymph vitellogenin (Vg), an indicator of vitellogenesis, and gonad histology using dsRNA-VIH and 5-HT/GnRH combinations. Based on our findings, we demonstrated that administration of dsRNA-VIH significantly increased the gonadosomatic index (GSI) on days 14 and 28 post-treatment. The combination cocktail, however, consisting of 5-HT + GnRH + dsRNA-VIH on days 14 and 28, and GnRH + dsRNA-VIH on day 28, was the most efficacious in increasing GSI and enhancing crab ovarian maturation. Upregulation of hemolymph Vg levels was observed solely on the 28th day following treatment with dsRNA-VIH, 5-HT + GnRH + dsRNA-VIH, and GnRH + dsRNA-VIH. Differential gene expression analysis using quantitative RNA-sequencing of the neural tissues (brain and ventral nerve cord), revealed a significant upregulation of certain receptors (5-HTR, GnRHR, LHR, and FSHR), neuropeptides (sNPF, NPF1, NPF2, SIFamide, AKH/Crz, CHH, and RPCH), downstream reproductive-related genes (FAMeT, ESULT, progesterone-like protein), and prostanoid-related genes (phospholipase A and C, COX, Thromboxane A synthase, prostaglandin D, E, and F synthases) following treatment, particularly dsRNA-VIH + GnRH and/or 5-HT-injected individuals. Upregulation of prostaglandin E synthase and estrogen sulfotransferase genes was confirmed by real-time PCR. Since the construction and propagation of dsRNA-VIH is costly, its lower dose application supplemented with synthetic GnRH and/or 5-HT may be an alternative approach to ensure that female S. olivacea attain sufficient reproductive fecundity in aquaculture. Furthermore, we propose that the administration of multiple hormones in crabs may better emulate the physiological conditions of crustaceans in their natural habitat.

The sea cucumber Holothuria scabra is both an economically important species in Asian countries and an emerging experimental model for research studies in regeneration and medicinal bioactives. Growth factors and their receptors are known to be key components that guide tissue repair and renewal, yet validation of their presence in H. scabra has not been established. We performed a targeted in silico search of H. scabra transcriptome data to elucidate conserved growth factor family and receptor genes. In total, 42 transcripts were identified, of which 9 were validated by gene cloning and sequencing. The H. scabra growth factor genes, such as bone morphogenetic protein 2A (BMP 2A), bone morphogenetic protein 5-like (BMP5-like), neurotrophin (NT) and fibroblast growth factor 18 (FGF18), were selected for further analyses, including phylogenetic comparison and spatial gene expression using RT-PCR and in situ hybridization. Expression of all genes investigated were widespread in multiple tissues. However, BMP 2A, BMP5-like and NT were found extensively in the radial nerve cord cells, while FGF18 was highly expressed in connective tissue layer of the body wall. Our identification and expression analysis of the H. scabra growth factor genes provided the molecular information of growth factors in this species which may ultimately complement the research in regenerative medicine.