Transcriptional silencing of vitellogenesis-inhibiting and molt-inhibiting hormones in the giant freshwater prawn, Macrobrachium rosenbergii, and evaluation of the associated effects on ovarian development

Abstract

Abstract In crustaceans, vitellogenesis-inhibiting hormone (VIH) plays a major regulatory role in vitellogenesis and the reproductive cycle, and molt-inhibiting hormone (MIH) controls both the molt cycle and the conditional pre-mating molt of mature Macrobrachium rosenbergii females occurring before every egg spawning event. In the current study, we investigated the effect of silencing the VIH gene alone or in combination with silencing the MIH gene on vitellogenesis and ovarian maturation in immature and mature M. rosenbergii females with the aim of determining the associated effects on ovarian development. The first step involved the construction of a dsRNA based on sequence alignment. Importantly, the sequence alignment and phylogenetic relationship between M. rosenbergii (Mr) VIH and Mr-MIH revealed that nucleotides 251–508 of VIH are almost identical to a sequence in Mr-MIH. However, nucleotides 9–250 – including the untranslated region (UTR; nucleotides 9–172) and the first 78 nucleotides of the open reading frame (ORF) - of VIH are completely different from the comparable MIH sequence. As a strategy to induce vitellogenesis and hence ovarian maturation, we used a single dsRNA retrieved from the above-mentioned conserved ORF (dsORF) sequence to silence VIH and MIH simultaneously. Another dsRNA retrieved from the distinct (dsDIS) sequence was used to silence VIH alone. Our results indicated that both dsRNAs are capable of inducing VIH silencing, as shown by a decline in mRNA levels. The results also indicated that VIH silencing is more effective in mature adults than in immature animals, leading to ovary maturation and vitellogenesis. Mature females injected with dsORF showed a decline in MIH levels. In addition, dsDIS also affected the expression of MIH, but the effect lagged behind that of dsORF by one week. We therefore concluded that dsDIS might be as effective as dsORF in silencing VIH, but without a marked influence on MIH and its mutual effects on reproduction and growth. The study thus lays down the foundation for the development of a unique novel tool for molecular manipulation of ovarian maturation and induction of the reproductive cycle in mature M. rosenbergii females. The suggested strategy might prove valuable for sustainable culture protocols not only in M. rosenbergii but also in other crustacean species. Further investigation is, however, needed to elucidate the regulation of the expression of VIH and MIH and how the neuroendocrine system controls growth molt, reproductive molt and reproduction in crustaceans.