Euryhaline crustaceans cope with external salinity changes by mechanisms of osmoregulation. In the current study, we first cloned and confirmed the ORF sequences of the ion-transportation-related genes Na⁺/K⁺-ATPase α subunit (NKAα), cytoplasmic carbonic anhydrase (CAc), and V-type H⁺-ATPase G subunit (VHA-G), and water channels of aquaporins (AQP3, AQP4, and AQP11) from kuruma shrimp (Marsupenaeus japonicus). Further tissue expression patterns showed a higher expression of MjAQP4, MjCAc, MjNKAα, and MjVHA-G in the gills, as well as a higher expression of MjAQP3 and MjAQP11 in the intestine and muscle, respectively. Then, qPCR analysis was used to assess the mRNA expression levels of those osmoregulatory genes in both post-larvae and adult shrimp when they were exposed to acute salinity stress or salinity acclimation. The results revealed significantly decreased expression levels of MjAQP3, MjAQP11, MjNKAα, and MjCAc, and higher expression levels of MjAQP4 and MjVHA-G when the post-larvae shrimp were directly subjected to 10‰ or 50‰ salinity. Moreover, similar expression patterns were also observed in the post-larvae shrimp during the accommodation to 10‰ or 50‰ salinity. As to the adult shrimp, significantly higher expression levels of those genes were observed in the gills after exposure to 10‰ salinity, whereas only the expression levels of MjAQP3, MjAQP11, and MjNKAα were up-regulated in the gills at 40‰ salinity. In contrast, the expression of MjVHA-G was significantly decreased at 40‰ salinity. Finally, during the acclimation to 10‰ salinity, the expression levels of MjAQP3, MjAQP11, and MjNKAα were also significantly elevated, while the expression of MjCAc was significantly decreased in the gills. In addition, the expression levels of MjAQP3, MjAQP4, MjCAc, and MjVHA-G were significantly decreased in the gills during the acclimation to 55‰ salinity. The findings of the study suggest that the examined genes are critical for the adaptation of aquatic crustaceans to changing environmental salinity. Our study lays as the foundation for further research on osmoregulation mechanisms in M. japonicus.

Neuropeptides are pivotal in regulating a broad spectrum of developmental, physiological, and behavioral processes throughout the life cycle of crustaceans. In this comprehensive study, we utilized a multiomics approach to characterize neuropeptide precursors and to assess the expression profiles of neuropeptide-encoding genes across various tissues and developmental stages in the Pacific white shrimp, Litopenaeus vannamei. Additionally, we explored the differential expression of neuropeptide genes in the eyestalk before and after the RNA interference-mediated suppression of crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH) gene expression. Our study identified a total of 125 neuropeptide-encoding genes in L. vannamei, with 54 of these genes previously uncharacterized in the genome. Notably, certain neuropeptide-encoding gene families showed significant expansion, as demonstrated by the discovery of 10 adipokinetic hormone/corazonin-like peptide (ACP) genes, 55 CHH superfamily genes, and 13 pigment-dispersing hormone (PDH) genes. Alternative splicing was also found to play a crucial role in generating functionally diverse neuropeptides; for example, the agatoxin and calcitonin genes undergo alternative splicing that leads to the production of three distinct agatoxin neuropeptides and two distinct calcitonin neuropeptides, respectively. Neuropeptide genes are predominantly expressed in neuroendocrine tissues, including the eyestalk, cerebral ganglia, thoracic ganglia, and ventral ganglia. During the embryonic development of L. vannamei, with the exception of the molt-inhibiting hormone (MIH) gene, all monitored genes display minimal expression from the zygote stage through to the larval in membrane (Lim) stage. In contrast, the majority of these genes exhibit a steady uptick in expression from the nauplius stage onwards, culminating in the post-larval stage. Furthermore, comparative transcriptomic analysis of the eyestalk revealed that the expression of the majority of neuropeptide genes was downregulated following the suppression of CHH and VIH gene expression. This downregulation was significantly associated with the enrichment of pathways related to amino acid metabolism and hormone synthesis. The findings of this study provide valuable insights for future research aimed at elucidating the role of neuropeptides in regulating physiological functions in L. vannamei, potentially leading to advancements in shrimp aquaculture practices.