This study was conducted to evaluate the effects of high salinity combined with stocking density on Litopenaeus vannamei. Three salinity gradients, namely, 28 g/L, 36 g/L, and 44 g/L, and two stocking densities, namely, 300 and 600 shrimp/m³, were used to analyze the synergistic effect of high salinity and stocking density on the growth performance, digestibility, and energy budgets of L. vannamei. The experimental testing period lasted 45 days. The research results showed that a salinity level of 36 g/L was the most suitable salinity level for shrimp growth under both high and low stocking densities. The body weight, specific growth rate, and relative weight gain of the shrimp in the 36 g/L salinity group were significantly higher than those in the other two salinity groups under both high and low stocking densities. The high-density farming group with 600 shrimp/m³ exhibited a significant inhibition of shrimp growth compared to the low-density group under the same salinity conditions. The activities of amylase, lipase, and protease in the high-density-group shrimp gradually decreased with an increase in salinity, and the three digestive enzymes had the same overall effect of changing trends. This indicates that under high-density farming conditions, the increase in salinity is not conducive to the digestive function of shrimps. At the same time, the proportion of respiratory energy to feeding energy gradually decreased in the high-density group and with the increase in salinity. However, under the same salinity conditions, the higher the stocking density, the higher the energy consumed by respiration compared to the low-density group. In addition, the expression of the growth-related gene’s small nuclear ribonucleoprotein polypeptide G (SNRPG) under high stocking density was significantly lower than that in the low-density group at a salinity of 28 g/L, and ribosomal protein L7 (RPL7) expression was also significantly lower under high stocking density than that in the low-density group at a salinity of 44 g/L. The expression levels of molting-related genes retinoid X receptor (RXR), ecdysone receptor (ECR), and ecdysone-induced protein 75 (E75) were significantly higher in the 36 g/L salinity group compared with the other two salinity groups under high-stocking-density treatment. The findings indicate that the synergistic effects of salinity and stocking density have a significant impact on the growth of L. vannamei, and excessive salinity would inhibit its growth in the process of high-density culturing.

The insulin receptor (IR) plays a crucial role in the growth and metabolism of animals. However, there are still many questions regarding the IR in crustaceans, particularly their role in shrimp growth and glucose metabolism. In this study, we identified a novel insulin-like receptor gene in Litopenaeus vannamei and cloned its full length of 6439 bp. This gene exhibited a highly conserved sequence and structural characteristics. Phylogenetic analysis confirmed it as an unreported RTK2-type IR, namely, LvRTK2. Expression pattern analysis showed that LvRTK2 is primarily expressed in female reproductive and digestive organs. Through a series of in vivo and in vitro experiments, including glucose treatment, exogenous insulin treatment, and starvation treatment, LvRTK2 was confirmed to be involved in the endogenous glucose metabolic pathway of shrimp under different glucose variations. Moreover, long-term and short-term interference experiments with LvRTK2 revealed that the interference significantly reduced the shrimp growth rate and serum glucose clearance rate. Further studies indicated that LvRTK2 may regulate shrimp growth by modulating the downstream PI3K/AKT signaling pathway and a series of glucose metabolism events, such as glycolysis, gluconeogenesis, glycogen synthesis, and glycogenolysis. This report on the characteristics and functions of LvRTK2 confirms the important role of RTK2-type IRs in regulating shrimp growth and glucose metabolism.

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.

The Pacific white shrimp (Litopenaeus vannamei), a euryhaline crustacean of significant economic importance, is widely cultivated for its adaptability to diverse salinity levels. Aquaporins (AQPs) are membrane channel proteins that mediate the transport of water and small solutes across biological membranes. Among them, aquaporin 11 (AQP11) is classified as a superaquaporin, and its physiological roles remain unclear. In this study, RNA interference (RNAi) was employed to silence AQP11 expression in L. vannamei, followed by RNA-seq analysis to investigate transcriptomic responses. Differentially expressed genes (DEGs) were identified by comparing dsAQP11 and control groups. The LvAQP11 knockdown significantly increased mortality to 76.7% under acute high-salinity stress (50‰) but not under low-salinity conditions (10‰). Transcriptomic analysis revealed that LvAQP11 deficiency disrupted amino acid metabolism pathways and triggered endoplasmic reticulum stress, as evidenced by the upregulation of proteasome subunits and unfolded protein response genes. Furthermore, silencing LvAQP11 delayed molting progression in the premolt stages, accompanied by the significant upregulation of molt-inhibiting hormone (LvMIH1/5) and downregulation of retinoic acid X receptor (LvRXR). The long-term silencing of LvAQP11 did not affect the weight gain rate (WGR) or the specific growth rate (SGR) but induced muscle fiber disorganization and significantly increased muscle water content. RNA sequencing identified enriched carbohydrate and chitin metabolism pathways, indicating disrupted cytoskeletal dynamics and extracellular matrix integrity. Through this study, we elucidate the crucial roles of LvAQP11 in osmoregulation, molting, and muscle integrity in L. vannamei, providing novel insights into the multifunctional nature of superaquaporins beyond water transport.

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.