Oocyte maturation and ovarian development are sequentially coordinated events critical to reproduction. In the ovaries of adult oviparous animals such as birds, bony fish, insects, and crustaceans, vitellogenin receptor (VgR) is a plasma membrane receptor that specifically mediates vitellogenin (Vg) transport into oocytes. Accumulation of Vg drives sexual maturation of the female crustaceans by acting as a pivotal regulator of nutritional accumulation within oocytes, a process known as vitellogenesis. However, the mechanisms by which VgR mediates vitellogenesis are still not fully understood. In this study, we first identified a unique VgR (Lv-VgR) and characterized its genomic organization and protein structural domains in Litopenaeus vannamei, a predominant cultured shrimp species worldwide. This newly identified Lv-VgR phylogenetically forms a group with VgRs from other crustacean species within the arthropod cluster. Duplicated LBD/EGFD regions are found exclusively among arthropod VgRs but not in paralogs from vertebrates and nematodes. In terms of expression patterns, Lv-VgR transcripts are specifically expressed in ovaries of female shrimps, which increases progressively during ovarian development, and rapidly declines toward embryonic development. The cellular and subcellular locations were For analyzed by in situ hybridization and immunofluorescence, respectively. The Lv-VgR mRNA was found to be expressed in the oocytes of ovaries, and Lv-VgR protein was found to localize in the cell membrane of maturing oocytes while accumulation of the ligand Vg protein assumed an even cytoplasmic distribution. Silencing of VgR transcript expression by RNAi was effective for stunting ovarian development. This present study has thus provided new insights into the regulatory roles of VgR in crustacean ovarian development.

Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the P_BAD or P_TAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli β2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3–12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species.

In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.

The Pacific white shrimp (Litopenaeus vannamei) is an important cultured crustacean species worldwide. However, little is known about the molecular mechanism of this species involved in the response to cold stress. In this study, four separate RNA-Seq libraries of L. vannamei were generated from 13°C stress and control temperature. Total 29,662 of Unigenes and overall of 19,619 annotated genes were obtained. Three comparisons were carried out among the four libraries, in which 72 of the top 20% of differentially-expressed genes were obtained, 15 GO and 5 KEGG temperature-sensitive pathways were fished out. Catalytic activity (GO: 0003824) and Metabolic pathways (ko01100) were the most annotated GO and KEGG pathways in response to cold stress, respectively. In addition, Calcium, MAPK cascade, Transcription factor and Serine/threonine-protein kinase signal pathway were picked out and clustered. Serine/threonine-protein kinase signal pathway might play more important roles in cold adaptation, while other three signal pathway were not widely transcribed. Our results had summarized the differentially-expressed genes and suggested the major important signaling pathways and related genes. These findings provide the first profile insight into the molecular basis of L. vannamei response to cold stress.