Insights into carbohydrate metabolism from an insulin-like peptide in Macrobrachium rosenbergii.

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Survival Characteristics and Transcriptomic Analyses Reveal the Adaptive Response of the Aquatic Pathogen Non-O1/O139 Vibrio cholerae to Starvation Stress.

Non-O1/O139 Vibrio cholerae is a pathogen of various aquatic organisms but requires major self-regulation to overcome environmental stress in the aquatic environment. However, its survival strategies under environmental stress are not well understood. The objective of this study was to describe the survival characteristics and changes in expression of stress resistance-related genes of non-O1/O139 V. cholerae after 6 months of starvation at room temperature. The results demonstrated that starved cells were still viable, exhibited shortened rods and shrinking surface, and maintained virulence to Macrobrachium rosenbergii. To investigate the changes in gene expression in non-O1/O139 V. cholerae under starvation stress, especially those involved in stress resistance, transcriptome profiles of starved and wild-type cells were determined. The differentially expressed genes (DEGs) in starved cells were identified, including 191 upregulated genes and 180 downregulated genes. Among these DEGs, the well-known stress resistance-related genes were upregulated significantly, including rpoS, rpoD, rpoN, rpoE, uspA, uspC, cspD, hslJ, etc. Gene Ontology (GO) analysis of the DEGs demonstrated that environmental adaptation-related categories, such as response to stimulus and signal transduction, were upregulated significantly in the starved cells, while cell motility was downregulated significantly. These DEGs were also enriched into 54 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including biofilm formation, two-component system, quorum sensing, flagellar assembly, bacterial chemotaxis stress resistance-related pathways, etc. The potential existence of long-starved non-O1/O139 V. cholerae bacteria in the aquatic environment may raise new concerns about this devastating pathogen in aquaculture. IMPORTANCE Non-O1/O139 V. cholerae is a causal agent of vibriosis that can be subject to nutrient insufficiency and cause high rates of mortality in aquatic animals. However, its molecular mechanisms of survival in response to starvation stress have been investigated only partially. Here, we demonstrate that under starvation stress, non-O1/O139 V. cholerae can survive over the long term and cause disease by dwarfing of the cell structure, upregulation of a series of stress resistance-related genes, and downregulation of flagellum assembly-related genes. This knowledge can help the development of intervention strategies to control non-O1/O139 V. cholerae infection in aquaculture.

Complete Genome Analysis of Highly Pathogenic Non-O1/O139 Vibrio cholerae Isolated From Macrobrachium rosenbergii Reveals Pathogenicity and Antibiotic Resistance-Related Genes.

Non-O1/O139 Vibrio cholerae is a highly virulent pathogen that causes mass mortalities of various aquatic animals. In the present study, we sequenced the whole genome of non-O1/O139 V. cholerae GXFL1-4, isolated from Macrobrachium rosenbergii, to reveal the pathogenicity and antibiotic resistance. The result showed its genome contained two circular chromosomes and one plasmid with a total size of 4,282,243 bp, which harbored 3,869 coding genes. Among them, 3,047, 2,659, and 3,661 genes were annotated in the Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. In addition, 372 potential virulence genes were predicted based on the Virulence Factor Database (VFDB) database, such as type II, III, IV, and VI secretion systems related genes, flagella genes, and pilus formation or motility-related genes. Blast results in the Comprehensive Antibiotic Resistance Database (CARD) database showed that the strain contained 148 antibiotic resistance-related genes belonging to 27 categories, such as efflux pump complex antibiotic resistance genes and antibiotic resistance gene cluster genes. The Pathogen-Host Interaction (PHI) database annotated 320 genes related to pathogen-host interaction, such as T3SS, virulence regulatory factors, transcriptional regulators, and two-component response regulator related genes. The whole-genome analysis suggested that the pathogenic non-O1/O139 V. cholerae strain GXFL1-4 might have a complex molecular mechanism of pathogenicity and antibiotic resistance. This study provides a wealth of information about non-O1/O139 V. cholerae genes related to its pathogenicity and drug resistance and will facilitate the understanding of its pathogenesis as well as the development of prevention and treatment strategies for the pathogen.

Investigation of growth retardation in Macrobrachium rosenbergii based on genetic/epigenetic variation and molt performance.

Giant freshwater prawn, Macrobrachium rosenbergii is an important freshwater aquaculture species worldwide, and China contributes the most to its global production. However, in recent years in China, many prawns have shown serious growth retardation, which is referred to as "iron prawn." To explore the mechanism behind this phenomenon, we compared the difference between these "iron prawns" and normal prawns in three aspects-changes in genetic diversity, DNA methylation, and transcriptomes-as well as comparing differences in their molt performance. The results are as follows: first, compared with normal prawns, "iron prawns" showed no significant decrease in genetic diversity, but they did show obvious genetic differentiation, and different DNA methylation levels were observed. The genetic and epigenetic variations that existed between "iron prawn" and normal prawn indicated the influence of germplasm on growth performance. Second, transcriptome analysis revealed 1813 differentially expressed genes (DEGs) between the "iron prawn" and normal prawn, and the DEGs mainly enriched the glucose metabolism- and immune-related pathways, such as in glycolysis/gluconeogenesis metabolism, insulin secretion, glucagon signaling pathway, antigen processing and presentation, as well as in complement and coagulation cascades. Enrichment analysis indicated the importance of the glucose level and pathogen attacks to growth performance in the "iron prawn." Finally, a comparison of the molt performance showed that the length of the molt cycle in the "iron prawn" was comparable to normal prawns with the same size, but the specific growth was much lower in the "iron prawn." This result suggested that lower body weight gain per molt cycle should be responsible for growth retardation in the "iron prawn," but not in the longer molt cycle. The results in this study provided fundamental information about the mechanism behind growth retardation in M. rosenbergii.