Single-Cell Ribonucleic Acid Sequencing Clarifies Cold Tolerance Mechanisms in the Pacific White Shrimp (Litopenaeus Vannamei)

To characterize the cold tolerance mechanism of the Pacific white shrimp (Litopenaeus vannamei), we performed single-cell RNA sequencing (scRNA-seq) of ∼5185 hepatopancreas cells from cold-tolerant (Lv-T) and common (Lv-C) L. vannamei at preferred and low temperatures (28°C and 10°C, respectively). The cells fell into 10 clusters and 4 cell types: embryonic, resorptive, blister-like, and fibrillar. We identified differentially expressed genes between Lv-T and Lv-C, which were mainly associated with the terms “immune system,” “cytoskeleton,” “antioxidant system,” “digestive enzyme,” and “detoxification,” as well as the pathways “metabolic pathways of oxidative phosphorylation,” “metabolism of xenobiotics by cytochrome P450,” “chemical carcinogenesis,” “drug metabolism-cytochrome P450,” and “fatty acid metabolism.” Reconstruction of fibrillar cell trajectories showed that, under low temperature stress, hepatopancreas cells had two distinct fates, cell fate 1 and cell fate 2. Cell fate 1 was mainly involved in signal transduction and sensory organ development. Cell fate 2 was mainly involved in metabolic processes. This study preliminarily clarifies the molecular mechanisms underlying cold tolerance in L. vannamei, which will be useful for the breeding of shrimp with greater cold tolerance.

Toxicity of The Microcystin-Producing Cyanobacterium Microcystis Aeruginosa To Litopenaeus Vannamei

Microcystis aeruginosa is a common kind of harmful bloom algae, which was also frequently found as a dominant microalgae specie in shrimp breeding ponds. And it was found that blooms always induced massive death of shrimp, but the toxic effects of M. aeruginosa on Litopenaeus vannamei are still not completely understood. In this paper, the toxicity of M. aeruginosa cells to L. vannamei was examined, and the toxic components in the cells were analyzed through high-pressure liquid chromatography (HLPC). In addition, the immune response of shrimp to the microalgal extract was assessed by measuring the activity of immune-related enzymes, as well as the transcription of the relevant genes. Overall, both M. aeruginosa cells and the algal extract resulted in a 100% mortality rate in shrimp, whereas the cell-free culture medium was ineffective. And HPLC analysis results revealed the presence of microcystin-LR (MC-LR) at a concentration of 190.40 mg/kg of cells. In addition, the activity and gene transcription of two immune related enzymes, SOD and LZM, were both significantly reduced in shrimp hepatopancreas (p<0.05) after injection with cell extract. However, reduced glutathione (GSH) content was slightly increased, but the ratio of GSH to GSSG was down, and the transcription of gst gene function as detoxification, was significantly downregulated (p<0.05). The results demonstrated that M. aeruginosa cell extract was highly toxic to L. vannamei, and exerted a negative effect on shrimp immunity including reduction of antioxidant capacity, antibacterial activity and detoxification activity, due to microcystin-LR.

A Glutathione Peroxidase Gene from Litopenaeus vannamei Is Involved in Oxidative Stress Responses and Pathogen Infection Resistance

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.

Bacterial composition and inferring function profiles in the biofloc system rearing Litopenaeus vannamei postlarvae at a low salinity

This study aimed to investigate the bacterial composition and inferring function profiles in the biofloc system rearing Litopenaeus vannamei postlarvae (PL) at a low salinity condition. PL (~ stage 15) were stocked in four parallel tanks filled in water with a salinity of 5.0‰ at a density of 4000 individuals per m3 for a 28-days culture experiment, during which glucose was added as carbon source with a C:N of 20:1. At the end of experiment, water was sampled from each tank and pooled to extract microbial DNA for high-throughput sequencing of V3-V4 region of 16S rRNA gene. Results showed that the bacterial community at 28 d was dominated by phyla of Proteobacteria (45.8%), Bacteroidetes (21.1%), Planctomycetes (13.5%), Chlamydiae (10.3%) and Firmicutes (6.8%). A proportion of 81% inferring KEGG functions of this bacterial community associated with metabolism. Among functions relating to nitrogen metabolism, 48.5% were involved in the conversion of ammonia to glutamate, but the proportion of those involved in transformation among ammonia, nitrite and nitrate was 18.0% in total, inferring higher protein-synthesis but lower inorganic nitrogen-transformation capacities of the bacterial community. At the same time (28 d), high levels of total nitrogen (231.3±6.0 mg L-1) and biofloc (127.0±63.0 mL L-1), but low concentrations of ammonia (0.04±0.01 mg L-1), nitrite (0.2±0.1 mg L-1) and nitrate (12.9±2.5 mg L-1) were observed. The results supply a novel insight for understanding the function of bacterial community in the biofloc system nursing L. vannamei PL at a low salinity.