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Apoptosis is programmed cell death that is strictly regulated by a series of related genes and is of great importance in resisting pathogen invasion and maintaining cell environment homeostasis. Among apoptotic proteins, the voltage-dependent anion channel protein (VDAC) plays a key role in the mitochondrial apoptosis pathway because of its close connection with changes in mitochondrial membrane potential. However, the role of VDAC in apoptosis and immune regulation in Procambarus clarkii is poorly understood. In this study, the VDAC gene in P. clarkii (PcVDAC) was cloned by rapid amplification of cDNA ends (RACE) technology. The gene was found to have a total length of 2277 bp, including a 194-bp 5'-UTR, 1234-bp 3'-UTR and 849-bp open reading frame (ORF), and to encode 282 amino acids. PcVDAC was expressed in all tissues tested, and its expression was upregulated after white spot syndrome virus (WSSV) infection (P 0.05). The RNA interference (RNAi) method was used to explore the role of PcVDAC in WSSV infection. The results showed that the number of WSSV copies in haemocytes was significantly reduced after RNAi (P 0.05), and the survival rate was significantly increased. In addition, after RNAi, the apoptosis rate was significantly increased (P 0.05), the mitochondrial membrane potential was reduced (P 0.01), and the expression of caspase-3 and other genes was upregulated (P 0.05). These results indicate that PcVDAC promotes the replication of WSSV in P. clarkii by inhibiting haemocytes apoptosis. Therefore, the results presented in this paper provide new insights into the immune response of P. clarkii infected with WSSV.

Trichlorphon, a common organophosphorus pesticide (OPs), is widely used in aquaculture to prevent aquatic insects from infecting cultured objects as well as to control the excessive proliferation of plankton in water bodies. However, its repeated use time can contaminate water bodies and impart direct/indirect toxicity to beneficial aquatic species. However, the underlying mechanism regarding toxicity and cellular metabolism remains unclear. Understanding the mechanism would enable the standardized use and management of OPs and their use in the aquatic environment. Here, low concentration of trichlorphon (5 × 10−5 g/L) was used to construct a hepatopancreatic transcriptional library 30 d, 60 d and 90 d after exposure using RNA-Seq. We detected 649, 148, and 2949 DEGs in the hepatopancreas of E. sinensis for the Tri01 vs. Ctr01, Tri02 vs. Ctr02 and Tri03 vs. Ctr03 library, respectively. The results of KEGG pathway enrichment analysis showed that DEGs were mainly enriched in signal transduction, carbohydrate metabolism, transport and catabolism, endocrine system, and digestive system. Also, under trichlorfon stress, DEGs of E. sinensis were enriched in thyroid hormone signaling pathways, protein digestion and absorption, cancer pathways, etc. The significant DEGs were mainly related to metabolism and the apoptosis and autophagy pathways. This study lays a foundation for further revealing the effects of long-term trichlorfon stress on E. sinensis as well as the potential physiological toxicity. The relevant transcriptome data could provide a reference for the molecular toxicological evaluation of trichlorfon in aquaculture.

The oriental river prawn, Macrobrachium nipponense (De Haan, 1849), an economically important species, can live in freshwater and brackish water environments, but molecular mechanism of adaptation to varying salinity levels is still poorly understood. We aimed to investigate the effects of four different salinities (0, 6, 12, and 18‰) on metabolic responses, histological structure, and expression patterns of acid phosphatase (ACP) and alkaline phosphatase (AKP) in the hepatopancreas. The full-length cDNAs of ACP and AKP of M. nipponense (designated MnACP and MnAKP, respectively) were obtained by rapid-amplification of cDNA ends (RACE)-PCR. Immunofluorescence experiments confirmed that MnAKP is located in the cell membrane of the hepatopancreas. Both genes were expressed in all seven studied tissues, with highest expression in the hepatopancreas by quantitative real-time PCR. The mRNA expression levels and enzyme activities of MnACP and MnAKP in the hepatopancreas significantly increased in 18‰ salinity group compared to other groups. Measurements of glycogen, triglycerides, and total cholesterol levels suggested that prawns can adjust the energy metabolism in a way to provide additional energy under unsuitable salinity. Besides, prawns at high salinity produced higher B-cells and lower number of R-cells in hepatopancreatic tubules, also suggesting enhance the metabolic function of the hepatopancreas under high salinity. Our results provide a better understanding for the mechanism regulating salinity acclimation in the oriental river prawn.

The peak period of morbidity and death in cultured Procambarus clarkii is around May each year and is called the "Black May" disease. The pathogen causing "Black May" disease is believed to be a white spot syndrome virus (WSSV). In 2018, a significant number of P. clarkii died in the pond culture of Xinglong Township, Xuyi County. Two sampling tests on the affected pond showed that, in addition to WSSV, a novel Dicistro-like virus (PcDV) was present. Genomic sequence analysis indicated that this new virus belongs to the Dicistroviridae family, Picornaviridaes order. A high number of spherical particles were detected in gill tissues of P. clarkii with "Black May" disease by electron microscopy, a finding consistent with viruses from the Picornaviridaes order. From October 2018 to September 2019, we took monthly samples from Hubei, Jiangsu and Anhui provinces, and tested for the presence of PcDV and WSSV in P. clarkii. The detection rates of PcDV in P. clarkii peaked from April to June, consistent with the onset of the "Black May" disease. In conclusion, we believe that the discovery of PcDV will provide new research directions for investigating the pathogens causing "Black May" disease in P. clarkii.

Neuropeptides and their G protein-coupled receptors (GPCRs) from the central nervous system regulate the physiological responses of crustaceans. However, in crustaceans, our knowledge regarding GPCR expression patterns and phylogeny is limited. Thus, the present study aimed to analyze the eyestalk transcriptome of the oriental river prawn Macrobrachium nipponense in response to salinity acclimation. We obtained 162,250 unigenes after de novo assembly, and 1,392 and 1,409 differentially expressed genes were identified in the eyestalk of prawns in response to low and high salinity, respectively. We used combinatorial bioinformatic analyses to identify M. nipponense genes encoding GPCRs and neuropeptides. The mRNA levels of seven neuropeptides and one GPCR were validated in prawns in response to salinity acclimation using quantitative real-time reverse transcription polymerase chain reaction. A total of 148 GPCR-encoding transcripts belonging to three classes were identified, including 77 encoding GPCR-A proteins, 52 encoding GPCR-B proteins, and 19 encoding other GPCRs. The results increase our understanding of molecular basis of neural signaling in M. nipponense, which will promote further research into salinity acclimation of this crustacean.

The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further.