A Meta-Transcriptomics Survey Reveals Changes in the Microbiota of the Chinese Mitten Crab Eriocheir sinensis Infected with Hepatopancreatic Necrosis Disease

Infection of the freshwater Chinese mitten crab Eriocheir sinensis with hepatopancreatic necrosis disease (HPND) has been a major problem in the crab-cultivated Chinese Province of Jiangsu since 2015. To explore the etiology of HPND, meta-transcriptomic libraries of the hepatopancreata from crabs with and without HPND were constructed. Comparison analyses showed that there were no statistically significant differences in viral and microsporidial communities in the hepatopancreata of diseased and healthy crabs. Bacteroidetes, Proteobacteria, and Firmicutes were the most dominant bacterial phyla in the hepatopancreata of healthy crabs, with a combined prevalence of 93%. However, a decrease in bacterial diversity and a striking shift in the microbial composition were found in the hepatopancreata of crabs infected with HPND. Tenericutes was the most prevalent bacterial phylum in diseased crabs (31.82%), whereas its prevalence was low in healthy crabs (0.02%). By contrast, the prevalence of Bacteroidetes was significantly lower in crabs with HPND (3.49%) than in crabs without HPND (41.04%). We also found that the prevalence of Actinobacteria was higher in crabs with HPND (16.70%) than in crabs without the disease (4.03%). The major bacterial family within the Tenericutes phylum in crabs with HPND was detected by polymerase chain reaction and determined to be Mycoplasmataceae. In conclusion, there were striking changes in the microbiota of diseased and healthy crabs. Specifically, the prevalence of bacteria belonging to Tenericutes and Actinobacteria phyla increased, whereas the prevalence of bacteria belonging to the Bacteroidetes phylum decreased in crabs with HPND, clearly pointing to an association with HPND

Cytochrome c Gene in Procambarus clarkii Inhibits WSSV Infection by Regulating the Apoptosis Pathway

Apoptosis is programmed cell death and is regulated by a series of related genes. It is of great significance in resistance to pathogen invasion and maintaining homeostasis in the environment. The release of cytochrome c (Cytc) from the mitochondria into the cytoplasm is a key step in the initiation of apoptosis. Increasing evidence from investigation of Cytc in cell apoptosis and immunity shows that it can participate in cell apoptosis induced by virus infection. For example, white spot syndrome virus (WSSV) stimulation can induce Cytc gene expression in Litopenaeus vannamei hepatopancreas and hemocytes, and the apoptosis of Epinephelus akaara hepatocytes induced by red-spotted grouper nervous necrosis virus (RGNNV) is related to the release of Cytc. However, the role of Cytc-mediated apoptosis in Procambarus clarkii WSSV infection has not yet been reported. Therefore, in this study, the full length of the the cytochrome c gene of P. clarkii (PcCytc) was cloned, and the role of PcCytc in P. clarkii was analyzed. Its expression in various tissues of P. clarkii proved that WSSV infection can induce the expression of PcCytc. The mechanism of PcCytc involvement in cell apoptosis during WSSV infection was also explored using RNA interference technology, to gain a deeper understanding of the potential role of apoptosis-related factors in the immune response of P. clarkii.In this study, PcCytc was cloned using RACE technology, with a total length of 897 bp, including the 163 bp 5′-UTR, 419 bp 3′-UTR, and 315 bp open reading frame; it encoded 104 amino acids. The structure prediction showed that PcCytc contained a conserved Cytochrom_C domain, proving that it is related to energy production and tends to be conserved in evolution.The results of the quantitative PCR showed that the PcCytc gene was expressed in all tissues of P. clarkii. The expression was lowest in the stomach and higher in the gills, intestines, and muscles, which showed, respectively, 9.46, 8.65 and 7.88 times greater PcCytc expression than that in the stomach. PcCytc showed relatively high expression in tissues with high energy consumption, such as the intestines and muscles, which is consistent with previous studies in Penaeus vannamei. The highest expression level was observed in the gills of the main immune and respiratory tissues of P. clarkii, indicating that PcCytc may be involved in the related biological processes. Based on the above results, we speculate that PcCytc may play different functions in different tissues.WSSV infection experiments showed that the expression level of PcCytc in the tested hepatopancreas, intestines, and muscle tissues increased after virus infection, and reached the highest value at 24 h (P < 0.01), after which it began to decrease until it returned to a normal level at 96 h; the overall performance was an induced expression pattern. This showed that PcCytc is involved in the process of WSSV infection. In addition, considering that PcCytc can participate in ATP production as a key element in the mitochondrial respiratory chain, the low expression of PcCytc leads to energy deficiency. We speculate that once the virus disrupts the energy metabolism of the host cell, the host may compensate for the loss by upregulating the expression of PcCytc.RNAi technology revealed the role of PcCytc in the process of WSSV infection. At 24 and 48 h after WSSV infection, the WSSV copies of the PcCytc RNAi group were significantly increased compared to the uninterrupted group (P < 0.01), and at 72 h were still significantly increased (P < 0.05). These results indicate that PcCytc plays an important role in inhibiting the replication of WSSV in P. clarkii and delays the infection process. To further confirm whether PcCytc mainly inhibits WSSV infection through the apoptotic pathway, we tested the expression changes of some important apoptosis-related genes (bcl-2, bax, and caspase-3). Among them, caspase-3 is an effector protein that regulates cell apoptosis, and its expression directly reflects the result of cell apoptosis. The ratio of bcl-2/bax is considered to be an indicator of the process of cell apoptosis; an increase in the ratio indicates that apoptosis has been affected. Inhibition (a decrease in the ratio) indicates that apoptosis was promoted. The test results were as follows: compared with the PBS group, the expression of bcl-2, bax, and caspase-3 genes of P. clarkii in the WSSV group was up-regulated to varying degrees, with a very significant difference in values (P < 0.01). This shows that WSSV can cause hemolymph apoptosis in P. clarkii, which is consistent with observations in mud crab and shrimp. In addition, the expression of caspase-3 in the dsCytc injection group was significantly downregulated (P < 0.01), indicating that apoptosis was inhibited after interfering with PcCytc. The value of bcl-2/bax in the dsCytc injection group was significantly increased (P < 0.01), which supported this conclusion.In summary, our results indicate that PcCytc can inhibit WSSV infection by regulating the apoptotic pathway. The results of this study provide new insights into the immune response of P. clarkii to WSSV infection

Mulberry Leaf Polysaccharides Attenuate Oxidative Stress Injury in Peripheral Blood Leukocytes by Regulating Endoplasmic Reticulum Stress

The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H2O2)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of Megalobrama amblycephala. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 μM of H2O2 for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 μg/mL) for 3 h, and then stimulated with 100 μM of H2O2 for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs’ antioxidant capacities. The 200 μg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H2O2-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (p p foxO1α), binding immunoglobulin protein (bip), activating transcription factor 6 (atf6), and C/EBP-homologous protein (chop)), Ca2+ transport-related genes (voltage-dependent anion-selective channel 1 (vdac1), mitofusin 2 (mfn2), and mitochondrial Ca2+ uniporter (mcu)), and interleukin 6 (il-6) and bcl2-associated x (bax) were significantly lower in the 200-MLP pre-treatment group than in the HP group (p p > 0.05). These results indicated that MLP pre-treatment attenuated H2O2-induced PBL oxidative damage in the M. amblycephala by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for M. amblycephala, as they protect against oxidative damage

Cloning and Expression Analysis of the Autophagy Related Gene PcAtg2 in Procambarus clarkii Under White Spot Syndrome Virus Stress

Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies

The Application of <i>Moringa oleifera</i> Leaf Meal and Its Fermentation Products in the Diet of <i>Megalobrama amblycephala</i> Juveniles

This study assessed the potential applicability of Moringa oleifera leaf meal (MO) and fermented Moringa oleifera leaf meal (FMO) as feed supplements for aquatic animals. Five experimental diets, including the basal diet (control), 2.2% and 4.4% MO-supplemented diets (MO2 and MO4), and 2.2% and 4.4% FMO-supplemented diets (FMO2 and FMO4), were prepared for feeding Megalobrama amblycephala juveniles. After the eight-week feeding trial, the growth performance, muscle nutritional composition, plasma and hepatic biochemistry indicators were measured. The results demonstrated that MO and FMO had no detrimental effects on the growth performance of M. amblycephala juveniles. The muscle crude protein, crude lipid, and total free amino acids contents were significantly enhanced in the FMO4 group (p p p p p M. amblycephala juveniles, while 2.2% FMO and 4.4% FMO improved the liver anti-inflammatory capacities

Integrative Analysis of Transcriptome and Metabolome Reveals Molecular Responses in <i>Eriocheir sinensis</i> with Hepatopancreatic Necrosis Disease

Hepatopancreatic necrosis disease (HPND) is a highly lethal disease that first emerged in 2015 in Jiangsu Province, China. So far, most researchers believe that this disease is caused by abiotic factors. However, its true pathogenic mechanism remains unknown. In this study, the effects of HPND on the metabolism and other biological indicators of the Chinese mitten crab (Eriocheir sinensis) were evaluated by integrating transcriptomics and metabolomics. Our findings demonstrate that the innate immunity, antioxidant activity, detoxification ability, and nervous system of the diseased crabs were affected. Additionally, metabolic pathways such as lipid metabolism, nucleotide metabolism, and protein metabolism were dysregulated, and energy production was slightly increased. Moreover, the IL-17 signaling pathway was activated and high levels of autophagy and apoptosis occurred in diseased crabs, which may be related to hepatopancreas damage. The abnormal mitochondrial function and possible anaerobic metabolism observed in our study suggested that functional hypoxia may be involved in HPND progression. Furthermore, the activities of carboxylesterase and acetylcholinesterase were significantly inhibited, indicating that the diseased crabs were likely stressed by pesticides such as pyrethroids. Collectively, our findings provide new insights into the molecular mechanisms altered in diseased crabs, as well as the etiology and pathogenic mechanisms of HPND

Genome-Wide Binding Site Analysis of FAR-RED ELONGATED HYPOCOTYL3 Reveals Its Novel Function in Arabidopsis Development

FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1), two transposase-derived transcription factors, are key components in phytochrome A signaling and the circadian clock. Here, we use chromatin immunoprecipitation–based sequencing (ChIP-seq) to identify 1559 and 1009 FHY3 direct target genes in darkness (D) and far-red (FR) light conditions, respectively, in the Arabidopsis thaliana genome. FHY3 preferentially binds to promoters through the FHY3/FAR1 binding motif (CACGCGC). Interestingly, FHY3 also binds to two motifs in the 178-bp Arabidopsis centromeric repeats. Comparison between the ChIP-seq and microarray data indicates that FHY3 quickly regulates the expression of 197 and 86 genes in D and FR, respectively. FHY3 also coregulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE5 and ELONGATED HYPOCOTYL5. Moreover, we uncover a role for FHY3 in controlling chloroplast development by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5, whose product is a structural component of the latter stages of chloroplast division in Arabidopsis. Taken together, our data suggest that FHY3 regulates multiple facets of plant development, thus providing insights into its functions beyond light and circadian pathways

In-Depth Transcriptome Analysis of the Red Swamp Crayfish <i>Procambarus clarkii</i>

<div><p>The red swamp crayfish <i>Procambarus clarkii</i> is a highly adaptable, tolerant, and fecund freshwater crayfish that inhabits a wide range of aquatic environments. It is an important crustacean model organism that is used in many research fields, including animal behavior, environmental stress and toxicity, and studies of viral infection. Despite its widespread use, knowledge of the crayfish genome is very limited and insufficient for meaningful research. This is the use of next-generation sequencing techniques to analyze the crayfish transcriptome. A total of 324.97 million raw reads of 100 base pairs were generated, and a total of 88,463 transcripts were assembled <i>de novo</i> using Trinity software, producing 55,278 non-redundant transcripts. Comparison of digital gene expression between four different tissues revealed differentially expressed genes, in which more overexpressed genes were found in the hepatopancreas than in other tissues, and more underexpressed genes were found in the testis and the ovary than in other tissues. Gene ontology (GO) and KEGG enrichment analysis of differentially expressed genes revealed that metabolite- and immune-related pathway genes were enriched in the hepatopancreas, and DNA replication-related pathway genes were enriched in the ovary and the testis, which is consistent with the important role of the hepatopancreas in metabolism, immunity, and the stress response, and with that of the ovary and the testis in reproduction. It was also found that 14 vitellogenin transcripts were highly expressed specifically in the hepatopancreas, and 6 transcripts were highly expressed specifically in the ovary, but no vitellogenin transcripts were highly expressed in both the hepatopancreas and the ovary. These results provide new insight into the role of vitellogenin in crustaceans. In addition, 243,764 SNP sites and 43,205 microsatellite sequences were identified in the sequencing data. We believe that our results provide an important genome resource for the crayfish.</p></div

Classification of SNPs identified in the crayfish transcriptome.

<p>Classification of SNPs identified in the crayfish transcriptome.</p