Interactions between the gut bacterial community of Exopalaemon carinicauda and infection by Enterocytozoon hepatopenaei.

To explore the relationship between the intestinal flora of Exopalaemon Carinicauda and infection by Enterocytozoo Hepatopenaei (EHP), we analyzed the species and richness of gut microbiota in infected individuals in different EHP load groups [i.e., control (C), high load (H), and low load (L)] using gene sequencing after infection. The results showed that the abundance of intestinal flora in the high-load EHP group was significantly lower than that in the healthy group. Based on the UPGMA cluster tree and PCoA analysis, with comparisons to healthy shrimp, the gut microbiota of the EHP high load and low load groups were clustered into one branch, which indicated that EHP infection changed the composition of the gut microbiota of infected shrimps. The heat map analysis of species abundance clustering revealed that the dominant bacteria in the low EHP load group and the control group were beneficial genera such as Lactococcus, Ligilactobacillius, and Bifidobacterium, but the dominant bacteria in the high EHP load group were harmful genera such as Pseudomonas, Photobacterium, and Candidatus hepatincola. The functions of the intestinal flora predicted that most genes related to metabolism were more abundant in healthy shrimp, most genes related to metabolism and the organisms' system were more abundant in the low EHP load group, and most genes related to diseases and environmental information processing were more abundant in the high EHP load group. After separation and purification, the dominant bacteria (Bifidobacterium animalis in healthy shrimp and Lactococcus garvieae in the low EHP load group) and the non-dominant bacteria (Macrococus caseolyticus in the low EHP load group) were obtained. Each of these isolated strains were used together with EHP to infect E. carinicauda, and the results showed that Bifidobacterium animali and Lactococcus garvieae significantly reduced the EHP load in EHP-infected individuals. At the same time, the morphology and structure of the hepatopancreas and intestinal tissue of EHP-infected E. carinicauda were improved. No improvement was seen in tissue that was infected with Macrococus caseolyticus.

Metagenomic dissection of the intestinal microbiome in the giant river prawn Macrobrachium rosenbergii infected with Decapod iridescent virus 1.

Microbiome in the intestines of aquatic invertebrates plays pivotal roles in maintaining intestinal homeostasis, especially when the host is exposed to pathogen invasion. Decapod iridescent virus 1 (DIV1) is a devastating virus seriously affecting the productivity and success of crustacean aquaculture. In this study, a metagenomic analysis was conducted to investigate the genomic sequences, community structure and functional characteristics of the intestinal microbiome in the giant river prawn Macrobrachiumrosenbergii infected with DIV1. The results showed that DIV1 infection could significantly reduce the diversity and richness of intestinal microbiome. Proteobacteria represented the largest taxon at the phylum level, and at the species level, the abundance of Gonapodya prolifera and Solemya velum gill symbiont increased significantly following DIV1 infection. In the infected prawns, four metabolic pathways related to purine metabolism, pyrimidine metabolism, glycerophospholipid metabolism, and pentose phosphate pathway, and five pathways related to nucleotide excision repair, homologous recombination, mismatch repair, base excision repair, and DNA replication were significantly enriched. Moreover, several immune response related pathways, such as shigellosis, bacterial invasion of epithelial cells, Salmonella infection, and Vibrio cholerae infection were repressed, indicating that secondary infection in M. rosenbergii may be inhibited via the suppression of these immune related pathways. DIV1 infection led to the induction of microbial carbohydrate enzymes such as the glycoside hydrolases (GHs), and reduced the abundance and number of antibiotic-resistant ontologies (AROs). A variety of AROs were identified from the microbiota, and mdtF and lrfA appeared as the dominant genes in the detected AROs. In addition, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration were the main antibiotic resistance mechanisms. Collectively, the data would enable a deeper understanding of the molecular response of intestinal microbiota to DIV1, and offer more insights into its roles in prawn resistance to DIVI infection.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

First report of Metschnikowia bicuspidata infection in Chinese grass shrimp (Palaemonetes sinensis) in China.

The Chinese grass shrimp (Palaemonetes sinensis) was found with a white turbidity appearance in the Panjin area. After dissection, typical symptoms of milky disease with hemolymph emulsification and noncoagulation were observed; however, the pathogen was unknown. In this study, we aimed to isolate the pathogen of the diseased P. sinensis. We found that the pathogen could grow on the fungal medium Bengal red, and microscopic examination showed that it reproduced by budding. Molecular identification of the isolated and purified yeast strain LNMB2021 based on 26S rDNA sequence showed that the pathogenic pathogen was Metschnikowia bicuspidata (GenBank OK094821), with 98.74% homology with M. bicuspidata strain LNES0119 (GenBank OK073903) and 98.56% with M. bicuspidata strain Liao (GenBank MT856369). The results of an artificial infection test showed that M. bicuspidata caused the same clinical symptoms in P. sinensis, and the isolated pathogen was still the same, which proved that P. sinensis was a new host of M. bicuspidata. Histopathological analysis showed that there were obvious pathological changes in the hepatopancreas and muscle tissue of the diseased P. sinensis. Identification of the pathogen is essential for the prevention and control of the disease and the healthy culture of P. sinensis. Furthermore, considering the transmissibility and cross-host transmission of M. bicuspidata, its risk of infecting other aquatic animals deserves high attention.

Functional and Stress Response Analysis of Heat Shock Proteins 40 and 90 of Giant River Prawn (Macrobrachium rosenbergii) under Temperature and Pathogenic Bacterial Exposure Stimuli.

The aims of this research were to perform molecular characterization and biofunctional analyses of giant river prawn Hsp40 and Hsp90 genes (Mr-hsp40 and Mr-hsp90) under various stress conditions. Comparisons of the nucleotide and amino acid sequences of Mr-hsp40 and Mr-hsp90 with those of other species showed the highest similarity scores with crustaceans. Under normal conditions, expression analysis using quantitative real-time RT-PCR (qRT-PCR) indicated that Mr-hsp40 was highly expressed in the gills and testis, and Mr-hsp90 expression was observed in all tissues, with the highest expression in the ovary. The expression patterns of Mr-hsp40 and Mr-hsp90 transcripts under Aeromonas hydrophila challenge and heat-cold shock conditions were examined in gills, the hepatopancreas and hemocytes, at 0, 3, 6, 12, 24, 48 and 96 h by qRT-PCR. Under bacterial challenge, Mr-hsp40 displayed variable expression patterns in all tissues examined during the tested periods. In contrast, upregulated expression of Mr-hsp90 was quickly observed from 3 to 12 h in the gills and hepatopancreas, whereas obviously significant upregulation of Mr-hsp90 was observed in hemocytes at 12-96 h. Under temperature shock conditions, upregulation of Mr-hsp40 expression was detected in all tested tissues, while Mr-hsp90 expression was quickly upregulated at 3-48 h in all tissues in response to 35 °C conditions, and conditions of 35 and 25 °C stimulated its expression in gills and the hepatopancreas at 12 and 48 h, respectively. Silencing analyses of these two genes were successfully conducted under normal, high-temperature (35 °C) and A. hydrophila infection conditions. Overall, knockdown of Mr-hsp40 and Mr-hsp90 effectively induced more rapid and higher mortality than in the PBS control and GFP induction groups in temperature and infectious treatments. Evidence from this study clearly demonstrated the significant functional roles of Mr-hsp40 and Mr-hsp90, which are crucially involved in cellular stress responses to both temperature and pathogenic bacterial stimuli.

Aeromonas hydrophila associated with red spot disease in Macrobrachium nipponense and host immune-related gene expression profiles.

In September 2018, a serious disease causing high mortality with red spot syndrome occurred in a Macrobrachium nipponense aquaculture farm in Jintan County, Jiangsu Province, China. In this study, a pathogenic isolate 5-S3 was isolated from diseased M. nipponense and was identified as Aeromonas hydrophila by phenotypically and molecularly. The pathogenicity of the isolate 5-S3 to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. hydrophila was pathogenic to M. nipponense, the LD50 was 9.58 × 104 CFU/mL, and histopathological analysis revealed that the hepatopancreas of infected M. nipponense exhibited obvious inflammatory responses to A. hydrophila infection. The isolate showed significant phenotypical activities such as the lecithinase, esterase, caseinase and hemolysin which are indicative of their virulence potential. Besides, virulence genes such as aerA, act, fla, ahpß, alt, lip, eprCAI, hlyA, acg and gcaT were detected in the isolate 5-S3. Subsequently, the immune-related genes expression in M. nipponense were evaluated by quantitative real-time PCR (qRT-PCR), and the results showed that the expression levels of dorsal, relish, crustin1, crustin2, anti-lipopolysaccharide factors 1 (ALF1), anti-lipopolysaccharide factors 2 (ALF2), hemocyanin, i-lysozyme and prophenoloxidase were significantly up-regulated in hepatopancreas of M. nipponense after A. hydrophila infection, the stat, p38, crustin3, anti-lipopolysaccharide factors 3 (ALF3) genes had no significant change during the infection. The present results reveal that A. hydrophila was an etiological agent causing red spot syndrome and mass mortality of M. nipponense and the influence of A. hydrophila infection on host immune genes.

Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge.

Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein for Toll-like receptor (TLR)-mediated signaling pathways and plays an important role in immune response. In this study, the full-length cDNA of MyD88 from Macrobrachium rosenbergii (MRMyD88) was cloned. The MRMyD88 cDNA is 1758 bp long and contains a 1398-bp open reading frame. Multiple sequence alignment and phylogenetic analysis revealed that the amino acid sequence of MRMyD88 shared high identity with the known MyD88 proteins. The MRMyD88 mRNA was widely expressed in all examined tissues, with highest level in intestine, followed by gonad and pleopod. Furthermore, the MRMyD88 promoter region, spanning 1622 bp, contains several transcription factor-binding sites, including nine GATA-1 box motifs. Electrophoretic mobility shift assay showed that Gfi-1, SRF, and Oct-1 bind to the upstream region of MRMyD88. Additionally, the results showed that the expression levels of TLR1, TLR2 and TLR3 were different in response to Vibrio anguillarum, Lactobacillus plantarum and Aeromonas hydrophila infections. However, these bacteria significantly increased the expression levels of MyD88 and prophenoloxidase. These data suggest that the TLR-mediated signaling pathway is MyD88-dependent in response to pathogenic and probiotic bacteria in M. rosenbergii.

Genome sequencing of cold-adapted Planococcus bacterium isolated from traditional shrimp paste and protease identification.

BACKGROUND: Psychrophiles have evolved to adapt to freezing environments, and cold-adapted enzymes from these organisms can maintain high catalytic activity at low temperature. The use of cold-adapted enzymes has great potential for the revolution of food and molecular biology industries. RESULTS: In this study, four different strains producing protease were isolated from traditional fermented shrimp paste, one of which, named Planococcus maritimus XJ11 by 16S rRNA nucleotide sequence analysis, exhibited the largest protein hydrolysis clear zone surrounding the colonies. Meanwhile, the strain P. maritimus XJ11 was selected for further investigation because of its great adaptation to low temperature, low salinity and alkaline environment. The enzyme activity assay of P. maritimus XJ11 indicated that the optimum conditions for catalytic activity were pH 10.0 and 40 °C. Moreover, the enzyme also showed an increasing activity with temperatures from 10 to 40 °C and retained more than 67% activity of the maximum over a broad range of salinity (50-150 g L-1 ). Genome sequencing analysis revealed that strain XJ11 possessed one circular chromosome of 3 282 604 bp and one circular plasmid of 67 339 bp, with a total number of 3293 open reading frames (ORFs). Besides, 21 genes encoding protease, including three serine proteases, were identified through the NR database. CONCLUSION: Cold-adapted bacterium P. maritimus XJ11 was capable of producing alkaline proteases with high catalytic efficiency at low or moderate temperatures. Furthermore, the favorable psychrophilic and enzymatic characters of strain P. maritimus XJ11 seem to have a promising potential for industrial application. © 2020 Society of Chemical Industry.

Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii.

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

Transcriptome analysis of the Macrobrachium nipponense hepatopancreas provides insights into immunoregulation under Aeromonas veronii infection.

The oriental river prawn Macrobrachium nipponense is a commercially important freshwater shrimp that is widely farmed in China. Aeromonas veronii is a conditional pathogen of farmed shrimp, which has caused huge economic losses to the industry. Therefore, there is urgency to study the host-pathogen interactions between M. nipponense and A. veronii to screen individuals with antimicrobial resistance. In this study, we examined the hepatopancreas of moribund M. nipponense infected with A. veronii and healthy individuals at both the histopathological and transcriptomic levels. We showed that A. veronii infection resulted in tubular necrosis of the M. nipponense hepatopancreas. Such changes likely affect assimilation, storage, and excretion by the hepatopancreas, which could ultimately affect the survival and growth of infected individuals. Among the 61,345 unigenes obtained through RNA sequencing and de novo transcriptome assembly, 232 were differentially expressed between the two groups. KEGG and GO analyses revealed that these differentially expressed genes were implicated in pathways, including PPAR, PI3K/AKT, and AMPK signaling. The results of this study will contribute to an analysis of the immune response of M. nipponense to A. veronii infection at the transcriptomic level. Furthermore, the RNA-seq data generated here provide an important genomic resource for research on M. nipponense in the absence of a reference genome.

Characterization and immune function of decapentaplegic (Dpp) gene from the oriental river prawn, Macrobrachium nipponense.

The Decapentaplegic (Dpp) gene, which belongs to the TGF-ß superfamily, is involved in multiple developmental processes in eukaryotic species. In this study, we firstly identified and characterized Dpp from Macrobrachium nipponense. Its full-length open reading frame (ORF) cDNA was 1332 bp, encoding 443 amino acids. The putative MnDpp protein contained a signal peptide, a TGF-ß propeptide region and a TGF-ß domain. Its TGF-ß domain was highly conserved from vertebrates to invertebrates, and exhibited highly similarity to Dpp derived from Bombyx mori. qRT-PCR analysis suggested that MnDpp expressed in all tested tissues and responded to both bacterial and virus pathogens, indicating MnDpp was involved in the innate immune response of M. nipponense. Knockdown of MnDppin vivo significantly increased bacteria growth and markedly decreased the expressions of NF-κB signaling genes including dorsal, relish, TAK1, TAB1, Ikkß and Ikkε as well as antimicrobial peptides (AMPs) including ALF2, ALF3, ALF4, ALF5, Cru1 and Cru2. Moreover, in vitro overexpression of MnDpp protein in HEK293T cells further demonstrated that it exerted antibacterial immune response by activation of NF-κB signaling cascade. In summary, these results indicated that MnDpp played an important role in the innate immunity in M. nipponense by modulating NF-κB signaling pathway, which might provide new insights about Dpp in crustaceans and paved the way for a better understanding of the crustacean innate immune system.

Leucine-rich repeat-containing G-protein-coupled receptor 2 (LGR2) can regulate PO activity and AMP genes expression in Macrobrachium nipponense.

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.

Identification of a dorsal transcription factor (MnDorsal) from Macrobrachium nipponense and its role in innate immunity.

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.

Differentially proteomic analysis of the hemocytes against Aeromonas hydrophila infection in oriental river prawn Macrobrachium nipponense by iTRAQ approach.

As the direct executors of biological function, the expression level of proteins in host will reveal the molecular mechanisms regulating bacteria infection more directly. In the present study, the differential proteomes of Macrobrachium nipponense hemocytes response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography electrospray ionization tandem mass spectrometry. The hemocyte proteins from the unchallenged and A. hydrophila challenged prawn, M. nipponense, at 12, 24 and 36 h post infection were compared. From this, a total of 3372 proteins were identified and 1014 proteins were considered differentially expressed, of which 117 common differentially expressed proteins were indicated between the time points. Hierarchical clustering, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the general characterization of overall enriched proteins. Cytoskeletal proteins including myosin heavy chain, myosin regulatory light chain, actin, tubulin alpha/beta chain, troponin I and troponin T as well as antioxidant enzymes such as catalase and cytosolic MnSOD were found significantly up-regulated in hemocytes, indicating that the phagocytosis process and ROS system were induced after challenge with A. hydrophila. And other proteins such as integrin ß, innexin inx2-like and heat shock protein 60 also participate in prawn immune response against bacteria. Parallel reaction monitoring analyses were carried out for validation of the expression levels of differentially expressed proteins, which indicated high reliability of the proteomic results. This is the first report on proteome of M. nipponense hemocytes against A. hydrophila infection, which contributes to better understanding on the molecular mechanisms of prawns.

Two Wnt genes regulate the expression levels of antimicrobial peptides during Vibrio infection in Macrobrachium nipponense.

The Wnt signal transduction pathway is involved in a wide variety of cellular processes, including cell proliferation, differentiation, apoptosis, and immunity against microbial infection. In the current study, we cloned and characterized two Wnt homologues (Mn-Wnt4 and Mn-Wnt16) in Macrobrachium nipponense. The full length cDNA of Mn-Wnt4 was 3144 bp with a 1074 bp open reading frame (ORF) that encoded a protein containing 358 amino acid residues. The full length cDNA of Mn-Wnt16 transcript was 2893 bp with a 1281 bp ORF that encoded a 427 amino acid protein. Mn-Wnt4 and Mn-Wnt16 proteins contained a highly conserved WNT1 domain. Tissue distribution analysis showed that Mn-Wnt4 and Mn-Wnt16 were highly expressed in the stomach. The transcriptional levels of Mn-Wnt4 and Mn-Wnt16 in the stomach were upregulated at most tested time points after bacterial (Staphylococcus aureus and Vibrio parahaemolyticus) and viral (White spot syndrome virus) infection. Moreover, the expression levels of some antimicrobial peptides (AMPs) (including anti-lipopolysaccharide factor [ALF] and crustin [CRU]) were upregulated after V. parahaemolyticus infection. We further used dsRNA-mediated RNA interference technology to explore the relationship between these two Wnt genes and the expression levels of AMPs during V. parahaemolyticus infection. Mn-Wnt4 knockdown could significantly inhibit the expression of ALF1 and CRU4 in the stomach of V. parahaemolyticus-injected prawns, whereas Mn-Wnt16 silencing could result in the inhibition of the expression level of CRU3 and CRU4 in the stomach of V. parahaemolyticus-infected prawns. These findings indicated that the Wnt gene family might participate in the body's innate immune response to Vibrio infection by regulating the synthesis of a variety of AMPs. Our study will help to understand the role of the Wnt signaling pathway in the immune response of crustaceans.

The composition of the microbial community associated with Macrobrachium rosenbergii zoeae varies throughout larval development.

The giant river prawn, Macrobrachium rosenbergii, is an economically important freshwater prawn. The cultivation of zoea larvae is crucial for the success of the M. rosenbergii industry. In this study, we surveyed the microbial community diversity and structure associated with M. rosenbergii zoeae at different stages of larval development. Samples of zoea larvae from different developmental stages were collected and subjected to high-throughput DNA sequencing. Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were the dominant phyla in all six sample groups. At the genus level, the relative abundance of Bacillus decreased, and that of Enterobacter increased with the growth of the zoeae. This may have been related to the intestinal development of the zoea larvae. The microbial diversity of M. rosenbergii zoea larvae decreased significantly with development. The beta diversity analysis showed that the closer the developmental stage of M. rosenbergii, the more similar the structure of the associated bacterial communities.

CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda.

During the immune defense reaction of invertebrate, a plenty of reactive oxygen species (ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids could function as scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda. EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed. Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis.

Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus, affecting PirAVP /PirBVP toxins production.

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirAVP /PirBVP toxins are mainly produced, while at 110 rpm PirAVP /PirBVP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

A Kruppel-like factor from Macrobrachium rosenbergii (MrKLF) involved in innate immunity against pathogen infection.

Kruppel-like factors (KLFs) belong to a family of zinc finger-containing transcription factors that are widely present in eukaryotes. In the present study, a novel KLF from the giant river prawn Macrobrachium rosenbergii (designated as MrKLF) was successfully cloned and characterized. The full-length cDNA of MrKLF was 1799 bp with an open reading frame of 1332 bp that encodes a putative protein of 444 amino acids, including three conserved ZnF_C2H2 domains at the C-terminus. Multiple alignment analysis showed that MrKLF and other crustacean KLFs shared high similarity. Quantitative real-time PCR analysis revealed that MrKLF mRNA was found in different tissues of prawns and detected in the gills, hepatopancreas, and intestines. After the challenge with Vibrio parahaemolyticus and Aeromonas hydrophila, different expression patterns of MrKLF in the gills, intestines, and hepatopancreas were observed. RNA interference analysis indicated that MrKLF was involved in regulating the expression of four antimicrobial peptides, namely, Crustin (Crus) 2, Crus8, anti-lipopolysaccharide factor (ALF) 1, and ALF3. These results help promote research on M. rosenbergii innate immunity.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.