Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

Ecytonucleospora hepatopenaei n. gen. et comb. (Microsporidia: Enterocytozoonidae): A redescription of the Enterocytozoon hepatopenaei (Tourtip et al., 2009), a microsporidian infecting the widely cultivated shrimp Penaeus vannamei.

The microsporidian Enterocytozoon hepatopenaei from Penaeus vannamei (EHPPv) was redescribed on the basis of spore morphology, life cycle, pathology, and molecular character. Compared with the Enterocytozoon hepatopenaei isolated from Penaeus monodon (EHPPm), described by Tourtip et al. in 2009, new features were found in EHPPv. Electron microscopy demonstrated that EHPPv was closely associated with the nucleus of host cell. The merogony and sporogony phages were in direct contact with the cytoplasm of host cells, whereas some of the sporoblasts and the spores were surrounded by the interfacial envelope. Mature spores of EHPPv were oval and monokaryotic, measuring 1.65 ± 0.15 µm × 0.92 ± 0.05 µm. Spores possessed many polyribosomes around a bipartite polaroplast and the polar filament with 4-5 coils in two rows. Phylogenetic analyses showed all Enterocytozoon hepatopenaei isolates shared a common ancestor. Based on the morphological and molecular analyses, we propose the establishment of a new genus Ecytonucleospora and transferring Enterocytozoon hepatopenaei to the genus Ecytonucleospora, retaining the specific epithet hepatopenaei that Tourtip et al. proposed in recognition of their first research, as the new combination Ecytonucleospora hepatopenaei n. comb. Furthermore, it was suggested Enterospora nucleophila, Enterocytozoon sp. isolate RA19015_21, and Enterocytozoon schreckii be assigned into this new genus.

Multifunctional chondroitin sulfate based hydrogels for promoting infected diabetic wounds healing by chemo-photothermal antibacterial and cytokine modulation.

Diabetic foot (DF) is difficult to heal due to the formation of drug-resistant bacterial biofilms and dysregulation of the wound microenvironment. To solve this problem, multifunctional hydrogels were prepared by in situ or spraying with 3-aminophenylboronic acid modified oxidized chondroitin sulfate (APBA-g-OCS), polyvinyl alcohol (PVA) and black phosphorus/bismuth oxide/ε-polylysine (BP/Bi2O3/ε-PL) as precursors for promoting infected diabetic wounds healing. The hydrogels display multiple stimulus responsiveness, strong adhesion and rapid self-healing ability owing to the dynamic borate ester bonds, hydrogen bonds and π-π conjugation cross-link points, remain synergistic chemo-photothermal antibacterial effect and anti-biofilm formation ability due to the doping of BP/ Bi2O3/ε-PL into the hydrogel by dynamic imine bonds crosslinking and possess anti-oxidation and inflammatory chemokine adsorption ability attributing to the presence of APBA-g-OCS. Most importantly, as a result of the above functions, the hydrogels can not only respond to the wound microenvironment to conduct combined PTT and chemotherapy for efficient anti-inflammation, but also improve the wound microenvironment by scavenging ROS and regulating the expression of cytokines, thus further accelerating collagen deposition, promoting granulation tissue formation and angiogenesis, finally promoting the healing of infected wounds in diabetic rats.

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii.

The microsporidian Enterocytozoon hepatopenaei (EHP) has become a critical threat to the global shrimp aquaculture industry, thus necessitating early detection by screening. Development of a rapid and accurate assay is crucial both for the active surveillance and for the assessment of shrimp with EHP infection. In the present study, a distinct strain of E. hepatopenaei (EHP Mr ) was found in Macrobrachium rosenbergii. The SWP1 gene analysis revealed it was a new genotype that differed with the common strain isolated from the Litopenaeus vannamei (EHP Lv ). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHP Mr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHP Mr strain and the EHP Lv strain. The new primers also had good specificity and high sensitivity, which may serve as an alternative for EHP genotyping. This study provided a method for detection of EHP in the biosecurity of Macrobrachium rosenbergii farming, and the developed protocol was proposed for the routine investigation and potential carrier screening, especially for molecular epidemiology.

Nucleospora hippocampi n. sp., an Intranuclear Microsporidian Infecting the Seahorse Hippocampus erectus From China.

The life cycle, ultrastructure, and molecular phylogeny of a new intranuclear microsporidian, Nucleospora hippocampi n. sp., infecting the intestine of the Hippocampus erectus, were described. The histopathology revealed an extensive infection, mainly in the columnar epithelium of the intestinal mucosa layer. The enterocytes were the important target cell for Nucleospora hippocampi n. sp. infection. Transmission electron microscopy results showed that this microsporidian developed directly within the host cell nucleoplasm. In the intranuclear life cycle, the transformation from meront to sporogonial plasmodium was recognized by forming electron-dense disc structures, which were considered the polar tube precursors. The microsporidian showed the typical morphological characteristics of the family Enterocytozoonidae in the formation and development of spore organelles prior to the division of the sporogonial plasmodium. According to wet smear observation, eight spores were generally formed in a single host nucleus. Mature spores were elongated ovoids that were slightly bent and measured 1.93 × 0.97 µm. The isofilar polar tube was arranged in 7~8 coils in one row. Phylogenetic analysis of its small subunit ribosomal DNA sequences demonstrated that the parasite belonged to the Nucleospora group clade. The histological, ultrastructural, and molecular data support the emergence of a new species in the genus Nucleospora. This is the first report of Nucleospora species in Asia and threatened syngnathid fishes.

Transcriptome analysis of Macrobrachium rosenbergii: Identification of precocious puberty and slow-growing information.

Macrobrachium rosenbergii is an important economic aquatic animal and has been cultivated worldwide. However, it has suffered a disease of precocious puberty and growth retardation. This disease was also called iron prawn syndrome (IPS) in recent years. However, the cause of this disease has thus far remained a mystery . The present work utilized transcriptome sequencing technology to acquire gene expression information of gonads and to find the differentially expressed genes(DEGs) between diseased and normal male prawn. Finally, 426 significantly expressed genes were identified(p less than 0.01, |log2FC|≥1), of which 171 genes were up-regulated and 255 were down-regulated. Furthermore, DEGs were annotated to 36 GO terms and 202 KEGG pathways. Enrichment analysis of DEGs resulted in 10 significantly enriched GO terms and a total of 12 significantly enriched KEGG pathways. Analysis of the transcriptome sequences and DEGs identified several unigenes and pathways involved in precocious puberty and growth retardation. Quantitative PCR was performed to validate accuracy of the RNA-seq and the expression level of 10 genes, calculated by two analysis method, was mostly consistent. This is the first time to report precocious puberty and growth retardation male M. rosenbergii by transcriptome sequencing. The data presented here reveals key insights into the genetic markers of precocious puberty and growth retardation of male M. rosenbergii.

LncRNA HLA-F-AS1 attenuates the ovarian cancer development by targeting miR-21-3p/PEG3 axis.

Dysregulated long noncoding RNA (lncRNA) HLA-F-AS1 is depicted in numerous cancers. However, its function in ovarian cancer has yet to be clarified. LncRNA HLA-F-AS1, miR-21-3p, and PEG3 expressions in ovarian cancer tissues and cells were measured via reverse transcription quantitative PCR. Scratch and CCK8 assays were performed to evaluate the cells' migratory and proliferative abilities, respectively. To assess the expressions of the apoptosis-related proteins Bax and Bcl-2, Western blotting was conducted. Anti-AGO2 RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were executed to study lncRNA HLA-F-AS1's and PEG3 3'UTR's interactions to miR-21-3p. Finally, the tumor growth in vivo was inspected by performing a xenograft experiment. Among the ovarian cancer tissues and cells, the expressions of PEG3 and lncRNA HLA-F-AS1 were depleted while an elevated miR-21-3p expression was observed. HLA-F-AS1's overexpression attenuated ovarian cancer development in vivo and in vitro . MiR-21-3p targeted PEG3 3'UTR while HLA-F-AS1 targeted miR-21-3p. HLA-F-AS1 overexpression mitigated the enhancement brought about by miR-21-3p mimic on ovarian cancer cells' proliferation and migration. Meanwhile, PEG3 overexpression abrogated miR-21-3p mimic's function as an oncogene in the progression of ovarian cancer. Ovarian cancer development is suppressed when lncRNA HLA-F-AS1 targets the miR-21-3p/PEG3 axis. This may possibly be a novel therapeutic target for ovarian cancer.

PvML1 suppresses bacterial infection by recognizing LPS and regulating AMP expression in shrimp.

Toll and Toll-like receptors (TLRs) play essential roles in the innate immunity of Drosophila and mammals. Recent studies have revealed the presence of Toll-mediated immune signaling pathways in shrimp. However, the recognition and activation mechanism of Toll signaling pathways in crustaceans remain poorly understood due to the absence of key recognition molecules, such as peptidoglycan recognition proteins. Here, a novel MD2-related lipid-recognition (ML) member named PvML1 was characterized in Penaeus vannamei. We found that PvML1 shared a similar 3D structure with human MD2 that could specifically recognize lipopolysaccharides (LPS) participating in LPS-mediated TLR4 signaling. PvML1 was highly expressed in hemocytes and remarkably upregulated after Vibrio parahemolyticus challenge. Furthermore, the binding and agglutinating assays showed that PvML1 possessed strong binding activities to LPS and its key portion lipid A as well as Vibrio cells, and the binding of PvML1 with bacterial cells led to the agglutination of bacteria, suggesting PvML1 may act as a potential pathogen recognition protein upon interaction with LPS. Besides, coating V. parahemolyticus with recombinant PvML1 promoted bacterial clearance in vivo and increased the survival rate of bacterium-challenged shrimp. This result was further confirmed by RNAi experiments. The knockdown of PvML1 remarkably suppressed the clearance of bacteria in hemolymph and decreased the survival rate of infected shrimp. Meanwhile, the silencing of PvML1 severely impaired the expression of a few antimicrobial peptides (AMPs). These results demonstrated the significant correlation of bacterial clearance mediated by PvML1 with the AMP expression. Interestingly, we found that PvML1 interacted with the extracellular region of PvToll2, which had been previously shown to participate in bacterial clearance by regulating AMP expression. Taken together, the proposed antibacterial model mediated by PvML1 might be described as follows. PvML1 acted as a potential recognition receptor for Gram-negative bacteria by binding to LPS, and then it activated PvToll2-mediated signaling pathway by interacting with PvToll2 to eliminate invading bacteria through producing specific AMPs. This study provided new insights into the recognition and activation mechanism of Toll signaling pathways of invertebrates and the defense functions of ML members.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Lateral flow colorimetric biosensor for detection of Vibrio parahaemolyticus based on hybridization chain reaction and aptamer.

Vibrio parahaemolyticus (V. parahaemolyticus) is the causative agent for acute hepatopancreatic necrosis disease (AHPND) of shrimp, and it is also a common seafood-borne pathogen for humans. Rapid and accurate identification of V. parahaemolyticus is helpful to diagnose the AHPND and ensure food safety. Common detection methods suffer the deficiency of time-consuming and complexed operation. Based on the increased development of aptamer and our previous study, a new detection assay of V. parahaemolyticus was introduced, in which the aptamer combined with magnetic nanoparticles (MNPs) was the recognizer, hybridization chain reaction (HCR) was the signal amplifier, and lateral flow nucleotide biosensor (LFNB) was the signal exporter. The assay possessed high specificity of distinguishing the target with other bacteria, and the calculated limit of detection was 2.6 × 103 cells. Furthermore, the whole process just needs 67 min, free of thermocycle instruments and signal readout instruments, which means it is suitable for low-resource laboratories or districts.

Effects of different salinity on the transcriptome and antibiotic resistance of two Vibrio parahaemolyticus strains isolated from Penaeus vannamei cultured in seawater and freshwater ponds.

The transcriptome and antibiotic resistance of Vibrio parahaemolyticus isolated from Penaeus vannamei cultured in seawater (strain HN1)and freshwater (strain SH1) ponds were studied at different salinity (2‰ and 20‰). At different salinity, 623 differentially expressed genes (DEGs) significantly upregulated and 1,559 DEGs significantly downregulated in SH1. In HN1, 466 DEGs significantly upregulated and 1,930 DEGs significantly downregulated, indicating high salinity can lead to the downregulation of most genes. In KEGG analysis, the expression of DEGs annotated to starch and sucrose metabolism pathway was higher at 2‰ salinity than at 20‰ salinity in HN1 and SH1, implying salinity affected bacterial growth mainly through this pathway. In the enrichment analysis of upregulated DEGs, two pathways (Valine, leucine, and isoleucine degradation, and Butanoate metabolism) were significantly enriched at different salinity. Antibiotic-susceptibility test discovered that SH1 isolated from P. vannamei cultured in freshwater was resistant to multiple drugs, including kanamycin, gentamicin, medemycin, and azithromycin, at a salinity of 2‰, whereas at 20‰ salinity, SH1 was not resistant to the drugs. The HN1 strain isolated from P. vannamei cultured in mariculture was resistant to polymyxin B and clindamycin at 20‰ salinity. Whereas, HN1 was intermediately susceptible to these two antibiotics at 2‰ salinity. These results indicate that the drug resistance of bacteria was affected by salinity. Furthermore, beta-lactam resistance was significantly enriched in SH1 at different salinity, and the inhibition zone of penicillin G was consistent with the results of a beta-lactam resistance pathway.

Transcriptome analysis of pacific white shrimp (Penaeus vannamei) intestines and hepatopancreas in response to Enterocytozoon hepatopenaei (EHP) infection.

Penaeus vannamei is the most economically important species of shrimp cultured worldwide. Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that severely affects the growth and development of shrimps. In this study, the transcriptome differences between EHP-infected and uninfected shrimp were investigated through next-generation sequencing. The unigenes were assembled with the reads from all the four libraries. The differentially expressed genes (DEGs) of intestines and hepatopancreas were analyzed. There were 2,884 DEGs in the intestines and 2,096 DEGs in the hepatopancreas. The GO and KEGG enrichment analysis indicated that DEGs were significantly enriched in signaling pathways associated with nutritional energy metabolism and mobilizing autoimmunity. Moreover, the results suggested the downregulation of key genes in energy synthesis pathways contributed greatly to shrimp growth retardation; the upregulation of immune-related genes enhanced the resistance of shrimp against EHP infection. This study provided identified genes and pathways associated with EHP infection revealing the molecular mechanisms of growth retardation.

Bacterial Flora in the Gill Tissues and Intestinal Tracts of Male and Female Chinese Mitten Crabs (Eriocheir sinensis) with Different Diets in a Mud Pond.

The Chinese mitten crab, Eriocheir sinensis, is an economically valuable aquaculture species. Prior to sale, farmed crabs are often fattened with pellet feed or wild fish. In this study, PacBio Sequel sequencing was used to determine the bacterial flora in the intestinal tracts and gill tissues of male and female E. sinensis fed with various diets. The flora was then compared with the microorganisms found in environmental samples. The results showed that Proteobacteria was the dominant phylum in both tissue and environmental samples. The relative abundances of Proteobacteria in the water grass surface flushing samples and water grass samples were the highest, at up to 95.68% and 67.85%, respectively. Beyond that, Bacteroidetes, Firmicutes, and Tenericutes were the dominant phyla (>1%) in the intestinal samples, whereas Bacteroidetes and Actinobacteria were the dominant phyla in the gills. In addition, different environment samples contained diverse bacterial phyla, indicating some differences in the community composition between the different sample groups. Heat map clustering and principal coordinate plot analyses indicated that intestinal samples, crab gill samples, and environmental samples clustered together, respectively. Furthermore, an unweighted pair-group method with arithmetic mean technique confirmed that the intestinal and gill samples of crabs with different diets separately clustered together, suggesting the microbial assemblages of the same tissues share a greater similarity than those from crabs of different sex and eating different diets. What's more, biomarker bacteria (LDA ≥ 4) from the different groups were identified. Pathogenic agents from the genus Aeromonas were abundant in the intestinal samples of crabs fed with pellet feed, and Vibrio species were prevalent in the intestinal samples of crabs fed with wild fishes.

Transcriptome analysis of Penaeus vannamei hepatopancreas reveals differences in toxicity mechanisms between phoxim and prometryne.

Due to overuse and terrestrial input, there are large quantities of phoxim and prometryne residues in some aquatic environments. In the present study, the effects of these compounds on Penaeus vannamei hepatopancreas were analysed at the transcriptome level to investigate toxicity in this nontarget aquaculture organism. Twelve normalised cDNA libraries were constructed using RNA from phoxim and prometryne treatment groups, and an untreated control group. A total of 667,750,902 clean reads were obtained. Analysis of differentially expressed genes (DEGs) identified 449 in control vs phoxim groups, 185 in control vs prometryne groups, and 183 in prometryne vs phoxim groups. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, arachidonic acid metabolism, pancreatic secretion, linoleic acid metabolism, and beta-alanine metabolism pathways were significantly enriched in control vs phoxim groups. In control vs prometryne groups, lysosome, pentose and glucuronate interconversion, antigen processing and presentation, and glycosaminoglycan degradation pathways were significantly enriched. In prometryne vs phoxim groups, protein digestion and absorption, extracellular matrix (ECM)-receptor interaction, PI3K-Akt signalling, cell adhesion molecule (CAM), AGE-RAGE signalling related to diabetic complications, focal adhesion, and renin secretion pathways were significantly enriched. In further detailed analysis, glutathione S-transferase (GST), glutathione peroxidase and basic phospholipase A2 were downregulated in the phoxim treatment group, indicating that phoxim damaged hepatopancreas. Upregulation of phospholipase A2 (secretory phospholipase A2-like) indicates possible inflammatory pathological injury to hepatopancreas caused by phoxim. Meanwhile, downregulation of CD63 indicates that prometryne affect the immune system.

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.

A novel ML domain-containing protein (SpMD2) functions as a potential LPS receptor involved in anti-Vibrio immune response.

The myeloid differentiation protein 2 (MD2)-related lipid-recognition (ML) proteins display diverse biological functions in host immunity and lipid metabolism by interacting with different lipids. Human MD2, an indispensable accessory protein in TLR4 signaling pathway, specifically recognizes lipopolysaccharides (LPS), thereby leading to the activation of TLR4 signaling pathway to produce many effectors that participate in inflammatory and immuneresponses against Gram-negative bacteria. Toll and immune deficiency (IMD) pathways are first characterized in Drosophila and are reportedly present in crustaceans, but the recognition and activation mechanism of these signaling pathways in crustaceans remains unclear. In the present study, a novel ML protein was characterized in mud crab (Scylla paramamosain) and designated as SpMD2. The complete SpMD2 cDNA sequence is 1114 bp long with a 465 bp open reading frame; it encodes a protein that contains 154 amino acids (aa). In the deduced protein, a signal peptide (1-21 aa residues) and a ML domain (43-151 aa residues) were predicted. SpMD2 shared a similar three-dimensional structure and a close evolutionary relationship with human MD2. SpMD2 was highly expressed in gills, hemocytes, intestine, and hepatopancreas and was upregulated in gills and hemocytes after challenges with bacteria, thereby suggesting its involvement in antibacterial defense. Western blot assay showed that SpMD2 possesses strong binding activities to different bacteria and two fungi. ELISA demonstrated that SpMD2 exhibits binding abilities to LPS, lipid A, peptidoglycan (PGN), and lipoteichoic acid (LTA). Its binding ability to LPS and lipid A were stronger than to PGN or LTA, implying that SpMD2 was an important LPS-binding protein in mud crab. Bacterial clearance assay revealed that the pre-incubation of Vibrio parahemolyticus with SpMD2 facilitates bacterial clearance in vivo and that knockdown of SpMD2 dramatically suppresses the bacterial clearance and decreases the expression of several antimicrobial peptides (AMPs). Furthermore, SpMD2 overexpression could enhance the promoter activity of SpALF2. These results revealed that SpMD2 affects bacterial clearance by regulating AMPs. Thus, by binding to LPS and by regulating AMPs, SpMD2 may function as a potential receptor, which is involved in the recognition and activation of a certain immune signaling pathway against Gram-negative bacteria. This study provides new insights into the diverse functions of ML proteins and into the antibacterial mechanisms of crustaceans.

Application of biofertilizers increases fluoroquinolone resistance in Vibrio parahaemolyticus isolated from aquaculture environments.

Antimicrobial resistance genes in aquaculture environments have attracted wide interest, since these genes pose a severe threat to human health. This study aimed to explore the possible mechanisms of the ciprofloxacin resistance of Vibrio parahaemolyticus (V. parahaemolytiucs) in aquaculture environments, which may have been affected by the biofertilizer utilization in China. Plasmid-mediate quinolone resistance (PMQR) genes, representative (fluoro)quinolones (FNQs), and ciprofloxacin-resistance isolates in biofertilizer samples were analyzed. The significantly higher abundance of oqxB was alarming. The transferable experiments and Southern blot analysis indicated that oqxB could spread horizontally from biofertilizers to V. parahaemolyticus, and two (16.7%) trans-conjugants harboring oqxB were provided by 12 isolates that successfully produced OqxB. To the best of our knowledge, this study is the first to report PMQR genes dissipation from biofertilizers to V. parahaemolyticus in aquaculture environments. The surveillance, monitoring and control of PMQR genes in biofertilizers are warranted for seafood safety and human health.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.