Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70) and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO), peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined.
Mild heat stress promotes thermotolerance and protection against several different stresses in aquatic animals, consequences correlated with the accumulation of heat shock protein 70 (Hsp70). The purpose of this study was to determine if non-lethal heat shock (NLHS) of the Asian green mussel, Perna viridis, an aquatic species of commercial value, promoted the production of Hsp70 and enhanced its resistance to stresses. Initially, the LT50 and LHT for P. viridis were determined to be 42°C and 44°C, respectively, with no heat shock induced death of mussels at 40°C or less. Immunoprobing of western blots revealed augmentation of constitutive (PvHsp70-1) and inducible (PvHsp70-2) Hsp70 in tissue from adductor muscle, foot, gill and mantel of P. viridis exposed to 38°C for 30 min followed by 6 h recovery, NLHS conditions for this organism. Characterization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that PvHsp70-1 and PvHsp70-2 respectively corresponded most closely to Hsp70 from P. viridis and Mytilus galloprovincialis. Priming of adult mussels with NLHS promoted thermotolerance and increased resistance to V. alginolyticus. The induction of Hsp70 in parallel with enhanced thermotolerance and improved protection against V. alginolyticus, suggests Hsp70 functions in P. viridis as a molecular chaperone and as a stimulator of the immune system.
Diseases have remained the major issue for shrimp aquaculture industry for decades by which different shrimp species demonstrated alternative disease resistance or tolerance. However, there had been insufficient studies on the underlying host mechanisms of such phenomenon. Hence, in this study, the main objective involves gaining a deeper understanding into the functional importance of shrimp STAT gene from the aspects of expression, sequence, structure, and associated genes. STAT gene was selected primarily because of its vital signalling roles in stress, endocrine, and immune response. The differential gene expressions of Macrobrachium rosenbergii STAT (MrST) and Penaeus monodon STAT (PmST) under White Spot Syndrome Virus (WSSV) and Vibrio parahaemolyticus/VpAHPND infections were identified through qPCR analysis. Notably, during both pathogenic infections, MrST demonstrated significant gene expression down-regulations (during either early or later post-infection time points) whereas PmST showed only significant gene expression up-regulations. Important sequence conservation or divergence was highlighted through STAT sequence comparison especially amino acid alterations at 614 aa [K (Lysine) to E (Glutamic Acid)] and 629 aa [F (Phenylalanine) to V (Valine)] from PmST (AY327491.1) to PmST (disease tolerant strain). There were significant differences observed between in silico characterized structures of MrST and PmST proteins. Important functional differentially expressed genes (DEGs) in the aspects of stress, endocrine, immune, signalling, and structural were uncovered through comparative transcriptomic analysis. The DEGs associated with STAT functioning were identified including inositol 1,4,5-trisphosphate receptor, hsp90, caspase, ATP binding cassette transmembrane transporter, C-type Lectin, HMGB, ALF1, ALF3, superoxide dismutase, glutathione peroxidase, catalase, and TBK1. The main findings of this study are STAT differential gene expression patterns, sequence divergence, structural differences, and associated functional DEGs. These findings can be further utilized for shrimp health or host response diagnostic studies. STAT gene can also be proposed as a suitable candidate for future studies of shrimp innate immune enhancement.
Shrimp aquaculture contributes significantly to global economic growth, and the whiteleg shrimp, Penaeus vannamei, is a leading species in this industry. However, Vibrio parahaemolyticus infection poses a major challenge in ensuring the success of P. vannamei aquaculture. Despite its significance in this industry, the biological knowledge of its pathogenesis remains unclear. Hence, this study was conducted to identify the interaction sites and binding affinity between several immune-related proteins of P. vannamei with V. parahaemolyticus proteins associated with virulence factors. Potential interaction sites and the binding affinity between host and pathogen proteins were identified using molecular docking and dynamics (MD) simulation. The P. vannamei-V. parahaemolyticus protein-protein interaction of Complex 1 (Ferritin-HrpE/YscL family type III secretion apparatus protein), Complex 2 (Protein kinase domain-containing protein-Chemotaxis CheY protein), and Complex 3 (GPCR-Chemotaxis CheY protein) was found to interact with -4319.76, -5271.39, and -4725.57 of the docked score and the formation of intermolecular bonds at several interacting residues. The docked scores of Complex 1, Complex 2, and Complex 3 were validated using MD simulation analysis, which revealed these complexes greatly contribute to the interactions between P. vannamei and V. parahaemolyticus proteins, with binding free energies of -22.50 kJ/mol, -30.20 kJ/mol, and -26.27 kJ/mol, respectively. This finding illustrates the capability of computational approaches to search for molecular binding sites between host and pathogen, which could increase the knowledge of Vibrio spp. infection on shrimps, which then can be used to assist in the development of effective treatment.
During our studies on Malaysian Laurencia species, brominated metabolites, tiomanene, acetylmajapolene B, and acetylmajapolene A were isolated from an unrecorded species collected at Pulau Tioman, Pahang along with known majapolene B and majapolene A. Acetylmajapolene A was a mixture of diastereomers as in the case of majapolene A. Tiomanene may be a plausible precursor for acetylmajapolenes B and A. In addition, three known halogenated sesquiterpenes and two known halogenated C(15) acetogenins were found from other two unrecorded species collected at Pulau Karah, Terengganu and Pulau Nyireh, Terengganu, respectively. Some of these halogenated metabolites showed moderate antibacterial activity against some marine bacteria.
Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production. Vibrio tritonius strain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2 production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production of V. tritonius strain AM2 to investigate the response to various NaCl concentrations. The modified Han-Levenspiel model reveals that salt inhibition in hydrogen production using V. tritonius starts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production of V. tritonius compared to that of Escherichia coli JCM 1649 reveals the marine-adapted fermentative hydrogen production system in V. tritonius. V. tritonius AM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)).
Aquaculture production of the Pacific white shrimp is the largest in the world for crustacean species. Crucial to the sustainable global production of this important seafood species is a fundamental understanding of the shrimp gut microbiota and its relationship to the microbial ecology of shrimp pond. This is especially true, given the recently recognized role of beneficial microbes in promoting shrimp nutrient intake and in conferring resistance against pathogens. Unfortunately, aquaculture-related microbiome studies are scarce in Southeast Asia countries despite the severe impact of early mortality syndrome outbreaks on shrimp production in the region. In this study, we employed the 16S rRNA amplicon (V3-V4 region) sequencing and amplicon sequence variants (ASV) method to investigate the microbial diversity of shrimp guts and pond water samples collected from aquaculture farms located in Malaysia and Vietnam. Substantial differences in the pond microbiota were observed between countries with the presence and absence of several taxa extending to the family level. Microbial diversity of the shrimp gut was found to be generally lower than that of the pond environments with a few ubiquitous genera representing a majority of the shrimp gut microbial diversity such as Vibrio and Photobacterium, indicating host-specific selection of microbial species. Given the high sequence conservation of the 16S rRNA gene, we assessed its veracity at distinguishing Vibrio species based on nucleotide alignment against type strain reference sequences and demonstrated the utility of ASV approach in uncovering a wider diversity of Vibrio species compared to the conventional OTU clustering approach.
The cytotoxicity of cell-free culture filtrates of 31 isolates of Vibrio cholerae O1 and O139, 5 reference strains and 26 clinical isolates, was tested on Madin Darby Bovine Kidney (MDBK) cells and Vero cells. The 3-[4,5-dimethylthiazol-2-y]-2, 5-diphenyltetrazolium bromide (MTT) test was used to detect the effect of the filtrates on the proliferation and viability of cultured cell populations. The filtrates were prepared from serial ten-fold dilutions of inoculated AKI and APW broth media with and without the addition of polymyxin B. The APW culture filtrates of both V. cholerae O1 and O139 with and without added polymyxin B showed greater toxicity to MDBK cells as compared to AKI filtrates. The cytotoxicity of AKI-grown V. cholerae O139 to MDBK cells was greater than that of V. cholerae O1 grown in the same medium. The cytotoxicity of APW filtrates on Vero cells was low and only noted when polymyxin was added to the medium.
The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.
The study compares the bacteriological quality on Asian seabass (Lates calcarifer) between ice and salt storage methods. The main objectives of the study were to identify different bacteria constituents and quantitative bacterial load in Asian seabass when preserved with ice and sea salt. For the purpose of this study, Asian seabass was stored in two different conditions of ice-chilled and salted for 2 days. All fish samples were analyzed by performing bacteriological analysis and the isolated bacteria were identified by using API identification system. In case of the quantity of bacteria in the flesh, Chilling and salting had no significant difference to the quantity of bacteria on fish flesh. As for the skin, salt-preserved fish showed higher quantity of bacteria than ice-preserved fish. Acinetobacter baumannii and Pseudomonas fluorescens had been identified from skin sample of ice-chilled fish. Besides P. fluorescens and A. baumannii other isolates identified include Vibrio and Myxobacteria. All bacteria were cocci-shaped except a few bacilli. In term of bacteria number and morphological characteristics, ice-chilled preserved fish was better than salt preserved fish. Overall, less number of bacteria was observed in both ice-chilled and sea salt preserved fish. The result of this study indicated that the quick preservation is a very important factor to control bacterial load in the preserved fish.
It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.
Three new halogenated tricyclic sesquiterpenes, omphalaurediol (1), rhodolaurenones B (2) and C (3) were isolated together with nine known haloganated sesquiterpenes such as rhodolaurenone A (4), rhodolaureol (5), isorhodolaureol (6), (-)-laurencenone D (7), elatol (8), (+)-deschloroelatol (9), cartilagineol (10), (+)-laurencenone B (11) and 2-chloro-3-hydroxy-α-chamigren-9-one (12) from a population of Bornean red algae Laurencia majuscula. The structures of three new metabolites were determined based on their spectroscopic data (IR, 1D and 2D NMR, and MS). These compounds showed antibacterial activity against three human pathogenic bacteria (Escherichia coli, Salmonella typhi and Vibrio cholera).
We report the first draft genome sequence of a Vibrio parahaemolyticus strain (VpAHPND), which causes acute hepatopancreatic necrosis disease (AHPND) in Penaeus monodon. The strain has a pVA1-like plasmid carrying pirAvp and pirBvp genes. Whole-genome comparisons revealed >98% similarity to VpAHPND isolates from Thailand, Mexico, and Vietnam.
We sequenced the genome of Vibrio parahaemolyticus strain ST17.P5-S1, isolated from Penaeus vannamei cultured in the east coast of Peninsular Malaysia. The strain contains several antibiotic resistance genes and a plasmid encoding the Photorhabdus insect-related (Pir) toxin-like genes, pirAvp and pirBvp, associated with acute hepatopancreatic necrosis disease (AHPND).
Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.
Ixotrophy is a process that enables certain microbes to prey on other cells. The ability of cells to aggregate or adhere is thought to be a significant initial step in ixotrophy. The gliding, multicellular filamentous bacterium Aureispira sp. CCB-QB1 belongs to the family Saprospiraceae and preys on bacteria such as Vibrio sp. in seawater. Adhesion and cell aggregation were coincident with preying and were hypothesized to play an important role in the ixotrophy in this bacterium. To test this hypothesis, experiments to elucidate the mechanisms of aggregation or adhesion in this bacterium were performed. The ability of Aureispira QB1 to adhere and aggregate to prey bacterium, Vibrio sp., required divalent cations, especially calcium ions. In the presence of calcium, Aureispira QB1 cells captured 99 % of Vibrio sp. cells after 60 min of incubation. Toluidine blue O, which binds acidic polysaccharides, bound to Aureispira QB1 and inhibited adhesion of Aureispira QB1. These results suggest that acidic polysaccharides are needed for aggregation or adhesion of Aureispira and that calcium ions play a significant role in these phenomena.
Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.
Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.