Evaluation of immune stimulatory products for whiteleg shrimp (Penaeus vannamei) by a metabolomics approach.

The use of probiotics, prebiotics and dietary fiber has become a common practice in shrimp aquaculture as alternatives to antibiotic treatment. However, not much is known about the metabolic mechanisms underlying the effects of probiotics and immunostimulant used in shrimp aquaculture. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomics approach was used to characterize metabolite profiles of haemolymph and gills of whiteleg shrimp (Penaeus vannamei) exposed to four treatments (cellulose fiber, probiotics with Vibrio alginolyticus, a combination of cellulose fiber and V. alginolyticus and a control treatment). The cellulose fiber was administrated as a feed additive (100 mg⋅Kg-1 feed), while the probiotics was applied in the water (105 UFC⋅mL-1 culture water). The results showed significant differences in haemolymph metabolite profiles of immune stimulated treatments compared to the control and among treatments. The combination of cellulose fiber and probiotics resulted in greater differences in metabolic profiles, suggesting a better immune stimulation with this approach. The changes in haemolymph metabolome of treated shrimp reflected several biochemical pathway modifications, including changes in amino acid and fatty acid metabolism, disturbances in energy metabolism and antimicrobial activity and stress responses. For gill tissues, significant differences were only found in lactic acid between the probiotic group and the control. Among the altered metabolites, the increases of itaconic acid in haemolymph, and lactic acid in both haemolymph and gill tissues of immune-stimulated suggest the potential use of these metabolites as biomarkers for health assessment in aquaculture.

Changes in metabolic profiling of whiteleg shrimp (Penaeus vannamei) under hypoxic stress.

Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studies have revealed important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regarding this stressor at the molecular level. In the present study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was employed to characterize the metabolic signatures and pathways underlying responses of Pacific white shrimp (Penaeus vannamei) to hypoxia and to identify associated candidate biomarkers. We compared metabolite profiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h). Dissolved oxygen levels were maintained above 85 % saturation in the control and before hypoxia, and 15 % saturation in the hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantly different between before and after hypoxia exposure. These metabolites were energy-related metabolites (e.g., intermediates of citric acid cycle, lactic acid, alanine), fatty acids and amino acids. Pathway analysis revealed 17 pathways that were significantly affected by hypoxia. The changes in metabolites and pathways indicate a shift from aerobic to anaerobic metabolism, disturbance in amino acid metabolism, osmoregulation, oxidative damage and Warburg effect-like response caused by hypoxic stress. Among the altered metabolites, lactic acid was most different between before and after hypoxia exposure and had the highest accurate value for biomarker identification. Future investigations may validate this molecule as a stress biomarker in aquaculture. This study contributes to a better understanding of hypoxia in shrimp and crustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.

Metabolic responses of whiteleg shrimp to white spot syndrome virus (WSSV).

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies.

Draft genome sequence of Bacillus velezensis strain 3TSA-3, a potential probiotic for Pacific white shrimp Penaeus vannamei postlarvae isolated from commercial hatchery tanks.

We report the draft genome of Bacillus velezensis strain 3TSA-3, isolated from Pacific white shrimp Penaeus vannamei postlarvae collected from a hatchery tank with high survival despite the presence of pathogenic Vibrio. The strain possesses genes encoding bacteriocins and lacks virulence factor genes, characteristics for a potential aquaculture probiotic.

Optimized swarming motility assay to identify anti-virulence products against Vibrio parahaemolyticus, a pathogen of farmed shrimp.

Swarming motility is a type of movement used by pathogenic flagellated bacteria as virulence factor to colonize surfaces and cause damage to the host. Vibrio parahaemolyticus is a pathogenic flagellated bacterium that increases its virulence by switching from swimmer to swarming cells. The hosts of pathogenic V. parahaemolyticus include farmed shrimp. Therefore, methods to detect and quantify this movement are important to control shrimp diseases caused by pathogenic V. parahaemolyticus strains. We developed an optimized swarming motility assay by identifying the most optimal type of agar, and drying time of the culture medium, agar concentration and volume of the bacterial culture to achieve the fastest swarming motility during the migration of V. parahaemolyticus on Petri dishes during a 24-hour incubation period. The method includes data analysis that could be used as a tool to identify potential anti-virulence products by comparing the slopes of the linearized diameters of the swarming halos of bacteria treated with the products, as they migrate on Petri dishes over a 24-hour incubation period. Here we report:•A simple method for detection and quantification of swarming motility halos of V. parahaemolyticus bacteria.•A method that could be used as a tool to identify potential anti-virulence products.

Genomic and Evolutionary Features of Nine AHPND Positive Vibrio parahaemolyticus Strains Isolated from South American Shrimp Farms.

Vibrio parahaemolyticus is a bacterial pathogen that becomes lethal to Penaeus shrimps when acquiring the pVA1-type plasmid carrying the PirABvp genes, causing acute hepatopancreatic necrosis disease (AHPND). This disease causes significant losses across the world, with outbreaks reported in Southeast Asia, Mexico, and South America. Virulence level and mortality differences have been reported in isolates from different locations, and whether this phenomenon is caused by plasmid-related elements or genomic-related elements from the bacteria remains unclear. Here, nine genomes of South American AHPND-causing V. parahaemolyticus (VPAHPND) isolates were assembled and analyzed using a comparative genomics approach at (i) whole-genome, (ii) secretion system, and (iii) plasmid level, and then included for a phylogenomic analysis with another 86 strains. Two main results were obtained from our analyses. First, all isolates contained pVA1-type plasmids harboring the toxin coding genes, and with high similarity with the prototypical sequence of Mexican-like origin, while phylogenomic analysis showed some level of heterogeneity with discrete clusters and wide diversity compared to other available genomes. Second, although a high genomic similarity was observed, variation in virulence genes and clusters was observed, which might be relevant in the expression of the disease. Overall, our results suggest that South American pathogenic isolates are derived from various genetic lineages which appear to have acquired the plasmid through horizontal gene transfer. Furthermore, pathogenicity seems to be a multifactorial trait where the degree of virulence could be altered by the presence or variations of several virulence factors. IMPORTANCE AHPND have caused losses of over $2.6 billion to the aquaculture industry around the world due to its high mortality rate in shrimp farming. The most common etiological agent is V. parahaemolyticus strains possessing the pVA1-type plasmid carrying the PirABvp toxin. Nevertheless, complete understanding of the role of genetic elements and their impact in the virulence of this pathogen remains unclear. In this work, we analyzed nine South American AHPND-causing V. parahaemolyticus isolates at a genomic level, and assessed their evolutionary relationship with other 86 strains. We found that all our isolates were highly similar and possessed the Mexican-type plasmid, but their genomic content did not cluster with other Mexican strains, but instead were spread across all isolates. These results suggest that South American VPAHPND have different genetic backgrounds, and probably proceed from diverse geographical locations, and acquire the pVA1-type plasmid via horizontal gene transfer at different times.

Microbial signature profiles of Penaeus vannamei larvae in low-survival hatchery tanks affected by vibriosis.

Vibriosis is caused by some pathogenic Vibrio and produces significant mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei larvae in commercial hatcheries. Acute hepatopancreatic necrosis disease (AHPND) is an emerging vibriosis affecting shrimp-producing countries worldwide. Zoea 2 syndrome is another type of vibriosis that affects the early stages of P. vannamei larvae. Although the pathogenesis of AHPND and zoea 2 syndrome is well known, there is scarce information about microbial composition and biomarkers of P.vannamei larvae affected by AHPND, and there is no study of the microbiome of larvae affected by zoea 2 syndrome. In this work, we characterized the microbiome of P. vannamei larvae collected from 12 commercial hatchery tanks by high-throughput sequencing. Seven tanks were affected by AHPND, and five tanks were affected by zoea 2 syndrome. Subsequently, all samples were selected for sequencing of the V3-V4 region of the16S rRNA gene. Similarity analysis using the beta diversity index revealed significant differences in the larval bacterial communities between disease conditions, particularly when Vibrio was analyzed. Linear discriminant analysis with effect size determined specific microbial signatures for AHPND and zoea 2 syndrome. Sneathiella, Cyclobacterium, Haliea, Lewinella, among other genera, were abundant in AHPND-affected larvae. Meanwhile, Vibrio, Spongiimonas, Meridianimaribacter, Tenacibaculum, among other genera, were significantly abundant in larvae affected by zoea 2 syndrome. The bacterial network at the phylum level for larvae collected from tanks affected by AHPND showed greater complexity and connectivity than in samples collected from tanks affected by zoea 2 syndrome. The bacterial connections inter Vibrio genera were higher in larvae from tanks affected by zoea 2 syndrome, also presenting other connections between the genera Vibrio and Catenococcus. The identification of specific biomarkers found in this study could be useful for understanding the microbial dynamics during different types of vibriosis.

Complete Coding Genome Sequence of Infectious Hypodermal and Hematopoietic Necrosis Virus Isolated from Penaeus (Litopenaeus) vannamei Shrimp in Ecuador.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV), recognized as Penaeus stylirostris penstyldensovirus 1 (PstDV1), has been associated with the runt-deformity syndrome (RDS) in cultured Penaeus (Litopenaeus) vannamei and Penaeus monodon shrimp. Here, we report the second published genome sequence of IHHNV, which was isolated from P. vannamei shrimp cultured in Ecuador.

Microbiome of Penaeus vannamei Larvae and Potential Biomarkers Associated With High and Low Survival in Shrimp Hatchery Tanks Affected by Acute Hepatopancreatic Necrosis Disease.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging bacterial disease of cultured shrimp caused mainly by Vibrio parahaemolyticus, which harbors the lethal PirAB toxin genes. Although Penaeus vannamei (P. vannamei) postlarvae are susceptible to AHPND, the changes in the bacterial communities through the larval stages affected by the disease are unknown. We characterized, through high-throughput sequencing, the microbiome of P. vannamei larvae infected with AHPND-causing bacteria through the larval stages and compared the microbiome of larvae collected from high- and low-survival tanks. A total of 64 tanks from a commercial hatchery were sampled at mysis 3, postlarvae 4, postlarvae 7, and postlarvae 10 stages. PirAB toxin genes were detected by PCR and confirmed by histopathology analysis in 58 tanks. Seven from the 58 AHPND-positive tanks exhibited a survival rate higher than 60% at harvest, despite the AHPND affectation, being selected for further analysis, whereas 51 tanks exhibited survival rates lower than 60%. A random sample of 7 out of these 51 AHPND-positive tanks was also selected. Samples collected from the selected tanks were processed for the microbiome analysis. The V3-V4 hypervariable regions of the 16S ribosomal RNA (rRNA) gene of the samples collected from both the groups were sequenced. The Shannon diversity index was significantly lower at the low-survival tanks. The microbiomes were significantly different between high- and low-survival tanks at M3, PL4, PL7, but not at PL10. Differential abundance analysis determined that biomarkers associated with high and low survival in shrimp hatchery tanks affected with AHPND. The genera Bacillus, Vibrio, Yangia, Roseobacter, Tenacibaculum, Bdellovibrio, Mameliella, and Cognatishimia, among others, were enriched in the high-survival tanks. On the other hand, Gilvibacter, Marinibacterium, Spongiimonas, Catenococcus, and Sneathiella, among others, were enriched in the low-survival tanks. The results can be used to develop applications to prevent losses in shrimp hatchery tanks affected by AHPND.

Microbial community characterization of shrimp survivors to AHPND challenge test treated with an effective shrimp probiotic (Vibrio diabolicus).

BACKGROUND: Acute hepatopancreatic necrosis disease (AHPND) is an important shrimp bacterial disease caused by some Vibrio species. The severity of the impact of this disease on aquaculture worldwide has made it necessary to develop alternatives to prophylactic antibiotics use, such as the application of probiotics. To assess the potential to use probiotics in order to limit the detrimental effects of AHNPD, we evaluated the effect of the ILI strain, a Vibrio sp. bacterium and efficient shrimp probiotic, using metabarcoding (16S rRNA gene) on the gastrointestinal microbiota of shrimp after being challenged with AHPND-causing V. parahaemolyticus. RESULTS: We showed how the gastrointestinal microbiome of shrimp varied between healthy and infected organisms. Nevertheless, a challenge of working with AHPND-causing Vibrio pathogens and Vibrio-related bacteria as probiotics is the potential risk of the probiotic strain becoming pathogenic. Consequently, we evaluated whether ILI strain can acquire the plasmid pV-AHPND via horizontal transfer and further cause the disease in shrimp. Conjugation assays were performed resulting in a high frequency (70%) of colonies harboring the pv-AHPND. However, no shrimp mortality was observed when transconjugant colonies of the ILI strain were used in a challenge test using healthy shrimp. We sequenced the genome of the ILI strain and performed comparative genomics analyses using AHPND and non-AHPND Vibrio isolates. Using available phylogenetic and phylogenomics analyses, we reclassified the ILI strain as Vibrio diabolicus. In summary, this work represents an effort to study the role that probiotics play in the normal gastrointestinal shrimp microbiome and in AHPND-infected shrimp, showing that the ILI probiotic was able to control pathogenic bacterial populations in the host's gastrointestinal tract and stimulate the shrimp's survival. The identification of probiotic bacterial species that are effective in the host's colonization is important to promote animal health and prevent disease. CONCLUSIONS: This study describes probiotic bacteria capable of controlling pathogenic populations of bacteria in the shrimp gastrointestinal tract. Our work provides new insights into the complex dynamics between shrimp and the changes in the microbiota. It also addresses the practical application of probiotics to solve problems with pathogens that cause high mortality-rate in shrimp farming around the world. Video Abstract.

An effective white spot syndrome virus challenge test for cultured shrimp using different biomass of the infected papilla.

White spot syndrome virus (WSSV) is one of the most virulent pathogens of cultured penaeid shrimp. Several control strategies are used commonly to mitigate the economic losses caused by the pathogen, such as application of antiviral products at farm level. One of the most practical method for the screening of potential anti-WSSV products is through challenge tests. Therefore, it is essential to develop simple, reproducible and effective bioassays able to simulate specific mortality levels. The purpose of this study was to develop a simple and reproducible bioassay that simulate different mortality levels by varying the proportion of WSSV-infected and noninfected shrimp tissues administered to susceptible shrimp during a per os challenge test. This method mimics one of the natural transmission routes of WSSV infection in shrimp and could be applied to identify potential antiviral products to different cultured shrimp species susceptible to WSSV. Here we report: •A simple and economic method to evaluate therapeutic antiviral products against WSSV through a challenge test, that uses different biomass amounts of WSSV-infected papilla.•Allows to simulate a wide and reproducible range of mortalities observed in shrimp farms.•A challenge test that simulates one mode of natural WSSV infection in shrimp.

Efficacy assessment of commercially available natural products and antibiotics, commonly used for mitigation of pathogenic Vibrio outbreaks in Ecuadorian Penaeus (Litopenaeus) vannamei hatcheries.

Bacterial diseases cause high mortality in Penaeus (Litopenaeus) vannamei postlarvae. Therefore, appropriate application of efficient therapeutic products is of vital importance for disease control. This study evaluated through in vitro analyses the antimicrobial effectiveness of commercial therapeutic products used for P. vannamei bacterial diseases and antibiotics against pathogenic Vibrio strains circulating in Ecuadorian hatcheries. Twenty strains were isolated from 31 larvae samples with high bacterial counts from 10 hatcheries collected during mortality events. The strains virulence was verified through challenge tests with Artemia franciscana nauplii and P. vannamei postlarvae. Through 16S rRNA sequence analysis, strains showed a great similarity to the Vibrio sequences reported as pathogens, with 95% belonging to the Harveyi clade. Through antibiograms and minimal inhibitory concentration (MIC) in vitro tests we found that furazolidone, ciprofloxacin, chloramphenicol, norfloxacin, nalidixic acid, florfenicol, fosfomycin and enrofloxacin inhibited the growth of all or most of the strains. Less efficient antibiotics were penicillin, oxytetracycline and tetracycline. A multiple antibiotic resistance (MAR) index of 0.23 showed some level of resistance to antibiotics, with two MAR prevalent patterns (Penicillin-Oxytetracycline and Penicillin-Oxytetracycline-Tetracycline). From a total of 16 natural products (five probiotics, nine organic acids and two essential oils), only three (one probiotic, one organic acid and one essential oil) were effective to control most of the strains. Shrimp producers can apply relatively simple in vitro analyses, such as those employed in this study, to help take adequate management decisions to reduce the impact of bacterial diseases and increase profit.

Draft Genome Sequence of a White Spot Syndrome Virus Isolate Obtained in Ecuador.

White spot syndrome virus (WSSV) is the most devastating viral disease affecting cultivated shrimp around the world. Currently, there is no reported genetic information on WSSV affecting Penaeus vannamei in Ecuador. Therefore, we determined the genome of a WSSV isolate from shrimp and compared it with the genomes of isolates from other geographical locations.

PirVP genes causing AHPND identified in a new Vibrio species (Vibrio punensis) within the commensal Orientalis clade.

Acute hepatopancreatic necrosis disease (AHPND) has extended rapidly, causing alarming shrimp mortalities. Initially, the only known causative agent was Vibrio parahaemolyticus carrying a plasmid coding for the mortal toxins PirVP. Recently, it has been found that the plasmid and hence the disease, could be transferred among members of the Harveyi clade. The current study performs a genomic characterization of an isolate capable of developing AHPND in shrimp. Mortality studies and molecular and histopathological analyses showed the infection capacity of the strain. Multilocus sequence analysis placed the bacteria as a member of the Orientalis clade, well known for containing commensal and even probiotic bacteria used in the shrimp industry. Further whole genome comparative analyses, including Vibrio species from the Orientalis clade, and phylogenomic metrics (TETRA, ANI and DDH) showed that the isolate belongs to a previously unidentified species, now named Vibrio punensis sp. nov. strain BA55. Our findings show that the gene transfer capacity of Vibrio species goes beyond the clade classification, demonstrating a new pathogenic capacity to a previously known commensal clade. The presence of these genes in a different Vibrio clade may contribute to the knowledge of the Vibrio pathogenesis and has major implications for the spread of emerging diseases.

Draft genome sequence of pathogenic bacteria Vibrio parahaemolyticus strain Ba94C2, associated with acute hepatopancreatic necrosis disease isolate from South America.

Vibrio parahaemolyticus is a pathogenic bacteria which has been associated to the early mortality syndrome (EMS) also known as hepatopancreatic necrosis disease (AHPND) causing high mortality in shrimp farms. Pathogenic strains contain two homologous genes related to insecticidal toxin genes, PirA and PirB, these toxin genes are located on a plasmid contained within the bacteria. Genomic sequences have allowed the finding of two strains with a divergent structure related to the geographic region from where they were found. The isolates from the geographic collection of Southeast Asia and Mexico show variable regions on the plasmid genome, indicating that even though they are not alike they still conserve the toxin genes. In this paper, we report for the first time, a pathogenic V. parahaemolyticus strain in shrimp from South America that showed symptoms of AHPND. The genomic analysis revealed that this strain of V. parahaemolyticus found in South America appears to be more related to the Southeast Asia as compared to the Mexican strains. This finding is of major importance for the shrimp industry, especially in regards to the urgent need for disease control strategies to avoid large EMS outbreaks and economic loss, and to determine its dispersion in South America. The whole-genome shotgun project of V. parahaemolyticus strain Ba94C2 have been deposited at DDBJ/EMBL/GenBank under the accession PRJNA335761.