Shrimp Aquaculture Research Articles

CRISPR-based, genotype-specific detection of yellow head virus genotype 1 with fluorescent, lateral flow and DNAzyme-assisted colorimetric readouts

The yellow head virus complex comprises 8 genotypes of single-stranded RNA viruses infecting penaeid shrimp, but only yellow head virus genotype 1 (YHV1) is capable of causing massive shrimp mortalities and severe economic losses. However, current molecular techniques could not detect YHV1 with high specificity, or require complicated protocols for this purpose. Here, we developed a combination of reverse-transcriptase recombinase polymerase amplification (RT-RPA) and CRISPR-Cas detection (“RR-Cas”) for the genotype-specific detection of YHV1. The method can be completed within 1.5 h at a constant temperature near 37 °C, after which the results can be visualized by the naked eye using fluorescent, lateral flow, and G-quadruplex DNAzyme-assisted colorimetric assays. The detection limit is 100 copies of plasmid DNA and 100 fg of total RNA from YHV1 infected shrimp, comparable to the WOAH-recommended one-step RT-PCR protocol. In the evaluation of clinical samples, RR-Cas was found to be less sensitive than nested RT-PCR, but displayed superior specificity, yielding no false positive results towards samples containing YHV2 and 3. Overall, our findings suggest that RR-Cas is a powerful addition to the toolkit for genotype-specific viral diagnosis in shrimp aquaculture, with the potential to greatly streamline disease surveillance and management in the shrimp industry.

Microbial biomarker detection in shrimp larvae rearing water as putative bio-surveillance proxies in shrimp aquaculture.

Aquacultured animals are reared in water hosting various microorganisms with which they are in close relationships during their whole lifecycle as some of these microorganisms can be involved in their host's health or physiology. In aquaculture hatcheries, understanding the interactions existing between the natural seawater microbiota, the rearing water microbiota, the larval stage and the larval health status, may allow the establishment of microbial proxies to monitor the rearing ecosystems. Indeed, these proxies could help to define the optimal microbiota for shrimp larval development and could ultimately help microbial management. In this context, we monitored the daily composition of the active microbiota of the rearing water in a hatchery of the Pacific blue shrimp Penaeus stylirostris. Two distinct rearing conditions were analyzed; one with antibiotics added to the rearing water and one without antibiotics. During this rearing, healthy larvae with a high survival rate and unhealthy larvae with a high mortality rate were observed. Using HiSeq sequencing of the V4 region of the 16S rRNA gene of the water microbiota, coupled with zootechnical and statistical analysis, we aimed to distinguish the microbial taxa related to high mortality rates at a given larval stage. We highlight that the active microbiota of the rearing water is highly dynamic whatever the larval survival rate. A clear distinction of the microbial composition is shown between the water harboring heathy larvae reared with antibiotics versus the unhealthy larvae reared without antibiotics. However, it is hard to untangle the effects of the antibiotic addition and of the larval death on the active microbiota of the rearing water. Various active taxa of the rearing water are specific to a given larval stage and survival rate except for the zoea with a good survival rate. Comparing these communities to those of the lagoon, it appears that many taxa were originally detected in the natural seawater. This highlights the great importance of the microbial composition of the lagoon on the rearing water microbiota. Considering the larval stage and larval survival we highlight that several genera: Nautella, Leisingera, Ruegerira, Alconivorax, Marinobacter and Tenacibaculum, could be beneficial for the larval survival and may, in the rearing water, overcome the r-strategist microorganisms and/or putative pathogens. Members of these genera might also act as probiotics for the larvae. Marivita, Aestuariicocccus, HIMB11 and Nioella, appeared to be unfavorable for the larval survival and could be associated with upcoming and occurring larval mortalities. All these specific biomarkers of healthy or unhealthy larvae, could be used as early routine detection proxies in the natural seawater and then during the first days of larval rearing, and might help to manage the rearing water microbiota and to select beneficial microorganisms for the larvae.

Protein-protein interaction network analysis on the whiteleg shrimp Penaeus vannamei and Vibrio parahaemolyticus host-pathogen relationship reveals possible proteins and pathways involved during infection

Whiteleg shrimp, Penaeus vannamei, is the prime candidate in shrimp aquaculture that significantly contributes to the economic growth of the global aquaculture sector. However, P. vannamei culture is constantly plagued with pathogenic infection of Vibrio spp., including Vibrio parahaemolyticus, which impairs shrimp production and consequently results in significant financial losses. Despite this persistent problem, the relationship between P. vannamei and V. parahaemolyticus remains unclear. The discovery of protein-protein interactions (PPIs) between host and pathogen might improve the understanding of bacterial infection in shrimp since most pathogenic bacteria infect hosts through multiple mechanisms. Hence, this study aims to identify the candidate effector proteins of V. parahaemolyticus and their targeted host proteins and potential pathways involved in its infection using a host-pathogen PPI approach. The P. vannamei – V. parahaemolyticus PPI (PVPPI) network was constructed using in silico methods, followed by clustering and pathway enrichment analyses. The constructed PVPPI network consisted of 448871 interactions between 4427 P. vannamei and 18119 V. parahaemolyticus proteins. Clustering analysis identified several effector proteins, i.e., secretion systems proteins, and their targeted proteins, including cell signalling proteins. The bacterial secretion system, mTOR signalling, and endocytosis were among the enriched pathways that might be involved during V. parahaemolyticus infection in P. vannamei. This study reports the first host-pathogen PPI network between P. vannamei and V. parahaemolyticus, where several hosts and pathogen proteins with potential pathways are highlighted. These findings offer new insights into the interaction between V. parahaemolyticus and P. vannamei, a vital aspect that could serve as a baseline for future disease prevention and treatment in the shrimp aquaculture industry.

Assessment of trace element content throughout the white shrimp (Litopenaeus vannamei) farming cycle

Environmental context Intensive aquaculture is the main source of fisheries products. Thus, investigating the food safety of these products and the environmental impacts of the farms on mangroves is necessary. A shrimp productive cycle was evaluated with a focus on trace element accumulation in shrimps and effluent. The results revealed secure levels of elements in the final product; however, the effluent produced in farms is an important source of contamination to mangrove environment. Rationale Aquaculture systems have increased in the last years due to the high demand for seafood consumption, this could impact the environment and subject fisheries to accumulation of toxic elements. To understand some parameters of food safety and environmental impact, the present study evaluated the concentration of trace elements (Al, As, Cd, Co, Cr, Cu, Mn, Mo, Pb, Se, V and Zn) throughout the production cycle of shrimp. Methodology About 50 shrimps per cycle were collected in a shrimp farm in Brazil and their trace element contents were determined by inductively coupled plasma–tandem mass spectrometry (ICP-MS/MS) and microwave-induced plasma–optical emission spectroscopy (MIP-OES). Results At their final lifecycle stage, shrimp samples present a content (mg g−1) of elements following the trend: Cu (102 ± 12) > Al (20.06 ± 4.24) > Zn (14.82 ± 2.46) > Mn (6.24 ± 0.94) > As (2.65 ± 0.42) > Se (0.932 ± 0.140) > Co (0.380 ± 0.05) > Mo (0.254 ± 0.03) > V (0.204 ± 0.02). Discussion The content of Cd and Pb are in allowance with Brazil and USA legislation for crustaceans, however, the content of As is 3–4-fold higher than that allowed by guidelines in all stages of the growth of the shrimp. The final effluent of the shrimp’s pond into the mangroves shows a high mass fraction of Zn and Mn that could be a source of contamination. Some correlations between some elements in the shrimp samples were found, such as As–Se, Se–Co and Se–V. This study was a scoping experiment to study the content of trace elements throughout the farming cycle of shrimps, encouraging the researcher to undergo a wide survey to evaluate the environmental impact of aquaculture shrimp farming.

The abundance of Microcystis sp. on intensive shrimp ponds

Microcytis sp is plankton species that is detrimental in shrimp pond ecosystems. The purpose of this study was to detect the abundance of plankton and the presence of Microcystis sp. in the waters of vaname shrimp (L. vannamei) aquaculture ponds. The research method used is descriptive quantitative and qualitative with added analysis of dynamic modeling systems. The results showed that pond water quality conditions were still good enough for shrimp farming activities with details of dissolved oxygen 4.01-6.17 mg/L, pH 7.7-7.9, salinity 20‰, temperature 27.050-28.250C, nitrate 0.01-0.15 mg/L, phosphate 0.05-0.20 mg/L, brightness 25-35 cm. For the classification of plankton found among them are from the Bacillariophyceae class, there are the genera Skeletonema sp., Cyclotella sp., Coscinodiscus sp., and Amphipora sp., from the Chlorophyceae class, there are the genera Chlorella sp., Oocystis sp., and Clamydomonas sp., from the Cyanophyceae class. the genus Oscillatoria sp., Microcystis sp., and Anabaenopsis sp. The plankton diversity index (H') was obtained between 0.01-0.33 and the uniformity index (E) was obtained between 0.10-0.50, meaning that the condition of the pond waters is still quite good and there are minimal indications of pollution. The abundance of Microcystis sp. obtained between 0.2x104-0.6x104 cell/ml. from the results of dynamic modeling analysis it is shown that the abundance of Microcystis sp. in pond waters is influenced by the value of the productivity level of the waters and the process of grazing organisms. The results of this study can be concluded that the plankton abundance rate found in ponds ranges from 0.4x104-1.9x104 cell/ml which consists of 10 genera and 3 classes of plankton. Then Microcystis sp. its presence was found in 3 ponds with abundance rate ranging from 0.2x104-0.6x104 cell/ml.Keywords:CultivationGenusGrazingModelWaters

Comparative life cycle assessment of whiteleg shrimp (Penaeus vannamei) cultured in recirculating aquaculture systems (RAS), biofloc technology (BFT) and higher-place ponds (HPP) farming systems in China

Recirculating aquaculture systems (RAS), biofloc technology (BFT), and higher-place ponds (HPP) are considered as alternative technologies in addressing major environmental challenges linked to conventional Whiteleg shrimp farming systems. Global concern about energy consumption and carbon emission in aquaculture industries, especially in countries like China. The application of life cycle assessment (LCA) in shrimp aquaculture, especially in comparing different shrimp production systems, is being used. In our study, surveys on three RAS, three BFT and nine HPP farms have been conducted, and the data inputs were used to establish a cradle to farm-gate LCA by means of software SimaPro 9.3.0.2 and CML-IA methods to evaluate the environmental performance. The methods are well suited to this LCA, as it focuses mainly on energy consumption input, pollution output, and ecological damage. A ton of live weight shrimp production did generate 4424.2, 4657.2, and 4965.6 kg CO2 eq; 21.8, 25.9, 36.2 kg PO4 eq; 38.7, 40.6 and 43.5 kg SO2 eq in RAS, BFT and HPP systems, respectively. Regarding the characterization and normalization results, eleven categories (abiotic depletion, acidification, eutrophication, etc.) were considered. In most impact categories, the results showed that HPP had the highest environmental impact per unit production when compared to RAS and BFT. Acidification potential (AP) and eutrophication potential (EP) were the top two contributing factors to RAS, BFT and HPP. And AP and EP is closely related to the production of electricity. Electricity was identified as primary contributors in all farming processes. Results indicate that BFT and RAS could be used in combination to reduce environmental impacts. In order to improve the environmental performance of these farming systems, feed ingredients substitution; feeding management and effluent treatment are among the suggested measures. In order to enhanced aquaculture LCI databases and support aquaculture LCA research, the life cycle inventory for shrimp farming were established.

The IUCN Global Standard for Nature-based Solutions™ as a tool for enhancing the sustainable development of marine aquaculture

This paper applies the IUCN Global Standard for Nature based Solutions™ self-assessment tool (published in 2020) to two aquaculture case studies. Data from the case studies were compiled by the authors. In Zanzibar, secondary data were obtained through a previous project, which included a stakeholder workshop in Zanzibar (in 2019) and one deliverable published by the IUCN on Zanzibar of their catalogue “Aquaculture and Marine Conservation”. In Indonesia, the original data were provided by the Blue Natural Capital Funding Facility (BNCFF) and the associated local teams. The analysis of the data, the information provided, and the scoring itself were done by the authors, in association with local teams in both areas. The results of the two assessments, discussed in the paper and presented in detail in the Supplementary materials, can be considered original research, never previously published in a scientific journal. The concept of Nature-based Solutions (NbS) was proposed by the International Union for Conservation of Nature (IUCN) to protect, restore, and sustainably manage natural and modified ecosystems for achieving a variety of societal benefits. The IUCN released the IUCN Global Standard for NbS™ to help design, assess, strengthen, and upscale NbS interventions. In the current context of growing uncertainties for the future of coastlines and oceans, aquaculture has been recognized as a positive activity for achieving sustainable development in coastal communities; supporting food security, poverty alleviation, and economic resilience; and contributing to the conservation of marine ecosystems in some cases. However, the sustainability of aquaculture systems has often been criticized. Aquaculture initiatives in coastal areas can achieve both nature conservation and sustainable development objectives, but reflection on the conditions under which this would happen is needed. This article examines aquaculture systems through the lens of the NbS concept and the IUCN Global Standard for NbS™, along with other sustainability concepts and instruments currently used in the context of aquaculture. The application of the IUCN Global Standard for NbS™’s to two case studies is explored: seaweed farming in Zanzibar in marine conservation areas and shrimp farming coupled with mangrove restoration in Indonesia. The results show that the NbS concept underpinning the IUCN Global Standard for NbS™ could help in the overall assessment of aquaculture systems and improve their sustainability by highlighting both their positive outcomes and issues requiring further examination in relation to marine biodiversity benefits, socio-economic development, and/or governance. The IUCN Global Standard for NbS™ could provide an operational framework to implement existing concepts, such as the Ecosystem Approach to Aquaculture, contribute to clarifying critical issues in aquaculture development, and provide guidance for the development of a new type of aquaculture project, specifically designed as NbS. This finding advocates the context-dependent exploration and promotion of aquaculture projects as NbS.

Evaluation of a synbiotic formulation for water remediation in a shrimp pond.

In recent years, the use of probiotic bacteria has attracted the interest of the marine shrimp farming industry. However, there are certain limitations pertaining to the practical application of many commercially available probiotics. Here, a thoroughly screened optimal consortium of three indigenous sulfur probiotics was tested for antibiotic susceptibility and was found to be safe, with each culture being sensitive to all the tested antibiotics. Further, de-potash vinasse (DPV), an environmental hazard, was tested for its prebiotic potential, and its 1% (w/v) concentration was found to be effective for long-term viability (> 66days) of the probiotic cultures and safe for Artemia. The synbiotic formulation was tested first in a lab-scale microcosm setup successfully and subsequently tried on a shrimp farm; it was observed that the product was congruent to the efficiency of a commercial probiotic regarding almost all physicochemical parameters, sulfide, nitrate-N, nitrite-N, phytoplankton sustenance, Pseudomonas count, coliform count, and heterotrophic count. In addition, it was significantly efficient in maintaining pH, reducing ammonia-N and phosphate-P, Vibrio and Aeromonas count, and a net increase in the yield of shrimp biomass by 625kg, thus proving to be a better alternative than one of the already available remediation methods.

Information practices for improved sustainability assurance in Vietnamese shrimp aquaculture

This paper explores the performance and embeddedness of information practices performed by smallholder farmers in Vietnamese shrimp aquaculture in response to private sustainability interventions. Using a social practice intervention framework, we analyse how existing information practices embedded in the wider social lives of farmers are affected by the adoption of both analogue and digital reporting methodologies prescribed by the Aquaculture Stewardship Council and VerifiK8. Our results demonstrate how these reporting methodologies are not aligned to the performance and embeddedness of existing information practices, and how this misalignment has undermined their function and performance. The paper concludes that understanding information practices offers important insights for redesigning these reporting interventions and, in doing so, better support both the goals of farmers and the improvement of shrimp sustainability. These observations hold implications for improving the impact of informational interventions for not only shrimp production but smallholder production in a range of food sectors.

Nutrient consumption of green microalgae, Chlorella sp. during the bioremediation of shrimp aquaculture wastewater

Aquaculture products are among the biggest contributor to food supplies to meet the global food demands of the growing population over these past few years. For aquaculture to continue developing, an effective wastewater treatment is required to lessen the environmental effects. This study examined the potential of Chlorella sp. to reduce nutrients in shrimp aquaculture wastewater and correlate with the growth kinetics of the algae during the bioremediation process. Six different Chlorella sp. inoculation dosages ranging from 0 to 60 % (v/v) were used in this study. Marine water wastewater (MW) and Freshwater wastewater (FW) where the two types of shrimp wastewater were employed. Results indicated that the 30 % (v/v) and 40 % (v/v) were the optimum dosage for MW and FW. During the treatment, microalgae cell density increased more than tenfold compared to the initial value. Moreover, batch culture resulted in the specific growth rate concentration of 0.18 k day−1 and 0.15 k day−1, respectively. Those dosage also resulting the highest removal efficiencies with removal of ammonia, nitrite and orthophosphate of 96.77 %, 82.07 %, 75.96 % and 90.10 %, 87.09 %, 95.60 %, respectively. The application of FTIR spectroscopy was employed in this study to analyze the functional group in the microalgae biomass. The results of the scanning electron microscopy (SEM) and Energy Dispersive Spectroscopy Analysis (EDS) also included to further illustrate how microalgae biomass was affected by the treatment in this study. Therefore, the research from this study could be used in design novel microalgae treatments that offer a thorough and environmentally beneficial method of treating shrimp aquaculture wastewater.

Exploring the Effect of Functional Diets Containing Phytobiotic Compounds in Whiteleg Shrimp Health: Resistance to Acute Hepatopancreatic Necrotic Disease Caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis (AHPND) is an emerging severe disease caused by strains of Vibrio parahaemolyticus (VpAHPND) in whiteleg shrimp (Litopenaeus vannamei). Mitigating its negative impact, and at the same time minimizing antibiotics treatments, is the major challenge in shrimp aquaculture. A sustainable strategy could be to include immunostimulants in diet. Phytobiotics, harmless plant extracts with immunostimulatory and biocidal activities, are promising candidates. In this study, we evaluated the effectiveness of two diets (E and F) supplemented with phytobiotics (functional diets) in terms of protecting shrimp against AHPND. For this purpose, groups of animals were fed functional or control diets for 4 and 5 weeks and, subsequently, they were challenged with VpAHPND by immersion. We compared the mortality in infected groups and estimated the percentage of carriers by using a specific qPCR in hepatopancreas tissue. The results showed that mortality was significantly lower in the group fed functional diet E and, after a 5-week feeding schedule. This group also showed the lowest percentage of carriers. The pathological effects were also reduced with diet F. Thus, feeding shrimp with phytobiotic-enriched diets in critical periods will be highly beneficial because it increases the host's resistance to AHPND pathology.

The political ecology of shrimp aquaculture in Tamil Nadu: A case study from Mayiladuthurai District

The primary purpose of this article is to analyze the impact of shrimp aquaculture in Tamil Nadu through a political ecology lens with a case study of a coastal village located in the Mayiladuthurai district of Tamil Nadu, India. The article firstly looks at the shrimp industry in the coastal village as a case that illustrates how global capitalism influence the local economy. Secondly, it identifies ecological degradation due to the commodification of nature and analyzes the social origins of degradation. Commodification alienates nature and people from each other, and its impacts are unevenly felt across social strata especially class, gender and caste lines. The article finally discusses the intersectionality of social difference and differentiates how the impacts of commodification are experienced and responded to.

An Indian Perspective on the Infection and Diagnostic Landscape of Shrimp Aquaculture

An Indian Perspective on the Infection and Diagnostic Landscape of Shrimp Aquaculture

Disinfection of Perinereis aibuhitensis eggs with peroxymonosulfate to eliminate covert mortality nodavirus (CMNV)

Perinereis aibuhitensis is an essential bait for prompting ovarian development and sexual maturation in aquaculture shrimp broodstock. It is common for shrimp to become infected with pathogens by ingesting feed containing potential viruses. To prevent P. aibuhitensis being live feeds for shrimp broodstock from transmitting pathogens, we evaluated the disinfection effect of peroxymonosulfate (PMS) against covert mortality nodavirus (CMNV) by carrying out Oryzias melastigma (marine medaka) challenge assay and testing CMNV-exposed P. aibuhitensis eggs. In the marine medaka injected with PMS (15 and 20 ppm (ppm))-inactivated CMNV, no CMNV was detected at 6 and 12 days post-infection (d.p.i.) by using quantitative reverse transcription PCR (RT-qPCR). As well, no CMNV probe positive signal and pathological signs caused by CMNV in these marine medaka were present in situ hybridization (ISH) and histopathology assays. CMNV was not detected in the nectochaeta larvae originated from CMNV-exposed P. aibuhitensis eggs treated with PMS (5, 10, 15 and 20 ppm) for 1 min. In addition, nereids treated with 15 ppm PMS for 1 min had higher hatchability. Even at low concentrations of PMS-treated eggs, the hatchability was higher than that of the no-PMS-treated group, and no adverse effects on larval development after hatching were observed. Our study showed that disinfection of CMNV-exposed eggs by PMS is feasible. Disinfection of P. aibuhitensis eggs with PMS could reduce the risk of transmission of CMNV. Within acceptable limits for hatchery spawn losses, the recommended disinfection dose of PMS is 15 ppm for 1 min. This study laid the foundation for the culture of specific pathogen-free P. aibuhitensis and to provide biological baits with no pathogenic risk to farmed shrimp.

WSSV exploits AMPK to activate mTORC2 signaling for proliferation by enhancing aerobic glycolysis

AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture.

Assessing pathways of heavy metal accumulation in aquaculture shrimp and their introductions into the pond environment based on a dynamic model and mass balance principle

The impact of heavy metals (HMs) on the quality of aquaculture products has attracted worldwide attention. Since Litopenaeus vannamei is a popular aquaculture product among consumers worldwide, it is of great importance to guarantee its dietary safety. An in-situ monitoring program lasting for three months in a typical Litopenaeus vannamei farm found that Pb (100 %) and Cr (86 %) in the adult shrimp were higher than the safety guidelines. In the meantime, Cu (100 %), Cd (100 %) in the water and Cr (40 %) in the feed exceeded the corresponding thresholds. Therefore, quantification of different exposure pathways of shrimp and contamination origins in pond is valuable to improve the dietary safety of the shrimp. Based on Optimal Modeling for Ecotoxicological Applications (OMEGA), Cu was primarily from the ingestion of feed, accounting for 67 % of bioaccumulation, while Cd, Pb and Cr primarily entered shrimp through the adsorption from overlying water (53 % for Cd and 78 % for Pb) and porewater (66 % for Cr), respectively. The HMs in the pond water were further tracked based on a mass balance analysis. The main source of Cu in the aquaculture environment was feed, being responsible for 37 % of the total input. Pb, Cd and Cr were primarily from the inlet water with contributions of 84 %, 54 % and 52 %, respectively. In summary, the proportions of different exposure pathways and origins of HMs in pond-cultured shrimp and its living environment varied widely. To keep end-consumers eating healthily, species specific treatment is required. Feed should be regulated more for Cu. Aimed pretreatments for Pb and Cd in influent water are needed and an additional immobilization for Cr in sediment porewater should be investigated. After implementation of these treatments, the food quality improvement could be further quantified based on our prediction model.

Decreased Severity of Acute Hepatopancreatic Necrosis Disease in White Shrimp (Litopenaeus vannamei) by Mixed Culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.

The objective of this study was to investigate the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium to control acute hepatopancreatic necrosis disease (AHPND) or EMS (Early Mortality Syndrome) in white shrimp Litopenaeus vannamei as a model. The infected shrimps with Vibrio parahaemolyticus AHPND strain were divided into tanks and different feeding of either B. subtilis, B. licheniformis, B. megaterium or all Bacillus strains. The infected shrimps that were fed with a mixed culture of Bacillus showed significantly highest survival rate and revealed lower percent detection of V. parahaemolyticus AHPND strain by Polymerase Chain Reaction (PCR) (57.14%) with a small amount of viability count in their hepatopancreas. In contrast, the infected shrimps that were fed with each of B. subtilis, B. licheniformis or B. megaterium, revealed the spread of V. parahaemolyticus AHPND strain in all tissue by PCR detection (86.67%-100%) with a large amount of viability count (3.53 - 4.24 × 103 CFU/g). This study indicated that the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium could control the dissemination of V. parahaemolyticus in shrimps, especially in hepatopancreatic that is the target tissue of AHPND in white shrimp (L. vannamei). The result of this study revealed the efficiency and mechanism of the mixed culture of B. subtilis, B. licheniformis and B. megaterium to control the virulence of AHPND and support the application of this mixed culture in aquaculture of shrimp farms to avoid chemical and antibiotic treatment by using it as a biological control.

In Silico and In Vitro Investigation of Phytochemicals Against Shrimp AHPND Syndrome Causing PirA/B Toxins of Vibrio parahaemolyticus.

In Southeast Asia, the penaeid shrimp aquaculture production faces a new pandemic bacterial disease called acute hepatopancreatic necrosis disease (AHPND). The highly profitable pacific white shrimp, Litopenaeus vannamei, has become a challenging species due to severe lethal infections. Recent research has identified a critical pathogen, Vibrio parahaemolyticus, which caused significant loss in the shrimp industry. The disease pathway involves a virulence plasmid encoding binary protein toxins (PirA/B) that cause cell death. The protein toxins were inherited and conjugatively transferred to other Vibrio species through a post-segregational killing system. In this study, "in silico" (Glide, 2021) analysis identified four phytocompounds as myricetin (Myr), ( +)-taxifolin (TF), (-)-epigallocatechin gallate (EGCG), and strychnine (STN) which could be most effective against both the toxins concerning its docking score and affinity. The interactions of complexes and the critical amino acids involved in docking were analyzed using the Discovery Studio (version 2016). Molecular dynamic studies showed lower root mean square deviations (RMSD) and improved stabilization of ( +)-taxifolin (TF) and (-)-epigallocatechin-3-gallate (EGCG) against both the protein toxins. The antibacterial potential of all four selected compounds had tested against pathogenic strains of V. parahaemolyticus through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The best MBC results were observed at concentrations of 1mg/mL for EGCG and 1.25mg/mL for TF. Moreover, the complete reduction of viable cell counts in the in vitro bactericidal activity had recorded after 24h of incubation.

Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture.

Shrimp aquaculture, especially during the hatchery phase, is prone to economic losses due to infections caused by luminescent vibrios. In the wake of antimicrobial resistance (AMR) in bacteria and the food safety requirements of farmed shrimp, aqua culturists are seeking alternatives to antibiotics for shrimp health management, and bacteriophages are fast emerging as natural and bacteria-specific antimicrobial agents. This study analyzed the whole genome of vibriophage-ϕLV6 that showed lytic activity against six luminescent vibrios isolated from the larval tanks of P. vannamei shrimp hatcheries. The Vibriophage-ϕLV6 genome was 79,862 bp long with 48% G+C content and 107 ORFs that coded for 31 predicted protein functions, 75 hypothetical proteins, and a tRNA. Pertinently, the vibriophage-ϕLV6 genome harbored neither AMR determinants nor virulence genes, indicating its suitability for phage therapy. There is a paucity of whole genome-based information on vibriophages that lyse luminescent vibrios, and this study adds pertinent data to the database of V. harveyi infecting phage genomes and, to our knowledge, is the first vibriophage genome report from India. Transmission electron microscopy (TEM) of vibriophage-ϕLV6 revealed an icosahedral head (~73 nm) and a long, flexible tail (~191 nm) suggesting siphovirus morphology. The vibriophage-ϕLV6 phage at a multiplicity of infection (MOI) of 80 inhibited the growth of luminescent V. harveyi at 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% salt gradients. In vivo experiments conducted with post-larvae of shrimp showed that vibriophage-ϕLV6 reduced luminescent vibrio counts and post-larval mortalities in the phage-treated tank compared to the bacteria-challenged tank, suggesting the potentiality of vibriophage-ϕLV6 as a promising candidate in treating luminescent vibriosis in shrimp aquaculture. The vibriophage-ϕLV6 survived for 30 days in salt (NaCl) concentrations ranging from 5 ppt to 50 ppt and was stable at 4 °C for 12 months.

Toxicological mechanism of ammonia-N on haematopoiesis and apoptosis of haemocytes in Litopenaeus vannamei

Ammonia, as an important pollutant, contributed to the reduction of immunity, disruption of physiology in animals. RNA interference (RNAi) was performed to understand the function of astakine (AST) in haematopoiesis and apoptosis in Litopenaeus vannamei under ammonia-N exposure. Shrimps were exposed to 20 mg/L ammonia-N from 0 to 48 h with injection of 20 μg AST dsRNA. Further, shrimps were exposed to 0, 2, 10 and 20 mg/L ammonia-N also from 0 to 48 h. The results showed that the total haemocytes count (THC) decreased under ammonia-N stress and the knockdown of AST resulted in a further decrease of THC, suggesting that 1) the proliferation was decreased through the reduction of AST and Hedgehog, the differentiation was interfered by Wnt4, Wnt5 and Notch, and the migration was inhibited by the decrease of VEGF; 2) oxidative stress was induced under ammonia-N stress, leading to the increase of DNA damage with the up-regulated gene expression of death receptor, mitochondrial and endoplasmic reticulum stress pathways; 3) the changes of THC resulted from the decrease of proliferation, differentiation and migration of haematopoiesis cells and the increase of apoptosis of haemocytes. This study helps to deepen our understanding of risk management in shrimp aquaculture.