Shrimp Stomach Research Articles

Influence of different percentages of water reuse with bioflocs on the composition of the microbiota in the stomach of Penaeus vannamei shrimp

The objective of this study was to analyze the stomach composition of Penaeus vannamei shrimp in biofloc cultures with varying percentages of reused bioflocs to identify the optimal culture conditions based on microorganism content. Four treatments were employed: clear water (CW), 25% biofloc inoculum (BF 25%), 50% biofloc inoculum (BF 50%), and 100% biofloc inoculum (BF 100%). Bioflocs were reused from mature bioflocs of previous cultures in biofloc treatments. Shrimp performance was monitored weekly through biometrics, and stomach samples were collected at the experiment's onset and conclusion. The samples underwent fixation in 4% formaldehyde for subsequent counting and identification of predominant microorganism groups using an inverted microscope. Bacterial abundance was assessed by fixing, filtering, and staining samples with acridine orange, followed by quantification with an epifluorescence microscope. Water quality parameters remained satisfactory in all treatments. Shrimp stomachs in biofloc systems contained Oocystis sp., ciliates, flagellates, diatoms, free coccoids, free and attached filamentous, Bacillus spp., and Vibrio spp. CW treatment exhibited the highest Vibrio spp. concentration. Overall, reusing bioflocs in all volume percentages effectively maintained water quality and colonized the animal's microbiota, with a recommended minimum reuse percentage of 25% for satisfactory results.

Characteristics and functional analysis of a novel mannose receptor in Penaeus vannamei

The mannose receptor (MR) plays a key role in the innate immune system as a pattern recognition receptor. Here, a novel type of mannose receptor, named PvMR2, was identified from Penaeus vannamei (P. vannamei). The PvMR2 coding sequence (CDS) obtained was 988 base pairs in length, encoding a protein consisting of 328 amino acids. This protein includes a signal peptide and two classical C-type lectin domains (CTLD). Quantitative real-time PCR showed that PvMR2 was distributed in all detected tissues, with the highest levels in the intestines and stomach. Following a bacterial challenge with Vibrio anguillarum (V. anguillarum), PvMR2 showed significant up-regulation in both the intestines and stomach of shrimp. To validate the function of PvMR2, recombinant proteins were extracted and purified using a His-tag. The resulting rPvMR2 demonstrated binding capability with lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner, affirming its binding affinity. The purified rPvMR2 demonstrated calcium-independent binding activity towards both Gram-positive bacteria (V. anguilliarum and Vibrio parahaemolyticus) and Gram-negative bacteria (Escherichia coli and Aeromonas Veronii). Antibacterial assays confirmed that rPvMR2 inhibits bacterial growth. Intestinal adhesion and adhesion inhibition experiments confirmed that the rPvMR2 can be used to reduce the adhesion capacity of harmful bacteria in the gut. Phagocytosis experiments have shown that rPvMR2 promotes phagocytosis in hemocytes and protects the host from external infection. Treatment with recombinant PvMR2 significantly bolstered bacterial clearance within the hemolymph and markedly augmented shrimp survival post-infection with V. anguillarum. These results suggest that PvMR2 has agglutination, growth inhibition, adhesion inhibition, clearance promotion, and phagocytosis effects on harmful bacteria, and plays a crucial role in the antimicrobial immune response of P. vannamei.

Investigating the impact of chlorine dioxide in shrimp-rearing water on stomach microbiome, gill transcriptome, and infection-related mortality in shrimp.

This study aimed to assess the effects of chlorine dioxide (ClO2) in water on whiteleg shrimp Penaeus vannamei, evaluating its impact on stomach microbiota, gill transcriptome, and pathogens. ClO2 was added to the aquarium tanks containing the shrimp. The application of ClO2 to rearing water was lethal to shrimp at concentrations above 1.2 ppm. On the other hand, most of the shrimp survived at 1.0 ppm of ClO2. Microbiome analysis showed that ClO2 administration at 1.0 ppm significantly reduced the α-diversity of bacterial community composition in the shrimp stomach, and this condition persisted for at least 7 days. Transcriptome analysis of shrimp gill revealed that ClO2 treatment caused massive change of the gene expression profile including stress response genes. However, after 7 days of the treatment, the gene expression profile was similar to that of shrimp in the untreated control group, suggesting a recovery to the normal state. This 1.0-ppm ClO2 significantly reduced shrimp mortality in artificial challenges with an AHPND-causing Vibrio parahaemolyticus and white spot syndrome virus, which were added to rearing water. The use of ClO2 at appropriate concentrations effectively eliminates a significant portion of the bacteria in the shrimp stomach and pathogens in the water. The results of this study provide fundamental knowledge on the disinfection of pathogens in water using ClO2 and the creation of semi germ-free shrimp, which has significantly decreased microbiome in the stomach.

Effect of Agricultural By-Products as a Carbon Source in a Biofloc-Based System on Growth Performance, Digestive Enzyme Activities, Hepatopancreas Histology, and Gut Bacterial Load of Litopenaeus vannamei Post Larvae

The present study evaluated the influence of different commercial agricultural by-products as a carbon source in a bifloc-based (BFT) culture system on growth performance, whole-body proximate composition, digestive enzyme activities, gut microbial abundance, and hepatopancreas histology of Pacific whiteleg shrimp, Litopenaeus vannamei post larvae (Pls). Three groups were designed, the first group was the control group, where the shrimp was reared in clear water (without carbon source addition and water exchange rate of 100% two times a week) and fed with a commercial diet, in the second and third groups shrimp were reared in BFT systems using two different carbon sources, sugarcane bagasse (SB) and rice bran (RB) without additional feeding or water exchange. The initial stocking density was 16 Pls/liter with an average individual shrimp weight of 0.01 ± 0.002 g and age (PL20). The experiment lasted 90 days. The water quality parameters were maintained at optimum levels during the experiment. The final body weight and specific growth rate of shrimp were significantly (p ≤ 0.01) higher in the control group than those reared in both SB and RB-based BFT. Meanwhile, the survival rate was significantly (p < 0.05) higher in BFT groups than in the control. The protease activity in shrimp stomach did not differ significantly. Meanwhile, protease, lipase, and amylase in the intestine showed a significant increase (p < 0.01) in BFT groups than those obtained in the control group. The total heterotrophic bacteria were significantly (p < 0.05) higher in BFT groups. Furthermore, the hepatopancreas histological status of shrimp reared in the SB-based BFT group showed an increase in the hepatopancreas tubules in the distal and B-cell zones (blister-like cells) by 16.83 and 34.89%, respectively, compared to the control. This study revealed that BFT could be used as a natural feed without artificial diets, which influenced the gut microbiota of shrimp, increased digestive enzyme activities, as well as improved the histological structure of the hepatopancreas of shrimp. However, the success of this conditions under high stocking density still needs more investigation.

Shrimp pmo-miR-750 regulates the expression of sarcoplasmic calcium-binding protein facilitating virus infection in Penaeus monodon

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and play crucial roles in antiviral responses. Penaeus monodon miR-750 (pmo-miR-750) was found to be strongly up-regulated in the late phase of white spot syndrome virus (WSSV) infection, but its function remains uncharacterized. Herein, the targets that were translationally down-regulated in the shrimp stomach following a pmo-miR-750 mimic injection were identified using two-dimensional gel electrophoresis. Sarcoplasmic calcium-binding protein (Scp) and actin1 (Act1) were revealed to be down-regulated protein spots. The genuine binding of pmo-miR-750 mimic to Scp but not Act1 mRNA was validated in vitro. In addition, a negative correlation between the Scp transcript and pmo-miR-750 expression level in WSSV-infected P. monodon stomach implies that pmo-miR-750 regulates Scp expression in vivo. When injected into WSSV-infected shrimp, the pmo-miR-750 mimic suppressed Scp expression but significantly increased the WSSV copy number. Consistent with the miRNA mimic-mediated Scp suppression, the loss of function assay of Scp in WSSV-challenged shrimp by RNA interference revealed a decreased survival rate with a dramatic increase in viral copy number. Besides that, apoptosis was activated in the hemocytes of the Scp knockdown shrimp upon WSSV infection. Collectively, our findings reveal that up-regulated pmo-miR-750 suppresses Scp expression at both the transcript and protein levels in the late stage of WSSV infection, which contributes to modulating apoptosis and eventually enabling viral propagation.

Litopenaeus vannamei peritrophin interacts with WSSV and AHPND-causing V. parahaemolyticus to regulate disease pathogenesis

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.

Bacterial and eukaryotic communities in pond water of whiteleg shrimp Litopenaeus vannamei and the bacterial communities of their stomach and midgut

Although microorganisms in pond water and shrimp gut have been thought to be important factors in shrimp health, knowledge of the microbiota surrounding the shrimp is limited. In this study, we analyzed bacterial and eukaryotic microbiota in the pond water of whiteleg shrimp Litopenaeus vannamei, and bacterial compositions in the stomach and midgut of shrimp that are reared in the ponds. Microorganisms were collected at three sites in each of two ponds using filter membranes for 7 consecutive days. Amplicons of 16S rDNA and 18S rDNA were generated. Shrimp were also collected from the ponds on the same days and 16S rDNA was amplified from their stomachs and midguts and analyzed. Results showed that the bacterial communities in the surface water were distributed uniformly and were relatively stable, and the differences between the ponds were small. However, a principal coordinate analysis indicated significant differences in the composition of bacteria in the water of each pond and the microbiota shifted continuously during the sampling period. The eukaryotic plankton in the water changed dynamically and consecutively including the alternations of the dominant families in 1 to 2 days. The bacterial compositions of the digestive tracts, especially the stomach, clearly differed between the two ponds. Also, a bacterium, classified as Candidatus Bacilloplasma in class Mollicutes which was found in the stomachs might be a novel lineage of Mollicutes. Analysis of the combination of bacterial and eukaryotic microbiota, and not only the intestine (midgut) but also the stomach microbiota would be useful to understand microbiome in shrimp aquaculture.

Fishing in troubled waters: Limited stress response to natural and synthetic microparticles in brown shrimp (Crangon crangon)

Marine invertebrates inhabiting estuaries and coastal areas are exposed to natural suspended particulate matter (SPM) like clay or diatom shells but also to anthropogenic particles like microplastics. SPM concentrations may reach 1 g per liter and more, comprising hundreds of millions of items in the size range of less than 100 μm. Suspension feeders and deposit feeders involuntarily ingest these particles along with their food. We investigated whether natural and anthropogenic microparticles at concentrations of 20 mg L−1, which correspond to natural environmental SPM concentrations in coastal marine waters, are ingested by the brown shrimp Crangon crangon and whether these particles induce an oxidative stress response in digestive gland tissue. Shrimp were exposed to clay, silica, TiO2, polyvinyl chloride (PVC), or polylactide microplastics (PLA) for 6, 12, 24, and 48 h, respectively. The activities of the anti-oxidative enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were measured. All five particle types were ingested by the shrimp along with food. The presence of the particles in the shrimp stomach was verified by scanning electron microscopy. The activities of the anti-oxidative enzymes did not vary between animals exposed to different types of microparticles and control animals that did not receive particles. The temporal activity differed between the three enzymes. The lack of a specific biochemical response may reflect an adaptation of C. crangon to life in an environment where frequent ingestion of non-digestible microparticles is unavoidable and continuous maintenance of inducible biochemical defense would be energetically costly. Habitat characteristics as well as natural feeding habits may be important factors to consider in the interpretation of hazard and species-specific risk assessment.

Effect of dietary complex Chinese herbal medicine on growth performance, digestive enzyme activities in tissues and expression of genes involved in the digestive enzymes and antioxidant enzymes and bacterial challenge in Litopenaeus vannamei

The purpose of this study was to evaluate the effect of complex Chinese herbal medicines (CCHM) on growth performance, digestive enzyme activity, immunity and metabolism and Vibrio parahemolyticus resistance of Litopenaeus vannamei. The 30-day feeding experimental results indicated that dietary CCHM obviously increased the survival rate (SR), weight gain rate (WGR) and specific growth rate (SGR) of shrimp. The feed conversion ratio (FCR) was obviously reduced by the CHMM. The supplementation of CCHM could significantly (p < 0.05) increase the activity of protease in the stomach, hepatopancreas and intestine of shrimp. The activities of amylase and lipase were enhanced significantly (p < 0.05) in the stomach, hepatopancreas and intestine of shrimp fed 10 g/kg CCHM diet. The number of haemocytes was increased significantly (p < 0.05) in the shrimp fed the diet with 10 and 15 g/kg CHMM. The activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in the hepatopancreas of shrimp fed with 10 g/kg CCHM were significantly (p < 0.05) higher than that of the control group. The gene expression levels of SOD, CAT, ATPase, TLR, chymotrypsin and GS in the hepatopancreas and intestine of prawns fed with 10 g/kg CCHM were significantly (p < 0.05) up-regulated. In challenge experiment, the relative protection rate was significantly (p < 0.05) increased in the shrimp fed CCHM-supplemental diet when challenged with V. parahaemolyticus. Comprehensive consideration of the current experimental results, the additive suitable dosage of the CCHM was 10 g/kg.

A novel C-type lectin LvCTL 4.2 has antibacterial activity but facilitates WSSV infection in shrimp (L. vannamei)

Glycan-binding protein C-type lectin (CTL), one of the pattern recognition receptors (PRRs), binds to carbohydrates on the surface of pathogens and elicits antimicrobial responses in shrimp innate immunity. The objective was to identify and characterize a novel C-type lectin LvCTL 4.2 in Litopenaeus vannamei. The LvCTL 4.2 protein consisted of a signal peptide at the N terminal and a carbohydrate-recognition domain (CRD) with a mutated mannose-binding (Glu-Pro-Ala; EPA) motif at the C terminal, and thereby has a putative secreted mannose-binding C-type lectin architecture. LvCTL 4.2 was highly expressed in nervous tissue and stomach. Infection with white spot syndrome virus (WSSV) induced expression of LvCTL 4.2 in shrimp stomach at 12 h post infection. Conversely, there was no obvious upregulation in expression of LvCTL 4.2 in stomach or hepatopancreas of shrimp with AHPND (acute hepatopancreas necrosis disease). Pathogen binding assays confirmed recombinant LvCTL 4.2 protein (rLvCTL 4.2) had significant binding ability with the WSSV virion, Gram-negative, and Gram-positive bacteria. Moreover, rLvCTL 4.2 had strong growth inhibition of Vibrio parahaemolyticus. Silencing LvCTL 4.2 suppressed WSSV replication, whereas pretreatment of WSSV with rLvCTL 4.2 facilitated viral replication in vivo. In conclusion, LvCTL 4.2 acted as a PRR that inhibited AHPND-causing bacteria, but facilitated WSSV pathogenesis.

Function of Litopenaeus vannamei RPL4A-L and RPS16 in WSSV infection

Ribosome, as basic components of the protein synthesis machinery, plays an important role in normal cellular physiology, cellular responses and pathogenesis. Ribosome consists of a large subunit (60S) and a small subunit (40S). In Litopenaeus vannamei (L. vannamei), the function of the 60S ribosome protein L4A-Like (LvRPL4A-L) and the 40S ribosome protein S16 (LvRPS16) in white spot syndrome virus (WSSV) infection was unclear. In the present study, the tissue distribution assay showed that LvRPL4-L and LvRPS16 were expressed in all selected tissues. The expressions of LvRPL4-L and LvRPS16 at the mRNA level were mainly distributed in the muscle, stomach, and hepatopancreas. Besides, the relative expressions of LvRPL4-L and LvRPS16 were up-regulated in the hepatopancreas and stomach of WSSV-challenged shrimp. In addition, after LvRPL4-L or LvRPS16 silencing and WSSV infection, the relative expressions of immediate early gene IE1 and late gene VP28 were significantly up-regulated. Meanwhile, compared with the control group, the viral copy number and cumulative mortality rate of the LvRPL4-L- or LvRPS16-silenced group were also significantly increased. We, for the first time, reported the function of LvRPL4-L and LvRPS16 in the antiviral immune response of L. vannamei.

Metabolic Alterations in Shrimp Stomach During Acute Hepatopancreatic Necrosis Disease and Effects of Taurocholate on Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), a recently emerged bacterial shrimp disease, has increased shrimp mortality and caused huge economic losses in many Asian countries. However, molecular factors underlying pathogenesis of this disease remain largely unknown. Our objective was to characterize metabolic alterations in shrimp stomach during AHPND and determine effects of taurocholate on AHPND-causing Vibrio parahaemolyticus. Based on metabolomics, pathways for lipid metabolism and for primary bile acid (BA) synthesis were majorly affected following AHPND infection. Bile acid metabolites, namely taurocholate, were downregulated in the metabolomics database. This prompted us to study effects of taurocholate on biofilm formation, PirABvp toxin release and biofilm detachment capabilities in AHPND-causing V. parahaemolyticus. Treatment of this bacterium with high concentration of taurocholate, a primary bile acid, induced biofilm formation, PirABvp toxin release and facilitated the dispersion of bacterial cells. Taken together, our findings suggest that AHPND infection can affect the lipid metabolites in shrimp stomach, and further suggest that the primary bile acid taurocholate is important for the virulence of AHPND-causing V. parahaemolyticus.

Sand grains in the stomach of brown shrimp, Crangon crangon: Crunchy garnish, supportive macerator, or simply dirt?

Brown shrimp, Crangon crangon, inhabit highly productive sandy and muddy grounds of the southern North Sea. The stomachs of the shrimp contain variable and often high numbers of sediment grains. The function of sediment grains inside the stomach and the purpose of their ingestion are only poorly understood. We tested in laboratory experiments whether sediment and associated organic material complement the natural food of C. crangon or if sand grains may be used by the shrimp to support trituration and maceration of ingested food. The shrimp showed no notable preference for sediment with natural organic content over sediment with reduced organic content, limited ingestion of sediment upon starvation, and no additional uptake of sand grains after feeding. Instead, C. crangon took up sediment only while feeding on regular food, suggesting that sand grains are not ingested intentionally but rather incidentally as a side effect of hasty gobbling. This conclusion is supported by the highly variable uptake of sand grains among individuals. Under experimental conditions, sand grains from sediments do not seem to have a crucial function in food processing and digestion in brown shrimp.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

Effect of sample type and the use of high or low fishmeal diets on bacterial communities in the gastrointestinal tract of Penaeus monodon.

In shrimp aquaculture, manufactured diets that include various supplements and alternative fishmeal ingredients are increasingly being used and their effect on the gastrointestinal (GI) microbiota studied. However, dietary effects on different shrimp GI samples are not known. We investigated how a high (HFM) or low (LFM) fishmeal diet affects bacterial communities from different sample types collected from Penaeus monodon gastrointestinal tract. Bacterial communities of the stomach, intestine tissue and intestine digesta were assessed using 16s rRNA gene sequencing. The feed pellets were also assessed as a potential source of bacteria in the GI tract. Results showed substantial differences in bacterial communities between the two diets as well as between the different sample types. Within the shrimp GI samples, stomach and digesta communities were most impacted by diet, while the community observed in the intestine tissue was less affected. Proteobacteria, Firmicutes and Bacteroidetes were the main phyla observed in shrimp samples, with enrichment of Bacteroidetes and Firmicutes in the LFM fed shrimp. The feed pellets were dominated by Firmicutes and were largely dissimilar to the shrimp samples. Several key taxa were shared however between the feed pellets and shrimp GI samples, particularly in the LFM fed shrimp, indicating the pellets may be a significant source of bacteria observed in shrimp GI samples. In summary, both diet and sample type influenced the bacterial communities characterised from the shrimp GI tract. Thus, it is important to consider the sample type collected from the GI tract when investigating dietary impacts on gut bacterial communities in shrimp. KEY POINTS: • Shrimp gastrointestinal communities are influenced by diet and sample type. • The low fishmeal diet enriched bacteria that aid in polysaccharide metabolism. • Feed pellets can be a source of bacteria-detected gastrointestinal tract of shrimp.

Effects of wheat flour and culture period on bacterial community composition in digestive tracts of Litopenaeus vannamei and rearing water in biofloc aquaculture system

In this study, we investigated the bacterial diversity in rearing water, shrimp (Litopenaeus vannamei) stomach and intestine based on temperature and exposure to wheat flour (carbon source) to determine the correlation between microbial community and surrounding environment in the zero-water exchange shrimp biofloc technology (BFT) system. Three temperature levels of 29 °C (optimal temperature for shrimp growth), 25 °C (rearing water temperature in winter) and 34 °C (summer) were used. Wheat flour was added to groups L (at late stage) and E (at early stage), and sodium bicarbonate was added to group N (wheat flour not added) to adjust the pH. During the operation of the system, the water quality was retained in groups E and N, while it seriously deteriorated in group L at all temperatures. After wheat flour addition in group L, all water quality parameters were recovered efficiently. Using next generation sequencing (NGS) analysis, a total of 26 phyla and 340 OTUs at the genus level were identified in rearing water (the number of samples, n = 31), shrimp stomach (n = 57), and intestine (n = 57). Five phyla, Proteobacteria, Planctomycetes, Actinobacteria, Verrucomicrobia, and Bacteroidetes and 13 bacterial groups related to Phaeobacter, Pirellulaceae, Verrucomicrobiaceae, Rhodobacteraceae, Flavobacteriaceae, Amaricoccus, Marinicellaceae, Acidimicrobiales, C111, Planctomyces, Nitrosomonadaceae, koll13, and Piscirickettsiaceae were found in all samples, representing the core microbiome. Alpha diversity and richness were higher in the shrimp digestive tract, in group E, and at 25 °C compared with the others under each condition. Based on Unifrac PCoA analysis, the samples were clearly classified according to habitat and culture period mostly. The correlation analysis showed that carbon source addition (wheat flour) had a positive correlation with alkalinity and pH, while it was negatively correlated with total ammonia nitrogen (TAN), and nitrate-N concentration in the rearing water (P < 0.05). Temperature was not directly correlated with other environmental factors. The correlation between the core bacterial groups and various environmental factors were estimated. In the rearing water, most core bacterial groups except for major groups in Proteobacteria were negatively correlated with the concentration of nitrogen compounds and positively with alkalinity, pH, and wheat flour (P < 0.05), while the Proteobacteria groups showed opposite patterns. In the shrimp digestive tract, Planctomycetes and Actinobacteria groups positively correlated with culture period (growth stage or weight), while Proteobacteria and Verrucomicrobia groups were negatively correlated. In all habitats, most Actinobacteria were positively correlated with temperature, while Bacteroidetes were negatively correlated (P < 0.05). The present study demonstrated the correlation between environmental factors and major bacterial groups in the shrimp BFT system, and provided useful information to enhance the research and development of a new probiotic strategy and BFT management.

Mitochondrial ATPase inhibitor factor 1, MjATPIF1, is beneficial for WSSV replication in kuruma shrimp (Marsupenaeus japonicus)

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

Penaeus vannamei serine proteinase inhibitor 7 (LvSerpin7) acts as an immune brake by regulating the proPO system in AHPND-affected shrimp

Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in aqua cultured shrimp that is caused by virulent strains of Vibrio parahaemolyticus (VP). Our previous study used transcriptomics to identify key pathogenic factors in the stomach of AHPND-infected shrimp (Litopenaeus vannamei), and here we used a different subset of the same data to construct a gene-to-gene expression correlation network to identify immune-responsive genes. LvSerpin7 was found to have the highest number of correlations after infection, and it also showed a significant increase in mRNA expression. LvSerpin7 is expressed in all tissues but its expression levels are highest in hemocytes. After successfully silencing LvSerpin7 transcript prior to AHPND challenge, mortality was significantly increased relative to the controls and reached 100% within 36 h post infection. Compared to the controls, the phenoloxidase (PO) activity also increased in both hemolymph and stomach. Recombinant LvSerpin7 inhibited shrimp PO activity in vitro, and we also found that rLvSerpin7 inhibited the growth of AHPND-causing bacteria. These results suggest that LvSerpin7 might reduce the toxic effects that result from unregulated activation of the PO defense system by AHPND-causing bacteria.

Bile acid and bile acid transporters are involved in the pathogenesis of acute hepatopancreatic necrosis disease in white shrimp Litopenaeus vannamei.

Acute hepatopancreas necrosis disease is a recently emerged shrimp disease that is caused by virulent strains of Vibrio parahaemolyticus. Although AHPND poses a serious threat to the shrimp industry, particularly in Asia, its underlying pathogenic mechanisms are not well characterized. Since a previous transcriptomic study showed upregulation of the apical sodium bile acid transporter (LvASBT), our objective here was to explore the role of bile acids and bile acid transporters in AHPND infection. We confirmed that mRNA expression of LvASBT was upregulated in the stomach of AHPND-infected shrimps. Bile acid concentrations were also higher in the stomach of AHPND-infected shrimp and correlated with high expression of pVA plasmid and Pir toxins. In vitro assays showed that bile acids enhanced biofilm formation and increased the release of PirABvp toxins in AHPND-causing V. parahaemolyticus, while in vivo inhibition of LvASBT by GSK2330672 reduced the copy numbers of pVA plasmid, Pir toxin and reduced the amounts of bile acids in AHPND-infected shrimp stomach. Transcriptomics data for AHPND-causing V. parahaemolyticus treated with bile acids showed upregulation of various genes involved in membrane transport, RND efflux pumps and a bacterial secretion system. Taken together, our results show that AHPND-causing V. parahaemolyticus virulence is positively regulated by bile acids and that LvASBT and bile acids in shrimp stomach have important roles in AHPND pathogenesis.

Ginger and its component shogaol inhibit Vibrio biofilm formation in vitro and orally protect shrimp against acute hepatopancreatic necrosis disease (AHPND)

Specific isolates of Vibrio parahaemolyticus bacteria that cause acute hepatopancreatic necrosis disease (AHPND) colonize and form biofilms on the chitin lining of the shrimp stomach where they release toxins that cause necrosis in the adjacent hepatopancreas. One potential strategy to prevent AHPND is to inhibit biofilm formation in the stomach using feed additives. Thus, we tested ethanolic extracts of ginger (0.2 mg/ml and 2 mg/ml) for their ability to inhibit biofilm formation by an AHPND-causing isolate of V. parahaemolyticus (3HP) using chitosan coated 96-well polystyrene plastic plates to mimic the shrimp stomach lining. The extract significantly (p < .05) inhibited biofilm formation in those plates but did not inhibit cell growth in broth culture in parallel uncoated plates. Subsequent in vivo immersion-challenge experiments using 3HP (106 CFU/ml) showed that use of extract-supplemented feed (0.2 mg/g and 2 mg/g) for 7 days prior to challenge protected whiteleg shrimp (Penaeus vannamei) against mortality (40%–60% higher survival) when compared to control shrimp fed un-supplemented feed. Extract supplementation had no apparent negative effect on feed palatability or on shrimp growth. Three potentially bioactive compounds known to occur in ginger extracts (6-gingerol, 8-gingerol, and 6-shogaol) were also tested for efficacy in biofilm inhibition in vitro, and 6-shogaol was found to be the most potent followed by the others in descending order. None were found to affect bacterial growth in broth. The results of this study suggested that the use of ginger-based or other feed additives that inhibit biofilm formation may constitute a practical approach to reduce the negative impact of AHPND in shrimp aquaculture.