Hepatopancreas Of Shrimp Research Articles

The Vibrio parahaemolyticus subunit toxin PirBvp recognizes glycoproteins on the epithelium of the Penaeus vannamei hepatopancreas.

Vibrio parahaemolyticus toxin PirABvp is the major virulence factor exotoxin that contributes to the disruption of the hepatopancreatic epithelium in acute hepatopancreatic necrosis disease in shrimp. The PirBvp subunit is a lectin that recognizes amino sugars; however, its potential role in recognition of the hepatopancreas has not been identified. In the present work, we identified the cellular receptor for PirBvp in the shrimp hepatopancreas. A ligand blot assay of hepatopancreas lysate showed that the PirBvp subunit recognizes two glycoprotein bands of 60 and 70kDa (Gc60 and Gc70). The hepatopancreas lysate was fractionated by anion-exchange chromatography, and the three main fractions obtained contained the recognized Gc60 and Gc70 protein bands. LC-MS/MS indicated that beta-hexosaminidases subunit beta and mucin-like 5AC corresponded to the 60 and 70kDa bands, respectively, which seem to be expressed in the epithelial cells of the hepatopancreas. Endoglycosidase treatment of hepatopancreas lysate with the O-glycosidase from Enterococcus faecalis, inhibits the binding of PirBvp. Altogether, these results suggest the relevance of the interaction of PirBvp with the hepatopancreas in the pathogenesis of acute hepatopancreatic necrosis disease in shrimp.

Histopathological of the hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) infected by white feces disease

The high production of Pacific white shrimp (Litopenaeus vannamei) in Pidie Jaya impacts water quality decline around the ponds due to aquaculture waste. This condition causes shrimp to be susceptible to diseases, one of which is white feces disease (WFD). The objective of the study is to determine the level of hepatopancreatic damage caused by WFD through histopathological performance. Four shrimp samples were taken randomly from two semi-intensive ponds in Meurah Dua District, Pidie Jaya Regency, Aceh, Indonesia. The results showed that the histopathology in the hepatopancreas was severely damaged. The damages include vacuolization, fat degeneration, and necrosis in ponds with higher ammonia concentrations. According to pH, temperature, salinity, and ammonia concentrations, ponds near residential areas were more polluted than ponds far from residential areas. This condition is projected by the more damaged histopathological obstruction of the shrimp’s hepatopancreas.

Experimental reproduction of White Feces Syndrome in whiteleg shrimp, Penaeus vannamei.

White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp (Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.

The Microbial Composition of Penaeid Shrimps' Hepatopancreas Is Modulated by Hemocyanin.

Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.

First Report of Enterocytozoon hepatopenaei Infection in Pacific Whiteleg Shrimp (Litopenaeus vannamei) Cultured in Korea.

Simple SummaryWe here report the first detection of Enterocytozoon hepatopenaei (EHP) in cultured Pacific whiteleg shrimp (Litopenaeus vannamei) with a growth disorder in Korea. The histopathology, electron microscopy and DNA phylogeny indicated that the EHP is a unique strain first recorded in Korea. This finding highlights the need for closer monitoring and surveillance to control EHP in aquaculture to prevent disastrous economic losses.The consumption of cultured crustaceans has been steadily increasing, and Pacific whiteleg shrimp (Litopenaeus vannamei) are major cultivated invertebrates worldwide. However, shrimp productivity faces a variety of challenges, mainly related to outbreaks of lethal or growth retardation-related diseases. In particular, hepatopancreatic microsporidiosis caused by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an important disease associated with growth retardation in shrimp. Here, we report the detection of EHP through histopathological, molecular and electron microscopy methods in the hepatopancreas of Pacific whiteleg shrimp with growth disorder in a South Korean farm. Phylogenetic analysis showed a clade distinct from the previously reported EHP strains isolated in Thailand, India, China and Vietnam. An EHP infection was not associated with inflammatory responses such as hemocyte infiltration. Although EHP infection has been reported worldwide, this is the first report in the shrimp aquaculture in Korea. Therefore, an EHP infection should be managed and monitored regularly for effective disease control and prevention.

Effects of a Single Cell Protein (Methylococcus capsulatus, Bath) in Pacific White Shrimp (Penaeus vannamei) Diet on Growth Performance, Survival Rate and Resistance to Vibrio parahaemolyticus, the Causative Agent of Acute Hepatopancreatic Necrosis Disease

The efficacy of a single cell protein (SCP) methanotroph (Methylococcus capsulatus, Bath) bacteria meal (FeedKind®, Calysta, Menlo Park, CA, United States), in Pacific white shrimp (Penaeus vannamei) diets was studied to determine growth performance, survival rate and disease resistance against Vibrio parahaemolyticus causing Acute Hepatopancreatic Necrosis Disease (AHPND). The growth trial was assigned in a completely randomized design (CRD) with four treatments and 5 replicates of each, T1: a fishmeal-based control containing 15% fish meal and 3 diets with graded levels of methanotroph bacteria meal, namely T2: 5% methanotroph bacteria meal, T3: 10% methanotroph bacteria meal, and T4: 15% methanotroph bacteria meal. Shrimp were fed ad libitum for 6 weeks on trial diets to assess growth. Subsequent to the growth trial, three replicates of the same groups were exposed to V. parahaemolyticus by a single bath challenge and held for a further 15 days on the same diets as the growth study to assess survival and resistance. No significant differences (p > 0.05) in survival or in growth performance, including final weight, weight gain, specific growth rate, feed consumption or feed conversion ratio of white shrimp fed feeds containing methanotroph bacteria meal or control diets for 6 weeks. Immune markers such as hemocyte counts, phenoloxidase, superoxide dismutase and lysozyme activity were similar across all groups after the 6-week feeding trial. In a V. parahaemolyticus challenge, methanotroph bacteria meal in the diet significantly promoted the survival rate, and the reduction of Vibrio sp. in the hepatopancreas of white shrimp. Hemocyte count and phenoloxidase activity showed no significant differences (p > 0.05) between diet treatment groups, but hemolymph protein was significantly higher (p < 0.05) in shrimp fed diets containing 15% methanotroph bacteria meal after challenge. The Vibrio colony counts from hepatopancreas in the treatment groups were all significantly lower than the control (p < 0.05). The findings show that methanotroph bacteria meal can entirely replace fishmeal in white shrimp diets and the 15% inclusion of methanotroph bacteria meal in shrimp diet shows no adverse effects on growth performance, feed utilization and survival rate. In addition, shrimp fed methanotroph bacteria meal diets exhibited improved survival rates to an AHPND challenge.

Effects of chitin from Daphnia similis and its derivative, chitosan on the immune response and disease resistance of white shrimp, Litopenaeus vannamei

Daphnia similis chitin and its derivative chitosan were prepared as immunostimulants to boost the immune response and determine the ability to control infectious disease caused by Vibrio alginolyticus in white shrimp, Litopenaeus vannamei. Three experimental diets supplemented with 0% chitin or chitosan (control) and 0.4% chitin or 0.4% chitosan were fed to shrimp for 56 days. Dietary inclusion of 0.4% chitosan accelerated shrimp growth compared to chitin and control. The survival and disease resistance of shrimp increased significantly when fed chitin and chitosan diets, after pathogenic injection, as indicated by the up-regulated immune responses in respiratory burst (RB), superoxide dismutase (SOD), and phagocytic activity (PA). There were no significant differences in the total haemocyte count (THC), phenoloxidase (PO)activity, and lysozyme (LYZ) activity among the groups. No significant differences were observed for prophenoloxidase system-related gene expressions among groups. However, shrimp fed chitin, and chitosan expressed significantly higher levels of antimicrobial proteins (penaeidin 3a, crustin, and anti-lipopolysaccharide factor 2) in the haemocytes than in control. The gene expressions of catalase and heat shock protein 70 increased in the hepatopancreas of shrimp fed chitosan diet compared to the chitin and control diet. The O-linked N-acetylglucosamine transferase (ogt) was significantly higher in the haemocytes of shrimp fed chitosan and chitin than the control, but ogt was only significantly higher in the hepatopancreas of shrimp fed chitosan. Dietary chitin and chitosan also showed positive effects on the transcription of peritrophin-like protein. These findings suggest that both chitin and chitosan from D. similis are efficacious at boosting the immunity of shrimp by preventing and controlling infectious diseases caused by Vibrio and have great potential to be used as a feasible immunostimulant that significantly contributes to the circular economy.

Characterization and functional study of nuclear factor erythroid 2-related factor 2 (Nrf2) in black tiger shrimp (Penaeus monodon)

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the Cap'n′collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants and chemopreventive agents. The full-length cDNA of Nrf2 from Penaeus monodon (PmNrf2; 2024 bp long with 729 bp coding region, GenBank accession no. MW390830) was cloned. The 242-amino-acid polypeptide encoded by this gene had a predicted molecular mass of 27.80 kDa. Sequence homology and phylogenetic analysis showed that PmNrf2 was similar to the insect Cap'n′Collar (CNC) transcription factor and mammalian Nrf2. Tissue expression profile analyzed by quantitative real-time RT-PCR (qRT-PCR) demonstrated that PmNrf2 was constitutively expressed in all examined tissues, with the highest expression observed in the intestines and the weakest expression observed in the hemocyte. PmNrf2 expression profiles were detected in the hepatopancreas of shrimp after bacterial challenge. The results suggested that PmNrf2 was involved in the responses to bacterial challenge, but the temporal expression pattern trend of PmNrf2 differed between the gram-negative and gram-positive bacterial challenges in the shrimp hepatopancreas. The recombinant PmNrf2 protein was expressed and purified through affinity chromatography. Furthermore, an anti-PmNrf2 polyclonal antibody was obtained, which was able to clearly detect PmNrf2 protein expression in the hepatopancreas of shrimp. Knockdown of PmNrf2 by RNA interference (RNAi) resulted in a reduction in the expression of PmGPx gene. Taken together, the results of our study indicated that PmNrf2 played a role in regulation the transcription of PmGPx antioxidant enzyme genes.

Systemic gene silencing from oral uptake of dsRNA in Litopenaeus vannamei requires both clathrin-mediated endocytosis and LvSID-1

RNA interference (RNAi) is a powerful technique that has been widely applied to inhibit both DNA and RNA viruses in shrimp. To accomplish RNAi in shrimp, dsRNA is often delivered by direct injection or feeding, although the latter method has a much lower efficacy. Therefore, understanding the molecular mechanisms underlying the cellular uptake of dsRNA after oral feeding may help in improving the potency of fed RNAi in shrimp. Chlorpromazine was used to block clathrin-mediated endocytosis before feeding a formulated food containing dsRNA specific to shrimp the Rab7 transcript for 6 days. Suppression of Rab7 expression was seen only in the hepatopancreas and gill tissues of the shrimp fed without drug, and suppression was lost when shrimp were pre-treated with chlorpromazine. The results indicate that clathrin-mediated endocytosis participates in dsRNA uptake from the diet into shrimp hepatopancreas. Up-regulation of LvSID-1 was also observed in the gill of Rab7-silenced shrimp fed with the dsRab7 diet, indicating that the RNAi signal derived from feeding was systemically spread by the hemolymph, possibly in the form of long dsRNA. Moreover, induction of LvSID-1 expression before ingestion of dsSTAT improved suppression levels of STAT mRNA in gill tissues, showing that the fed RNAi signal was taken up into gill cells via the LvSID-1 protein. This study reports for the first time on the involvement of both clathrin-mediated endocytosis and LvSID-1 in systemic gene silencing after oral uptake of dsRNA in shrimp, which could help in improving the potency of antiviral strategies in shrimp by fed with RNAi in the future.

Limosilactobacillus fermentum SWP-AFFS02 Improves the Growth and Survival Rate of White Shrimp via Regulating Immunity and Intestinal Microbiota

White shrimp Litopenaeus vannamei is an important species of farmed shrimp. Intestinal bacterial composition and immune activity play important roles in regulating the health condition of shrimp. Lactic acid bacteria Limosilactobacillus fermentum SWP-AFFS02 was isolated from the intestine of sea fish Rachycentron canadum, and the potential of its effect on growth, immunity, and intestinal microbiota of L. vannamei shrimp was investigated. Shrimps received feed with or without the addition of 8 log CFU/g L. fermentum SWP-AFFS02 thrice a day for 8 weeks. After 8-week treatment, weight gain, feed conversion rate, and survival rate of shrimp were greater in the L. fermentum SWP-AFFS02-feed group than in the control group. L. fermentum SWP-AFFS02 treatment increased the number of granular cells and semi-granular cells and decreased hyaline cell number when compared to the control group. L. fermentum SWP-AFFS02 promoted prophenoloxidase (PO) activity through increasing immune-associated gene expression in the hepatopancreas of shrimp. In addition, administration of feed containing L. fermentum SWP-AFFS02 regulated intestinal microbiota via decreasing the ratio of pathogenic bacteria, such as Vibrionaceae and Enterobacteriaceae, in the intestine of shrimp. This study demonstrated that administration of L. fermentum SWP-AFFS02 effectively prevented infection of L. vannamei shrimp by regulating intestinal microbiota and enhancing immunity in shrimp to increase the growth and improve their health status, which acted as a probiotic and provided beneficial effects on shrimp.

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.

Effects of dietary short‐chain fatty acid salts on the growth performance, digestive, antioxidant and immune enzyme activities, immune‐related gene expression and resistance to Vibro parahaemolytics infection in juvenile ridgetail white prawn ( Exopalaemon carinicauda )

An 8-week feeding trial was conducted to study the effects of dietary short-chain fatty acid salts, sodium acetate (SA), sodium propionate (SP) and sodium butyrate (SB), on the growth performance, digestive ability, immune parameters and disease resistance of juvenile Exopalaemon carinicauda. Shrimps (14.60 ± 1.48 mg) were fed with 10 experimental diets containing 0.0 (control), 0.25%, 0.5% and 1% of SA, SB and SP in triplicate. The highest survival rate was observed in the SP (0.5%) group (p < 0.05). The final weight, specific growth rate and feed efficiency was highest in the SP (0.25%) group (p < 0.05). Shrimps that were fed SP (0.25%) had greater intestinal digestive enzyme (chymotrypsin) activity. Shrimps in the SA (0.5%) group had the highest activities of glutathione peroxidase, catalase, lysozyme, acid phosphatase and alkaline phosphatase, which was accompanied by the highest relative percent survival after a Vibro parahaemolyticus challenge (p < 0.05). The expression of immune-related genes (prophenoloxidase, C-type lectin and mannose binding lectin) in the hepatopancreas of shrimps fed SA (0.5%) and SP (0.25%) was higher than that of the control group (p < 0.05). These data suggest that SA (0.5%) and SP (0.25%) are beneficial to the growth performance and health of E. carinicauda.

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.

Effects of replacing fishmeal with dietary dried distillers grains with solubles on growth, serum biochemical indices, antioxidative functions, and disease resistance for Litopenaeus vannamei juveniles

This research was conducted to assess the effects of replacing fishmeal (FM) with dried distillers grains with solubles (DDGS) at various levels (D0 (0 %), D2 (2 %), D4 (4 %), D6 (6 %), D8 (8 %), D12 (12 %), and D16 (16 %)) on the growth, proximate body composition, serum biochemical indices, antioxidant activities, histology, and disease resistance in juvenile Pacific whiteleg shrimp. Forty shrimp per tank were stocked in seven different tanks with three replicates of each treatment group with an initial weight of 0.23 ± 0.00 g. Their respective experimental diets were used to feed shrimp for 56 days. The results showed an enhancement in shrimp fed dietary DDGS (D8) diets on the growth performance, including final weight, weight gain, acid phosphatase, alkaline phosphatase, glucose, and total antioxidant capacity in the serum (P < 0.05). Decreased FCR and High-Density Lipoprotein Cholesterol were observed in shrimp fed DDGS (D8) diets in serum compared to D0. No severe changes and significant differences in villus height and intestinal wall thickness were observed in the shrimp hepatopancreas and mid-gut among treatment groups. Furthermore, disease resistance increased in shrimp fed the DDGS diet, with D8 obtaining the lowest mortality rate, 50.9 %, compared to the control group (D0), 68.3 %. In summary, regarding all the factors, replacing FM with DDGS at D8 (8 %) in the shrimp diet could modulate and cater to overall growth performance, health and immunity, histology and disease resistance in shrimp.

Effect of Noni, Morinda citrifolia fruit extract supplementation on the growth performances and physiological responses of the hepatopancreas of Whiteleg shrimp, Penaeus vannamei Post Larvae

This study focused on the effect of Noni, Morinda citrifolia fruit extract supplementation on the growth and the hepatopancreas function of Whiteleg shrimp, Penaeus vannamei Post Larvae (PL). The crude extract of M. citrifolia fruit was obtained through drying and methanol extraction followed by concentration through rotary evaporation. The extracts were incorporated into commercial shrimp pellet at the concentrations of 0%, 1%, 2%, 3%, 4% and 5%. Feeding trial was done for 30 days and shrimp samples were collected at 5 days interval for growth performance determination and physiology analysis on the hepatopancreas. Despite the inconsistency in growth performances during the early feeding period, the extract supplementation did improved the growth of P. vannamei PL. Significant differences in the digestive enzymes and antioxidant enzymes activities between treatments were observed. The correlation results showed only amylase were correlated with M. citrifolia fruit extract supplementation. In terms of the relationship with growth, amylase and lipase showed correlation with the BW changes of P. vannamei PL while trypsin otherwise. Antioxidant enzymes were strongly correlated with the concentration of M. citrifolia fruit extract supplemented and growth of P. vannamei PL. This indicates that the growth of shrimp was improved with increased antioxidant enzyme activities. The number of hepatopancreatic cells (B, R & E cells) also increased significantly with the increasing concentration of M. citrifolia supplementation during the 30-day feeding trial. Similarly, strong correlation was observed between the hepatopancreatic cells and the growth of P. vannamei PL. The appearance of cell atrophies and haemocytic infiltration in the hepatopancreas might indicate the stimulation of innate defense mechanism in the hepatopancreas by the extract supplementation.

Behavioural and biochemical responses in adult Pacific white shrimp,Litopenaeus vannamei, exposed to the essential oil ofCymbopogon citratus

This study evaluated the effects of continuous exposure to the essential oil of Cymbopogon citratus (EOC) on behavioural and biochemical parameters in shrimp Litopenaeus vannamei. Adult shrimp were randomly placed in aquaria and divided into the following groups: control (pure seawater), ethanol (360 μl L−1 of ethanol) and 5 or 10 μl L−1 EOC for 6 h. Shrimp movements were recorded using a camera for 5 min at the following timepoints: 0, 1, 2, 4 and 6 h of exposure. Light sedation and behavioural changes were observed in shrimp in the 10 μl L−1 EOC group. The total antioxidant capacity against peroxyl radicals (ACAP) in the gills and hepatopancreas of shrimp exposed to 10 µl L−1 EOC was higher than control, whereas, in the muscle, it was lower in those exposed to 5 µl L−1 EOC. In the gills and hepatopancreas, reduced glutathione (GSH) was increased in the control group. Sulphhydryl groups associated with protein (P-SH) were decreased in the gills of shrimp exposed to 10 μl L−1 EOC. Thiobarbituric acid reactive substance levels, indicative of lipid peroxidation, were higher in the gills and hepatopancreas of shrimp exposed to ethanol, which indicates ROS formation. It is concluded that EOC reduced the swimming behaviour of L. vannamei and improved their tolerance with up to 6 h of exposure.

Bioaugmentation of biofloc system with enzymatic bacterial strains for high health and production performance of Penaeus indicus

The beneficial effects of two probiotic bacterial strains Marinilactibacillus piezotolerans and Novosphingobium sp. during the culture of Indian white shrimp, Penaeus indicus, under biofloc and clear water system were evaluated. The experimental variation were CW1 (M. piezotolerans in clear water), BFT1 (biofloc + M. piezotolerans), CW2 (Novosphingobium sp. in clear water), BFT2 (biofloc + Novosphingobium sp.) and control (without bacterial strains and biofloc). Growth and survival considerably increased in probiotic bio-augmented treatments. Probiotic incorporation significantly improved water quality, especially ammonia reduction. Microbiota analysis from gut samples taken from different treatments revealed varied microbial population structure among clear water culture, biofloc culture and control. Proteobacteria and Firmicutes were the top phyla observed in the treatments which were significantly higher in bio-augmented systems than the control. Vibrio genera were predominantly observed in control and clear water system compared to that of biofloc systems. Immune genes were significantly altered in response to probiotic gut microbial supplementation than the control. Higher gene expression profile of important immune genes was observed in the biofloc reared shrimps. Expression of digestive enzyme related genes such as trypsin, chymotrypsin, cathepsin L, cathepsin B and alpha amylase were also upregulated significantly in probiotic supplementation especially in the biofloc treatments. Proteomic analysis of hepatopancreas of shrimps from different treatments was carried out by using 2D gel electrophoresis and MALDI-TOF analysis. The proteins were mostly related to growth and stress tolerance. Eukaryotic initiation factor 4E binding protein was expressed in all the groups and it was high in biofloc treated animals followed by animals treated solely with probiotics compared to those of control groups. The results concludes that biofloc already proved as an effective culture method for healthy shrimp production and supplementation of probiotic bacterial strains registered additional benefit for growth, survival, microbial, immunological status of P, indicus culture.

Identification and characterization polar tube protein 2 (PTP2) from Enterocytozoon hepatopenaei and its potential effect on shrimp microsporidian germination activity evaluation

Microsporidia are obligate, intracellular, spore-forming parasites, whose hosts involve almost all animals from invertebrates to vertebrates. In the last decade, the microsporidium Enterocytozoon hepatopenaei (EHP) mainly infects the hepatopancreas of shrimp to hinder the host's growth, causing substantial economic losses in farms. Polar tube, as an important taxonomy index, is a unique highly specialized invasion apparatus of microsporidia. It is composed of polar tube proteins (PTPs), and more than six polar tube proteins have been identified from various microsporidia. In this study, we have successfully identified the polar tube protein 2 of EHP. EHPPTP2, rich in lysine, encoded 284 amino acids, and it contained a signal peptide of 15 amino acids at the N-terminus. The sequence alignment analysis revealed that EHPPTP2 was homologous with other polar tube protein 2 from microsporidia Ent. bieneusi, Encephalitozoon cuniculi, Enc. hellem, Enc. intestinalis and Enc. romaleae. Additionally, EHPPTP2 was expressed in mature EHP spores. Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM) results showed that EHPPTP2 was localized on the whole polar tube of germinated spores. This is the first study to identify and characterize the polar tube protein from aquatic microsporidia, and these results could serve for the following epidemiological study and provide a reliable target for EHP diagnosis.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP)

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Co‐infection of infectious myonecrosis virus and Enterocytozoon hepatopenaei in Penaeus vannamei farms in the east coast of India

Diseases caused by infectious pathogens are common in grow-out shrimp culture. The emergence of two shrimp diseases viz. infectious myonecrosis virus (IMNV) and hepatopancreatic microsporidiosis which is caused by Enterocytozoon hepatopenaei (EHP), has resulted in substantial economic losses to shrimp farmers in India. Based on previous reports of IMNV disease outbreaks in a small cluster of farms, an attempt was made to study the horizontal spread of this disease along the east coast of India, by active surveillance in four districts of Andhra Pradesh and Tamil Nadu. The present investigation reports the cases of co-infection of IMNV and EHP in Penaeus vannamei shrimp farms in India. Among the 12 ponds investigated, shrimp in four ponds were infected only with IMNV; a single pond was infected with EHP and seven ponds with shrimp harbouring both the pathogens. Shrimp affected by IMNV exhibited white discolouration on the abdominal segments. The EHP-positive pond did not exhibit any apparent clinical signs. The co-infected shrimp exhibited clinical lesions of both IMNV and EHP. Histopathology revealed that the hepatopancreas of shrimp with signs of slow growth, showed cytoplasmic developmental stages of EHP in the tubular epithelial cells and sloughing of infected cells, releasing spores in the tubular lumen whereas, IMNV affected shrimps had shredding of muscle fibres, coagulative necrosis with haemocytic infiltration in striated muscles and hypertrophy of lymphoid organ (LO). Both the infections were further confirmed by PCR as well as sequencing.