Control Shrimp Research Articles

Ammonia nitrogen stress induces dysbiosis of the intestinal bacterial community and facilitates the enrichment of pathogenic bacteria in intestines of shrimp

Environmental stress can disrupt the intestinal microbiota of aquatic animals, thereby impacting their health status. In this study, we aimed to elucidate the dynamic changes in the bacterial community within shrimp intestines under short-term ammonia nitrogen stress and investigate the subsequent ecological risks associated with it. Our findings revealed a significant reduction in the Chao1 diversity index within shrimp intestines of the ammonia nitrogen stress group compared to the Control group. Furthermore, distinct disparities in the bacterial community structures were observed between the two groups at 72 h and 120 h post-stress. Additionally, ammonia nitrogen stress significantly altered the abundances of specific bacterial taxa within shrimp intestines. Specifically, the abundance of Bacillus subtilis increased while that of Lactococcus garvieae decreased in the intestine under stress at 72 h. Additionally, Roseivivax jejudonensis exhibited an increase, whereas uncultured_o_Saccharimonadales and Hyphomicrobium sp. showed a decrease at 120 h. Notably, opportunistic pathogens such as Vibrio parahaemolyticus experienced a significant increase at 72 h post-stress, potentially due to a substantial contribution from stochastic processes in shaping the assembly of the intestinal community. Additionally, the intestinal bacterial community of shrimp displayed a noticeable decrease in complexity of bacterial co-association networks under stress conditions. Thus, short-term exposure to ammonia nitrogen disrupts the integrity of shrimp's intestinal community, indicating potential ecological risks within their microhabitat. Confirmatively, we conducted the immersion challenge experiment of V. parahaemolyticus on shrimp post community disturbance under stress, and observed a significantly higher copy numbers of V. parahaemolyticus in host intestines of stressed compared to control shrimp. These findings can provide novel insights into the interactions among ammonia nitrogen stress, intestinal microbiota and host health of aquatic animals, and guide the practices for shrimp healthy culture.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

Effect of soybean meal replacement with corn gluten meal on the survival, biochemical and metabolic responses, and disease resistance of Pacific white shrimp (Litopenaeus vannamei)

Abstract This study delves into the potential of replacing soybean meal (SBM) with corn gluten meal (CGM) in the diet of Litopaeneaus vannamei. We aimed to investigate the effects of a dietary soybean meal replacement on various aspects of shrimp, including survival, biochemical indices, digestive enzymes, metabolomics, and disease resistance against Vibrio harveyi. To conduct the study, we fed 840 juvenile Pacific shrimp seven different diets, each containing varying levels of CGM. The control group received a diet with 0% CGM; the other diets contained 3%, 5%, 7%, 9%, 12%, and 14% CGM. Each group comprised three replicates, and the shrimp were fed their diets for eight weeks. Our findings revealed that the survival rate of the shrimp ranged from 90.83% to 97.50% and did not differ significantly between the control and those fed diet treatments 2–5. Additionally, there were no significant differences in crude protein, moisture, and lipid content. However, we observed that total antioxidant capacity content increased with the dietary inclusion of CGM. Furthermore, malondialdehyde content decreased with increasing CGM levels, while superoxide dismutase increased, indicating no obvious oxidative damage was observed in CGM treatment groups. Interestingly, shrimp fed diets 2 and 3 had considerably lower alanine aminotransferase activity than shrimp in the control group. The disease resistance in shrimp was improved across all treatment groups, with a notably higher CGM inclusion having the highest level of mortality during the challenge test. Finally, we analyzed the metabolomics data and found PCA score plots clearly separated the control group and shrimp fed CGM diets. Our study revealed that a 3% substitution rate of SBM with CGM can enhance survival and immunity, regulate metabolites and improve disease resistance.

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

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

SESBANIA GRANDIFLORA LEAF EXTRACT ENHANCES GROWTH AND NON-SPECIFIC IMMUNE RESPONSES OF SHRIMP (Penaeus monodon)

This study determined the effects of Sesbania grandiflora (SG) leaf extract on the growth, feed utilization efficiency and non-specific immunological responses in black tiger shrimp (Penaeus monodon). Three diets were prepared by mixing the methanol extract of SG onto a pellet feed (34% protein) at concentrations of 0% (control), 0.05% (T1) and 0.1% (T2), respectively. Ten shrimps (mean weight 2.1g), were reared in each tank of 80 L, and the weight and immunological reaction of shrimp were received after a feeding trail of 28 days. The dietary extract treated shrimp had better growth performance (WG, SGR), and feed utilization efficiency (FCR, PER) compared to the control group. The treated shrimp exhibited enhanced immunological responses; the total haemocyte count, prophenoloxidase activity, superoxide dismutase activity, and shorter haemolymph clotting time compared to the control shrimp. In conclusion, a lower level of SG (0.05%) was found to be most effective in terms of immune response enhancement; hence, this plant could be a good source for feed additives for enhancing sustainable shrimp production.

Long noncoding RNA profiling in hepatopancreas of Pacific white shrimp and its role in response to white spot syndrome virus infection

Long noncoding RNA (lncRNA) is a potential regulator of biological processes, including immunity, reproduction, and development. Although several transcriptome studies have focused on responses of viral infections in several organisms, the role of lncRNAs in viral responses in shrimp is still unclear. Therefore, this work aimed to identify putative lncRNAs and study their role in white spot syndrome virus (WSSV) infection in white shrimp. The hepatopancreas transcriptome from WSSV infected shrimp was analyzed in silico to identify putative lncRNAs. Among 221,347 unigenes of the de novo assembled transcriptome, 44,539 putative lncRNAs were identified, 32 of which were differentially expressed between WSSV-infected and control shrimp. Five candidate lncRNAs were validated for their expressions in shrimp tissues and in response to WSSV infection. Lnc164 was chosen for further investigation of its role in WSSV infection. Knockdown of lnc164 prolonged survival of shrimp when challenged with WSSV, suggesting a role in shrimp immunity. In addition, lnc164 was not directly involved in the control of total hemocytes and viral loads in hemolymph of WSSV-infected shrimp. A set of lnc164-regulated genes was obtained by RNA sequencing among which 251 transcripts were differentially expressed between lnc164 knockdown and control shrimp. Six immune-related genes were validated for their expression profiles. Our work sheds light on lncRNA profiles in L. vannamei in response to WSSV infection and paves the way to a functional study of lnc164 in host antiviral response.

Optimization of ultrasonic-assisted extraction of flavor compounds from shrimp by-products and characterization of flavor profile of the extract

This study aimed to optimize the ultrasonic-assisted extraction conditions of flavor compounds from white shrimp heads (WSHs). The effects of sonication amplitude, sonication time, and solvent-to-solid ratio on the extraction yield (EY) of flavor compounds and the degree of protein modification (DPM) were evaluated by Box-Behnken design and response surface methodology (RSM). The optimum EY (40.87 %) and DPM (26.28 %) were obtained at amplitude, time, and solvent-to-solid ratios of 63.2 %, 20.5 min, and 20.8 mL/g, respectively. The optimum DPM value indicates that sonication markedly influenced the protein denaturation, as evidenced by the higher TCA soluble protein content. Further, we also investigated the taste active composition of ultrasonic extract of shrimp head (USH). Results show that the equivalent umami concentration (EUC) value was significantly (p < 0.05) increased in the ultrasonic extract of shrimp head (USH) (55.44 ± 3.25 g MSG/100 g) compared to the control shrimp head extract (CSH) (8.32 ± 1.07 g MSG/100 g). This study also deals with the development of shrimp flavor concentrate (SFC) using USH by the conventional heating process. Sensory evaluation of SFC revealed that the shrimp-like aroma and umami taste characteristics were predominant. Thus, USH's improved umami taste composition demonstrates its potential utilization for producing SFC with higher EUC.

Survival and Immune Responses of F1 Transgenic Tiger Shrimp Penaeus monodon Against White Spot Syndrome Virus (WSSV)

Transgenic technology has been applied to tiger shrimp Peneus monodon to produce a resistant strain to white spot syndrome virus (WSSV) by antiviral gene overexpression. The founders (F0) transgenic tiger shrimp have been successfully bred to produce the first generation (F1). The present study aimed to evaluate the survival and immune responses of F1 transgenic tiger shrimp against WSSV. The F1 transgenic and non-transgenic shrimp (control) with an average weight of 7.93±1.49 g were collected from the controlled brackish water ponds and stocked in a 20 L fiberglass tank with a density of 5 shrimp/tank. The transgenic shrimps were confirmed by a PCR assay. The shrimp were intramuscularly injected with WSSV to evaluate the performance between transgenic and non-transgenic. The survival (SR) was observed daily after the challenge test. Measurement of immune responses, namely total haemocyte count (THC), differential haemocyte count (DHC), prophenoloxidase (proPO) activity, and RNA content in haemolymph was conducted on before challenge, and on the 1st, 3rd, and 5th days after challenge. The survival and immune responses were statistically analysed by the t-student test. The results showed that the SR of transgenic (52.0%), with a relative percentage survival of 47.82%, was greater (P&lt;0.05) than the control shrimp (8.0%). The THC, proPO, and RNA content of the transgenic shrimp was higher (P&lt;0.05) than the non-transgenic shrimp. The results suggested that the transgenic tiger shrimp, due to antiviral gene overexpression, increased the resistance against WSSV.

Identification and profiling of long non-coding RNAs during molt cycle: An involvement of lnc1182 in the molt of white shrimp, Litopenaeus vannamei

Molt is an essential physiological process for crustacean growth. It is known to be controlled by factors such as hormones and steroids, but the role of long non-coding RNA (lncRNA), a potential regulatory molecule, has not been studied. In this study, we investigated the role of lncRNA in the molt process of white shrimp, Litopenaeus vannamei. The 9437 of 58,142 unigenes of de novo assembled transcriptome were identified as putative lncRNAs, and 768 lncRNAs were differentially expressed during molt cycles. Five differentially expressed lncRNAs were selected to determine their expressions in different tissues and molt stages. Most were expressed more in gills and epidermis than other tissues. Expressions of lnc1182 and lnc336 significantly decreased during premolt stages and increased at postmolt in gills and epidermis, while lnc179 expression increased during premolt stages. RNA interference was used to investigate the role of lnc1182 in the molt process. The molt of lnc1182 knockdown shrimp progressed to early premolt faster than shrimp injected with dsGFP, and also shortened molt duration. In addition, total sugar level in hemolymph was higher in the lnc1182 knockdown shrimp than the control shrimp, whereas gill Na+/K+ ATPase activity, total hemocyte count, and expression of molt-inhibiting hormone (MIH) were not changed. These suggested that lnc1182 is likely involved in the control of sugar metabolism during molting but not in ion exchange activity and MIH expression. Furthermore, lnc1182 and 20-HE probably regulated each other. RNA sequencing was used to investigate the set of genes associated with lnc1182. Fifty-five transcripts were differentially expressed in gills of lnc1182 knockdown shrimp. Expressions of candidate metabolism and immune-related genes were validated for their differential expressions in lnc1182 knockdown shrimp. Our work produced lncRNA profiles and indicated possible functions of lnc1182 in metabolism and immunity during the molt cycle in shrimp.

Antibacterial sachet from the β‐Cyclodextrin/lemongrass oil inclusion complex for shrimp freshness

SummaryAntibacterial sachets are one of the technological approaches to maintaining food freshness by releasing active compounds through the headspace to inhibit bacterial growth. In this study, an antibacterial sachet was developed from the inclusion complex of β‐cyclodextrin/lemongrass oil (β‐CD/LO) to maintain the freshness of shrimp (Litopenaeus vannamei). The β‐CD/LO inclusion complex was prepared using the co‐precipitation method. The antibacterial sachet was prepared by filling a nonwoven fabric bag with 1 g of the β‐CD/LO inclusion complex, inserted inside the shrimp packaging, and stored for 30 h at room temperature. The total volatile base nitrogen (TVBN) value was monitored as a spoilage indicator caused by bacterial activity during storage. The inclusion complex had antimicrobial activity against E. coli ATCC 8739 and S. aureus ATCC25923 which correlates with its ability to inhibit shrimp spoilage due to bacterial activity. The release study suggested that the inclusion complex exhibited a sustainable release of active compounds, ensuring protection against evaporation and maintaining the active compound for a long time. The TVBN value of the control shrimp had exceeded the threshold (&gt;25 mg/100 g) after 18 h of storage, whereas in the packaged shrimp with the addition of antibacterial sachets, the same TVBN levels were reached after 30 h. The shelf life of shrimp can be extended up to 12 h at room temperature (28 ± 2 °C) because the active compounds reach the surface of the shrimp in a controlled manner so that bacterial growth can be inhibited.

Absorption of sulfur dioxide gas in moistened porous material on a suspended gold leaf electrochemical sensor

This work describes an unconventional voltammetric detection in a gas medium using a suspended electrode unit. A paper moistened with the supporting electrolyte is used to absorb the gas. This concept was demonstrated for the in-situ detection of SO2 gas to quantify sulfite residue in samples of frozen shrimp. The gas is generated by the acidification of an aqueous suspension of shrimp tissues. The porosity of facial tissue paper is employed to provide a liquid layer of supporting electrolyte for the operation of the gold leaf electrochemical sensor. The absorbed gas is also dissolved in this liquid layer. The gold leaf sensor is reusable by replacing the tissue disc and rinsing the electrodes with water. The electrochemical sensor does not require refrigeration or pre-conditioning as with commercial biosensors and is thus suitable for point-of-need purposes. Production of the gold leaf sensor is only ca. 0.2 USD per unit and can be used regularly for up to 3 months. The linear calibration range of spiked negative control shrimp samples is from 3 to 240 mg kg−1 SO2 for a 30 mL solution of an extract of 5.0 g homogenized defrosted shrimp. The limit of detection (3 ×SDintercept/slope) for spiked samples is 2 mg kg−1 SO2. This is the first electrochemical sensor in a gas medium exploiting the entrapment of electrolyte solution inside a porous substrate.

Comparison of Immune Response of Litopenaeus vannamei Shrimp Naturally Infected with Vibrio Species, and after Being Fed with Florfenicol

The outbreaks caused by Vibrio spp. are a notable threat to the potential growth of the economy of penaeid culture, which is still controlled by the administration of antibiotics. At first, the infected group was subjected to phenotypic bacteriological examination with subsequent molecular identification via 16S rRNA gene sequencing, which confirmed four strains of Vibrio spp., V. atlanticus, V. natriegens, V. alginolyticus, and V. harveyi, from moribund-infected shrimp during mortality events in an Egyptian hatchery. To better understand the defense mechanism of the most effective antibiotic against Vibrio strains, the immune responses were compared and evaluated in infected Litopenaeus vannamei broodstock after being fed 5 mg kg−1 of florfenicol antibiotic, which was first determined through in vitro antibiogram tests. Therefore, our study aimed to determine the immune response of L. vannamei during Vibrio spp. infection in Egyptian hatcheries and after antibiotic medication. The parameters assessed were the total and differential hemocyte count (THC), granular cells (GC), semi-granular cells (SGC), and hyaline cells (HC). As well as the metabolic and immune enzymes: alanine aminotransferases (ALT), aspartate aminotransferases (AST), alkaline phosphatase (ALP), acid phosphatase (ACP), and lysozyme activity; an antioxidant index, such as superoxide dismutase (SOD) and glutathione (GSH); a phagocytic assay; changes in reactive oxygen species (ROS); and bactericidal activity in the hemolymph of the control, infected, and treated groups. Further evaluation of the mRNA expression levels of the prophenoloxidase (LvproPO), toll-like receptor 1 (LvToll1), and haemocyanin (LvHc) genes were performed in the hepatopancreas of the same groups. A significant drop in the THC, GC, SGC, and HC counts, as well as lysozyme and bactericidal activities, phagocytic assay, ROS, SOD, and GSH index, were represented in infected shrimp compared to control shrimp; however, a marked increase in the activity of ALT, AST, ALP, and ACP was observed. These activities were significantly restored in the treated shrimp compared to the infected shrimp. Nevertheless, no significant changes were noted in the transcriptional levels of the LvproPO and LvToll1 genes in the treated shrimp when compared to the infected shrimp; however, a significant suppression of the LvHc gene was noted. Our study aimed to determine the immune response of L. vannamei during Vibrio spp. infection in Egyptian hatcheries and after antibiotic medication. We concluded that florfenicol in medicated feed could be effective in controlling vibriosis and ameliorating the immune response of shrimp.

White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

Polystyrene Microparticles and the Functional Traits of Invertebrates: A Case Study on Freshwater Shrimp Neocardina heteropoda

The excessive worldwide production of plastic materials results in omnipresent microplastic pollution. Scientific studies dealing with the impacts of microplastics on aquatic ecosystems focus mainly on the marine environment, documenting the effect on the functional traits of various organisms. Polystyrene, one of the most commonly used plastics, has become a widely used model in this respect. In our study, freshwater shrimps (Neocardina heteropoda) were exposed to virgin polystyrene particles (size 0.5 mm; nominal concentration 8 mgL−1), and their behavioral and physiological responses were compared to control shrimp. The exposed shrimps exhibited modified activity patterns (greater speeds, accelerations and distances moved), accompanied by a lowered standard metabolic rate (SMR). The observed effects differed in their progression from the 7th to 14th day of exposure, from undetectable changes (distance, SMR) to significant differences (speed, acceleration). Significant differences were also detected in the behavioral syndromes expressed by the exposed and controlled shrimps, indicating that the microplastics influence not only the particular traits, but also their functional relationships. As such, our study contributes to the integration of behavioral ecotoxicology in risk assessment, documenting the adverse performance of freshwater invertebrates exposed to microplastics with the potential to transpose the problem to higher levels of the food web.

Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrioparahaemolyticus

Vibrio parahaemolyticus (Vpara) is the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS) in shrimp. Shrimp, like other invertebrates, lack an adaptive immune system and depend solely on innate immunity against invading pathogens. To better understand the defense mechanisms of shrimp to this problematic pathogen, we evaluated the changes in hematology, immunology and biochemical values of the hemolymph from shrimp challenged with V. parahaemolyticus up to 8 days post-challenge. Thirty-six shrimp (12 g) were distributed in 9 tanks (75 L), divided into three groups (non-challenged, challenged with 5 × 102 cfu/shrimp and challenged with 1 × 103 cfu/shrimp) in triplicate. Pacific white shrimp, Litopenaeus vannamei, were administered an inoculum of V. parahaemolyticus under the shell between the 5th and 6th abdominal segment to assess cellular and humoral immune responses. Total hemocyte count (THC) significantly decreased in shrimp challenged with Vpara at 6 h, 12 h and 24 h-post infection. Hemocyte lysate phenoloxidase (PO) activity in Vpara-challenged shrimp at 48 h post challenge was significantly increased compared to that of control shrimp. No significant differences were observed in total plasma protein between plasma from control and Vpara-challenged shrimp. However, shrimp challenged with 5 × 102, and 1 × 103 cfu/shrimp had significantly lower hemocyanin at 6 h and 48 h sampling point, respectively. At 24 h post-challenge, the ≥140 kDa and 70 kDa bands from SDS-PAGE of hemocyanin-concentrated hemolymph lysate samples showed a higher and lower intensity, respectively, in Vpara-challenged group than those of the control group. Plasma from Vpara-challenged shrimp at 6 h and 12 h-post infection significantly suppressed V. parahaemolyticus growth. However, significantly less bacterial growth suppression was observed in plasma of shrimp challenged with higher dose compared to control shrimp at the 192 h post-challenge point. Plasma chemistry parameters did not significantly differ among treatments. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp.

High nutrient induces virulence in the AHPND-causing Vibrio parahaemolyticus, interpretation from the ecological assembly of shrimp gut microbiota

Shrimp diseases frequently occur during the later farming stages, when the rearing water is eutrophic. This observation provides clue that the virulence of pathogens could be induced by elevated nutrient, whereas the underlying ecological mechanism remains limited. To address this pressing knowledge, we explored how gut microbiota responded to the infection of oligotrophic (OVp) or eutrophic (EVp) pre-cultured Vibrio parahaemolyticus, a causing pathogen of shrimp acute hepatopancreatic necrosis disease (AHPND). Resulted revealed that OVp and EVp infections caused dysbiosis in the gut microbiota and compromised shrimp immunity, while the later infection led to earlier and higher mortality. Significant associations were detected between the gut microbiota and each of the measured immune activities. Neutral community model showed that the assembly of gut microbiota was more strongly governed by deterministic processes in EVp infection, followed by EVp infected and control shrimp. Additionally, there were significantly lower temporal turnover rate and average variation degree in the gut microbiota in EVp infected shrimp compared with control individuals. Notably, we identified 22 infection-discriminatory taxa after ruling out the ontogenic effect. Using profiles of the 22 indicators as independent variables, the diagnosis model accurately distinguished (an overall 85.9% accuracy) the infected status (control, OVp or EVp infected shrimp), with 81.3% accuracy at the initial infection stage. The convergent and deterministic gut microbiota in EVp infected shrimp could partially explain why it is challenge to cure APHND from an ecological viewpoint. In addition, we provided a sensitive approach for diagnosing the onset of infection, when disease symptom is unobservable.

Effect of dietary supplementation with Moringa oleifera leaf extract and Lactobacillus acidophilus on growth performance, intestinal microbiota, immune response, and disease resistance in whiteleg shrimp (Penaeus vannamei)

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 107 CFU/g of diet (ME0+P, ME2.5 + P, and ME5.0 + P, respectively). Growth performance was measured after 60 days of the rearing period. On the final day, the shrimp were sampled to assess enzyme activity, intestinal and hepatopancreatic histology, and gut microbiota. Shrimp hemocytes were examined on Days 0, 1, 2, 4, 7, 14, 21, and 28 to measure the immune response in terms of the total hemocyte count, phenoloxidase activity, phagocytosis, and superoxide anion production. Furthermore, the shrimp were challenged with V. alginolyticus and V. parahaemolyticus. The results revealed that ME2.5 + P significantly increased (P < 0.05) final weight, weight gain, specific growth rate, enzyme activities, and villi height compared with ME2.5 and control. Wall thickness was increased in the shrimp fed diet supplemented with moringa and L. acidophilus compared with the control shrimp. Hepatopancreatic histology revealed that R cells were more abundant in the shrimp fed diet containing moringa and L. acidophilus compared with those fed diet containing moringa alone (P < 0.05) at the same concentration. High-throughput sequencing analysis indicated that the dietary supplementation with moringa and L. acidophilus affected the gut microbiota composition. All gene functions, members of KEGG level 2, related to metabolism were increased in diet supplemented with moringa with or without L. acidophilus compared with the control group. The immune assay revealed that the total hemocyte count, phenoloxidase activity, phagocytic rate, superoxide anion production, and immune-related gene expression (including those of prophenoloxidase II, alpha-2-macroglobulin, penaeidin2, antilipopolysaccharide factor, crustin, lysozyme, glutathione peroxidase, and superoxide dismutase) were higher in the experimental groups than in the control group on several observed days; however, the increases were observed more often in the ME2.5 + P group than in the other treatment groups. Furthermore, the ME2.5 + P group exhibited a significantly higher survival rate (P < 0.05) in the challenge test against V. alginolyticus and V. parahaemolyticus. In conclusion, supplementation with dietary moringa and L. acidophilus at ME2.5 + P improved growth performance, immune system, and resistance against Vibrio in the shrimp.

Growth, Health, and Gut Microbiota of Female Pacific White Shrimp, Litopenaeus vannamei Broodstock Fed Different Phospholipid Sources.

Phospholipids have an important antioxidant effect on animals. The effects of different dietary phospholipid sources on the growth, antioxidant activity, immunity, and gut microbiota of female broodstock of Pacific white shrimp Litopenaeus vannamei were investigated. Four isoproteic and isolipid semi-purified diets containing 4% soybean lecithin (SL), egg yolk lecithin (EL), or krill oil (KO) and a control diet without phospholipid supplementation were fed to female broodstock of L. vannamei (34.7 ± 4.2 g) for 28 days. The growth performance, antioxidative capacity, and innate immunity of the female broodstock fed phospholipid supplemented diets were improved regardless of sources compared with the control shrimp. The effects on growth and antioxidant capacity in female shrimp fed the KO diet were highest. The innate immunity of female shrimp fed the EL and KO diets were significantly higher than shrimp fed the SL diet. Dietary phospholipid supplementation increased gut microbiota diversity and richness, and the Chao1 and ACE values in the KO group were significantly higher than in the control group. The richness of Proteobacteria, Photobacterium, and Vibrio decreased, whereas the richness of Firmicutes and Bacteroidetes increased in the shrimp fed the KO diet compared with the shrimp fed the SL and EL diets. The interactions of gut microbiota in shrimp fed the KO diet were the most complex, and the positive interaction was the largest among all the treatments. The functional genes of gut microbiota in shrimp fed the KO diet were significantly enriched in lipid metabolism and terpenoid/polyketide metabolism pathways. Spearman correlation analysis showed that Fusibacter had significantly positive correlations with antioxidant activity (total antioxidant capacity, superoxide dismutase, glutathione peroxidase), immune enzyme activity (phenoloxidase and lysozyme), and immune gene expression (C-type lectin 3, Caspase-1). All findings suggest that dietary phospholipids supplementation can improve the growth and health status of female L. vananmei broodstock. Krill oil is more beneficial in improving the antioxidant capacity and innate immunity than other dietary phospholipid sources. Furthermore, krill oil can help establish the intestinal immune barrier by increasing the richness of Fusibacter and promote the growth of female shrimp. Fusibacter may be involved in iron metabolism to improve the antioxidant capacity of female shrimp.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Gut bacterial profile associated with healthy and diseased (AHPND) shrimp Penaeus vannamei

The effect of Vibrio parahaemolyticus IPNGS16 on the bacterial profile of the gut of Penaeus vannamei was assessed by 16S metagenomic analysis. The V3 hypervariable region of the bacterial 16S rDNA was amplified by PCR. Sequencing reads were generated using the 2×150 (300 cycles) for the base-read length chemistry of the Illumina MiniSeq platform. The web-based Shaman and MicrobiomeAnalyst platforms were used to analyze the sequences. The phyla Proteobacteria, Bacteroidetes, and the genera Vibrio, Ruegeria, Nautella, and Pseudoalteromonas were found among the most abundant taxonomic ranks in control, diseased, and healthy shrimp. Alpha and beta indices showed significant differences between shrimp survival in the control condition and dying shrimp (lower diversity). Metabolism (carbohydrate and amino acid metabolism-related genes and, to a lesser extent, energy, lipid, and cofactors and vitamin metabolism-related genes) of dying and surviving shrimp was affected by Vibrio infection. The top metabolic functions (cell cycle, glycine, serine, threonine, cysteine, methionine, purine, pyrimidine, pyruvate, and quorum sensing) in dying and surviving shrimp were affected by Vibrio, especially quorum sensing. The interaction network analysis showed fewer interactions in dying shrimp than control and surviving shrimp. Proteobacteria, Bacteroidetes, Vibrio, and Ruegeria predominated in all samples, and Vibrio changed bacterial diversity and metabolism in the intestine of P. vannamei. Ruegeria and Pseudoalteromonas showed negative interactions with Vibrio, suggesting their use as probiotics. This study sheds light on the Vibrio infection in the gut microbiota of shrimp.