Decoding the role of HIF-1α in immunoregulation in Litopenaeus vannamei under hypoxic stress.

Hypoxia poses a significant challenge to aquatic organisms, especially Litopenaeus vannamei (L. vannamei), which play a vital role in the global aquaculture industry. Hypoxia-inducible factor 1α (HIF-1α) is a pivotal regulator of the organism's adaptation to hypoxic conditions. To understand of how HIF-1α affects the immunity of L. vannamei under hypoxic conditions, we conducted a thorough study involving various approaches. These included observing tissue morphology, analyzing the expression of immune-related genes, assessing the activities of immune-related enzymes, and exploring immune-related pathways. Our study revealed that RNA interference (RNAi)-mediated knockdown of HIF-1α markedly reduced HIF-1α expression in the gill (75-95 %), whereas the reduction ranged from 2 to 43 % in the hepatopancreas. Knockdown of HIF-1α resulted in increased damage to both gill and hepatopancreatic tissues in hypoxic conditions. Additionally, immune-related genes, including Astakine (AST), Hemocyanin (HC), and Ferritin (FT), as well as immune-related enzymes such as Acid Phosphatase (ACP), Alkaline Phosphatase (AKP), and Phenoloxidase (PO), exhibited intricate regulatory patterns in response to hypoxia stress following the knockdown of HIF-1α. Transcriptome analysis revealed that HIF-1α knockdown significantly impacts multiple signaling pathways, including the JAK-STAT signaling pathway, Th17 cell differentiation pathways, PI3K-Akt signaling pathway, ErbB signaling pathway, MAPK signaling pathway, chemokine signaling pathway, ribosomal pathways, apoptosis, lysosomes and arachidonic acid metabolism. These alterations disrupt the organism's immune balance and interfere with normal metabolic processes, potentially leading to various immune-related diseases. We speculate that the weakened immune response resulting from HIF-1 inhibition is due to the reduced metabolic capacity, and the existence of a direct regulatory relationship between them requires further exploration. This study greatly advances our understanding of the vital role that HIF-1α plays in regulating immune responses in shrimp under hypoxic conditions, thereby deepening our comprehension of this critical biological mechanism.

Effects of myo-inositol on growth and biomarkers of environmental stress and metabolic regulation in Pacific white shrimp (Litopenaeus vannamei) reared at low salinity.

This study explored the role of myo-inositol in alleviating the low salinity stress of White Shrimp (Litopenaeus vannamei). Juvenile shrimp (0.4 ± 0.02 g) in low salinity (salinity 3) water were fed diets with myo-inositol levels of 0, 272, 518, 1020 and 1950 mg/kg (crude protein is 36.82 %, crude lipid is 7.58 %), fed shrimp in seawater at a salinity of 25 were fed a 0 mg/kg myo-inositol diet as a control (Ctrl). The experiment was carried out in tanks (50 L) with satiety feeding, and the experiment lasted for 6 weeks. After sampling, the serum was used to measure immune function, the hepatopancreas homogenate was used to measure the antioxidant capacity and hepatopancreas damage state, the hepatopancreas was used for transcriptomics analysis, and the gills were used for qPCR to measure osmotic pressure regulation. The results showed that the final weight and survival of the shrimp in the 1020 mg/kg group increased significantly compared with those in the other low salinity groups, but the final weight and biomass increase were significantly lower than those in the Ctrl group. Dietary myo-inositol improved the antioxidant capacity of shrimp under low salinity. B-cell hyperplasia and hepatic duct damage were observed in the hepatopancreas in the 0 mg/kg group. The results of transcriptome analysis showed that myo-inositol could participate in the osmotic pressure regulation of shrimp by regulating carbohydrate metabolism, amino acid metabolism, lipid metabolism and other related genes. Myo-inositol significantly affected the expression of related genes in ion transporter and G protein-coupled receptor-mediated pathways. This study demonstrated that myo-inositol can not only act as an osmotic pressure effector and participate in the osmolar regulation of shrimp through the phosphatidylinositol signaling pathway mediated by G protein-coupled receptors but also relieve low salinity stress by mediating physiological pathways such as immunity, antioxidation, and metabolism in shrimp. The binomial regression analysis of biomass increases and survival showed that the appropriate amount of myo-inositol in the L. vannamei diet was 862.50-1275.00 mg/kg under low salinity.

Dietary supplementation of Pseudoalteromonas piscicida 1UB and fructooligosaccharide enhance growth performance and protect the whiteleg shrimp (Litopenaeus vannamei) against WSSV and Vibrio harveyi coinfection.

P. piscicida 1Ub and FOS were evaluated for their potential synbiotic effects on growth, immunological responses, and disease resistance against white spot syndrome virus and V. harveyi coinfection, the major pathogen in whiteleg shrimp aquaculture. Four different supplemented diets were used to feed the experimental shrimp for 40 days: control (control, no probiotic, and prebiotic), probiotic (PRO, P. piscisida 1UB 108 CFU mL-1), prebiotic (PRE, FOS 0.5% w/w), and the synbiotic (SYN, PRO + PRE). Shrimp's body weight, weight gain, specific growth rate, feed conversion ratio, survival, digestive enzyme activity, and metabolism-related gene expression were all evaluated on day 40. After 40 days, shrimp were infected with WSSV as the primary infection and V. harveyi as the secondary infection 24 h later. Shrimp were then grown for seven days and fed with a control diet. Survival, total hemocyte count (THC), differential hemocyte, phenol-oxidase (PO), respiratory burst activity (RB), and immune-gene expression were all analyzed at 0, 3, and 7 days after infection. The results showed that the PRO, PRE, and SYN supplementation improves whiteleg shrimp growth performance, immune responses, and protection against WSSV and V. harveyi coinfection. The increased activity of digestive enzymes and metabolism-related genes correlates with higher growth performance. The increase in THC, PO, RB, and immune-related gene expression after coinfection was associated with a significant reduction in shrimp mortality. Our findings also suggest that supplementing with synbiotics improves the overall performance of whiteleg shrimp significantly more than probiotics or prebiotics only.

Effects of Compound Feed Attractants on Growth Performance, Feed Utilization, Intestinal Histology, Protein Synthesis, and Immune Response of White Shrimp (Litopenaeus Vannamei).

To investigate the effects of compound attractants on the growth performance, feed utilization, intestinal morphology, protein synthesis, and immune response of Litopenaeus vannamei, the following seven diets were formulated: a positive control (P), a negative control (N), and five diets with compound attractants which were labeled as A, B, C, D, and E, each with four of five tested attractants (yeast extract, squid visceral powder, fish soluble, and squid paste, shrimp paste), respectively. Shrimp (0.71 ± 0.00 g) were distributed to seven groups of four replicates and fed for 7 weeks. Results showed that the final body weight, feed intake, specific growth rate, and weight gain of shrimp in the B and D groups were the greatest. Hemolymph total antioxidant capacity of shrimp in the B, D, and E groups reached the highest level. In the hepatopancreas, the activity of total nitric oxide synthase, malondialdehyde content, the expression levels of sod, myd88, eif4e2, and raptor in shrimp fed the B diet were the highest, and the highest levels of dorsal and relish were observed in the C group. In the intestine, intestinal muscle thickness and expression levels of toll and eif2α in the C group were the highest, while the highest expression level of sod and relish occurred in the B group. In summary, the B and E diets promoted the feed intake, growth performance and the antioxidant enzyme activity of L. vannamei. The C diet enhanced the protein synthesis of shrimp. Regression analysis indicated that the WG and FI of shrimp were increased as the dietary inclusion levels of squid paste and shrimp paste increased, while they were decreased as the dietary inclusion levels of yeast extract and fish soluble increased.

Inflammation and apoptosis pathways mediated the stress response of Litopenaeus vannamei to acute cold and air exposure during waterless live transportation: Based on ultrastructure and transcriptome.

the combination of acute cold (AC) and waterless duration (WD) constitutes the major environmental stress and induces the damage or even mortality to shrimp L. vannamei during live transport, whereas the responding mechanism to AC + WD at molecular level remains unknown. The present study aims to clarify the responding mechanism of L. vannamei to AC + WD stress by ultrastructural observation and transcriptomic analysis on hepatopancreas tissue. The results showed that the dramatical oxidative stress induced by AC + WD significantly mediated the alteration of amino acids and energy metabolism. Furthermore, KEGG pathway enrichment analysis revealed that the genes including DDO, GOT1, IDH1 and BBOX1 involved in energy metabolism and were significantly down-regulated, while some apoptosis- and inflammation-related genes such as DRONC, AP-1, and COX-2 were significantly up-regulated under AC + WD stress in comparison with those at normal control (all p < 0.05 or 0.01). These findings suggested that metabolic processes mediate the stress-induced damages of L. vannamei during waterless transport. Moreover, the significant overexpression of apoptosis-and inflammation-related proteins, and levels of inflammation cytokines in serum of shrimps strongly demonstrated the implication of inflammation and apoptosis pathways in stress-induced ultrastructural damage. These findings deepen our understanding into the response mechanisms of L. vannamei to AC + WD stress and provide the potential controlling biomarkers for transportation management.

Isolation and characterization of Vibrio owensii from Palk Bay and its infection study against post larvae of Litopenaeus vannamei.

Vibrio is heterotrophic ubiquitous marine bacteria that plays dual role as putative halobiont and potential pathogen. Environment and diseases are inextricable hence the role of vibrio as a potential pathogen in the natural environment must be comprehended. Hence the present study aims at investigating the pathogenicity of Vibrio owensii on the post larvae of Litopenaeus vannamei. V. owensii isolated from the marine natural habitat of the Palk Bay province in India was highly resistant to ampicillin, methicillin, tetracycline and vancomycin. The strain also lacked pathogenicity against the post larvae of L. vannamei due to the absence of major virulence factors viz. Chitinase, phospholipase and hemolytic activity. Presumably this is the first report on the occurrence of V. owensii in the Indian waters therefore there arises a need to carry out more serious research on the pathogenicity of this species on other commercial crustaceans reared in the Indian aquaculture settings in order to apprehend its role as potential pathogen or the contrary.

Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae.

The emergence of Vibrio diseases, including acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp., had resulted in heavy losses in global shrimp production. Biofloc technology is a closed aquaculture system developed as one of the sustainable solutions to increase system resilience in the shrimp industry. In this study, biofloc was formed externally (ex situ biofloc) with probiotics Bacillus sp. strain BME and Bacillus sp. strain BCE, diatom microalgae Chaetoceros calcitrans, and a consortium of nitrifying bacteria, in the ratio of 1:1:6:6 as a starter. The study showed that the ex situ biofloc supplementation in Pacific whiteleg shrimp (L. vannamei) postlarvae culture can increase the shrimp culture performance (shrimp survival and growth), reduce Vibrio counts in the water and shrimp body, and provide stimulation of the shrimp immune response through humoral immune responses, such as pattern recognition protein (C-type lectin) and melanization process (proPO). Overall, the results indicate that the supplementation of ex situ biofloc provided protection to shrimp under Vibrio infection, regardless of the timing of addition (before, simultaneously, or after addition of Vibrio sp. strain VPA). This suggests that the ex situ biofloc can be effective as a preventive and a supportive treatment against potential AHPND infection in L. vannamei postlarvae culture. Taken together, the ability of the ex situ biofloc to modulate immune-related gene expression and resistance of L. vannamei against potentially AHPND-causing Vibrio sp. strain makes it an effective aquaculture technology for infectious disease control in shrimp production with high-density and minimal water exchange culture. KEY POINTS: • Supplementation of ex situ produced biofloc in shrimp postlarvae culture. • Ex situ biofloc reduces Vibrio counts in the water and shrimp body. • Ex situ biofloc stimulates shrimp humoral immune responses and survival.

The Combined Use of Pediococcus pentosaceus and Fructooligosaccharide Improves Growth Performance, Immune Response, and Resistance of Whiteleg Shrimp Litopenaeus vannamei Against Vibrio parahaemolyticus.

In this study, we evaluated the effect of probiotic bacteria Pediococcus pentosaceus supplemented at different inclusion levels in a control diet [basal diet containing 0.5% fructooligosaccharide (FOS)] on the growth performance, feed conversion ratio, immune response, and the disease resistance of whiteleg shrimp Litopenaeus vannamei juveniles against Vibrio parahaemolyticus. A control diet with 0.5% FOS but without P. pentosaceus supplementation (Control) was prepared. In addition, three other test diets were also formulated: control diet supplemented with P. pentosaceus at (i) 1 × 106 cfu g-1 diet (P1), (ii) 1 × 107 cfu g-1 diet (P2), or (iii) 1 × 108 cfu g-1 diet (P3). After a 60-day feeding trial, the experimental shrimps were challenged with V. parahaemolyticus. The results showed that dietary supplementation of P. pentosaceus significantly improved the growth performance and immune responses of L. vannamei juveniles. The juveniles that were fed with a P2 or P3 diet recorded the maximum increase in the final body weight, final length, weight gain, and survival rate. The total hemocyte counts, phenoloxidase, and lysozyme activity of shrimp fed with either of these two diets were significantly enhanced. The results also showed that juveniles fed with a P2 or P3 diet exhibited significantly lower mortality when challenged with V. parahaemolyticus. Overall results suggested that a combination of P. pentosaceus at the inclusion level of 1 × 107 cfu g-1 diet (P2) and 0.5% FOS could be considered as a potential synbiotic formulation for improving the growth, health, and robustness of L. vannamei.

Energy metabolism response of Litopenaeus vannamei to combined stress of acute cold exposure and waterless duration: Implications for physiological regulation and waterless live transport.

Maintaining the homeostasis of energy metabolism is crucial for organism's stress tolerance and survival. Acute cold exposure (AC) and waterless duration (WD) represent the two predominate abiotic stressors during waterless live transport of Litopenaeus vannamei. Although previous reports have explored the physiological response of L. vannamei to combined stress AC + WD, the roles of energy metabolism response in regulation of stress tolerance remains unknown. The present study comparatively examined the variations of energy metabolism-related indicators in hemolymph (cortisol, hemocyanin, glucose and lactate), hepatopancreas and muscle tissues (levels of lactate and glycogen, activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase), and ATP levels). Combined stress significantly disturbed the homeostasis of energy metabolism with the increase in levels of hemocyanin, glucose and lactate, and decrease in glycogen and ATP content (P < 0.05). In addition, the activities of HK, PFK, PK, and SDH initially elevated and then decreased with the prolongation of combined stress from 3h to 9h duration, while the activity of lactate dehydrogenase (LDH) remained gradual elevation and ATPase activity decreased in a duration time dependent manner throughout the experiment. These alterations revealed that exposure to combined stress could accelerate anaerobic metabolism at initial stage and inhibit aerobic metabolism in a duration time-dependent manner, following with the reduction of energy biosynthesis and the disturbance of energy metabolism equilibrium. On the other hand, the progressive impairment on hepatopancreas tissue was observed under combined stress. In summary, the deficiency of ATP supply and histopathological injures on hepatopancreas tissue might the underlying mechanisms inducing mortality of L. vannamei during live transport.

Purification, Identification and Molecular Docking of Immunomodulatory Peptides from the Heads of Litopenaeus vannamei.

In order to realize the high-value utilization of Litopenaeus vannamei (L. vannamei) heads, immunomodulatory peptides were prepared from the enzymatic hydrolysate of L. vannamei heads, and the action mechanism of immunomodulatory peptides was determined by molecular docking. The results showed that six proteases were used to hydrolyze L. vannamei head proteins, with the animal protease hydrolysate exhibiting the highest macrophage relative proliferation rate (MRPR). The enzymatic products were then sequentially purified by ultrafiltration, Sephadex G-15 gel chromatography, identified by liquid chromatography-mass spectrometry (LC-MS/MS), and finally selected for six immunomodulatory peptides (PSPFPYFT, SAGFPEGF, GPQGPPGH, QGF, PGMR, and WQR). These peptides maintained good immune activity under heat treatment, pH treatment, and in vitro gastrointestinal digestion. Molecular docking analysis indicated that these peptides showed great binding to both toll-like receptor 2 and 4 (TLR2 and TLR4/MD-2), leading to immunomodulation. The discarded L. vannamei heads in this article are considered to be promising food-borne immunomodulators that contribute to enhancing the immune function of the body.

Combined stress of acute cold exposure and waterless duration at low temperature induces mortality of shrimp Litopenaeus vannamei through injuring antioxidative and immunological response in hepatopancreas tissue.

High mortality is a frequent occurrence during live transport of shrimp species and the biochemical mechanism remains unknown. This study aimed to explore the influence of combined stress of acute cold exposure (AC) and waterless duration (WD) on survivability and biochemical response of shrimp L. vannamei during live transport. The shrimps in NC and AC groups remained the total survivability throughout the experiment while the shrimps exposed to AC + WD stress exhibited significantly higher mortality since 6h afterwards (P < 0.05) and the median survival time was calculated at 10.46 h. Moreover, the typical combined stress points at AC + WD3h, AC + WD6h and AC + WD9h were assigned for exploring the immunological and antioxidative responses. For immunity response, the total hemocyte counts (THC) decreased with the prolongation of duration time and the activities of non-specific immunity enzymes such as phenol oxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine transaminase (ALT) were significantly elevated in AC + WD9h groups (P < 0.05). Moreover, compared with that in NC group, the significant accumulation of reactive oxygen species (ROS) was observed in AC group and then reduced in combined stress groups (P < 0.05), with the highest level of malonaldehyde (MDA) in AC and AC + WD3h groups. Overall, the significant elevation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) was detected in AC + WD9h group (P < 0.05). Furthermore, the accumulative pathological impairment on hepatopancreas tissue revealed the cytoskeleton degradation. In addition, correlation analyses visualized the correlation between oxidative stress and biochemical response. This study not only deepens our understanding on the biochemical mechanism of shrimp mortality induced by combined stress, but also provides a potential strategy for improving the management of L. vannamei during live transport.

Doing More with Less: A Comparison of 16S Hypervariable Regions in Search of Defining the Shrimp Microbiota.

The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

Litopenaeus vannamei CK2 is involved in shrimp innate immunity by modulating hemocytes apoptosis.

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

Carbohydrate sources deferentially influence growth performances, microbial dynamics and immunomodulation in Pacific white shrimp (Litopenaeus vannamei) under biofloc system.

This study aims to evaluate the influence of different carbohydrate sources on water quality, growth performance and immunomodulation in pacific white shrimp and to find an alternate for molasses in biofloc system. The experiment consists of 8 biofloc treatments with different carbon sources, C1 (maida flour), C2 (wheat flour), C3 (gram flour), C4 (millet flour), C5 (rice flour), C6 (corn flour), C7 (molasses), C8 (multigrain flour) and un-supplemented control C0 was conducted in 200 L tank system for 120 days. Shrimp juveniles of average weight 1 g were stocked at the rate of 300 nos/m3. Shrimp reared in C8, C7 and C4 treatments had similar growth, survival rate, and disease resistance and were significantly higher (P < 0.05) than other treatments including control. Immune parameters like total hemocyte count (THC) and prophenoloxidase (ProPO) activity showed significantly higher (P < 0.05) levels in biofloc treatment groups. The genes targeting the proPO cascade (PX, BGBP) and antioxidant defense systems (SOD, MnSOD, CAT) revealed significant upregulation in the transcript levels indicating an enhancement in the immune-regulatory functions in the BFT groups. The results suggest that millets and multigrain flour can effectively replace molasses as the carbohydrate source for biofloc system and the biofloc system offers higher growth, survival, and immunomodulation than control.

Detoxification and immunoprotection of Zn(II)-curcumin in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxin B1.

Aflatoxins, which was produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing or storage, could cause severe health problems and reduction of yield during shrimp cultures. To evaluate toxic effects of aflatoxin B1 (AFB1) in juvenile Pacific white shrimp (Litopenaeus vannamei) and potential protective effect of Zn(II)-curcumin (Zn-CM), four experimental diets (control, 500 µg/kg AFB1, 500 µg/kg AFB1+100 mg/kg Zn-CM, 500 µg/kg AFB1+200 mg/kg Zn-CM) were formulated in quadruplicate to feed the shrimp for 8 weeks. The results revealed that AFB1 could induce significant decrease in final body weight (FBW), weight gain (WG, %) and visible variations of the hepatopancreas structures in L.vannamei. Compared with AFB1 group, AFB1+100 mg/kg Zn-CM group significantly ameliorated the toxic effects of AFB1 on growth performance, while AFB1+100 mg/kg Zn-CM group had no effect on growth performance. Dietary AFB1+100 mg/kg Zn-CM enhanced phenoloxidase (PO) (P < 0.05) activity. Both dietary AFB1+100 mg/kg Zn-CM and AFB1+200 mg/kg Zn-CM reduced inducible nitric oxide synthase (iNOS) activity and glutathione (GSH) level, decreased the content of malondialdehyde (MDA) (P < 0.05) in hepatopancreas compared with AFB1 group. Transmission electron microscopy (TEM) analysis demonstrated that Zn-CM could relieve the microvilli transformation and mitochondria accumulation reduction caused by AFB1. Consequently, the results demonstrated that suitable Zn-CM could mitigate the AFB1-induced hepatotoxicity and immunotoxicity effects on L.vannamei.

Isolation of Potential Bacteria as Inoculum for Biofloc Formation in Pacific Whiteleg Shrimp, Litopenaeus vannamei Culture Ponds.

BACKGROUND AND OBJECTIVE: A new green technology to reduce environmental damages while optimizing production of Pacific Whiteleg shrimp, Litopenaeus vannamei was developed known as "Biofloc technology". Microbial communities in biofloc aggregates are responsible in eliminating water exchange and producing microbial proteins that can be used as supplemented feed for L. vannamei. This study aimed to isolate and identify potential bioflocculant-producing bacteria to be used as inoculum for rapid formation of biofloc. MATERIALS AND METHODS: For the purpose of this study, bacterial communities during 0, 30 and 70 days of culture (DOC) of L. vannamei grow-out ponds were isolated and identified through phenotypic and 16S rDNA sequences analysis. Phylogenetic relationships between isolated bacteria were then evaluated through phylogenetic tree analysis. One-way analysis of variance (ANOVA) was used to compare the differences of microbial communities at each DOC. RESULTS: Out of 125 bacterial isolates, nine species of bacteria from biofloc were identified successfully. Those bacteria species were identified as Halomonas venusta, H. aquamarina, Vibrio parahaemolyticus, Bacillus infantis, B. cereus, B. safensis, Providencia vermicola, Nitratireductor aquimarinus and Pseudoalteromonas sp., respectively. Through phylogenetic analysis, these isolates belong to Proteobacteria and Firmicutes families under the genera of Halomonas sp., Vibrio sp., Bacillus sp., Providencia sp., Nitratireductor sp. and Pseudoalteromonas sp. CONCLUSION: In this study, bioflocculant-producing bacteria were successfully identified which are perfect candidates in forming biofloc to reduce water pollution towards a sustainable aquaculture industry. Presence of Halomonas sp. and Bacillus sp. in all stages of biofloc formation reinforces the need for new development regarding the ability of these species to be used as inoculum in forming biofloc rapidly.

Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures.

The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (<12.5 µM) neutralized the toxic effects of IMNV at loads sufficient to kill 50% of shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

Immune gene expression for diverse haemocytes derived from pacific white shrimp, Litopenaeus vannamei.

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-ß-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to ß-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated.

RNA-Seq analysis reveals genes associated with resistance to Taura syndrome virus (TSV) in the Pacific white shrimp Litopenaeus vannamei.

Outbreak of Taura syndrome virus (TSV) is one of the major pathogens of the Pacific white shrimp Litopenaeus vannamei. Although selective breeding for improvement of TSV resistance in L. vannamei has been successfully developed and has led to a great benefit to the shrimp farming industry worldwide. The molecular mechanisms underlying the viral resistance in shrimp remain largely unknown. In the present study, we conducted the first transcriptomic profiling of host responses in hemolymph and hemocytes in order to identify the differentially expressed genes associated with resistance to TSV in L. vannamei. High-throughput RNA-Seq was employed, obtaining 193.6 and 171.2 million high-quality Illumina reads from TSV-resistant and susceptible L. vannamei lines respectively. A total of 61,937 contigs were generated with an average length of 546.26 bp. BLASTX-based gene annotation (E-value < 10(-5)) allowed the identification of 12,398 unique proteins against the NCBI non-redundant NR database. In addition, comparison of digital gene expression between resistant and susceptible strains revealed 1374 significantly differentially expressed contigs (representing 697 unigenes). Gene pathway analysis of the differentially expressed gene set highlighted several putative genes involved in the immune response activity including (1) pathogen/antigen recognition including immune regulator, adhesive protein and signal transducer; (2) coagulation; (3) proPO pathway cascade; (4) antioxidation; and (5) protease. The expression patterns of 22 differentially expressed genes involving immune response were validated by quantitative real-time RT-PCR (average correlation coefficients 0.94, p-value < 0.001). Our results provide valuable information on gene functions associated with resistance to TSV in L. vannamei.

Uso de efluentes da carcinicultura de águas interiores na irrigação do arroz / Use of inland shrimp farm effluent for rice irrigation

O trabalho objetivou avaliar a produção de arroz e as alterações químicas do solo, em resposta à irrigação com o efluente da carcinicultura de águas interiores e comparar os resultados com aqueles obtidos com a irrigação convencional, utilizando água do Rio Jaguaribe. A produção de grãos obtida com o uso do efluente da carcinicultura foi semelhante àquela obtida com o uso da água de rio, quando foi utilizada na adubação uma dose de N-P-K equivalente a 100% da dose recomendada para a cultura. O uso do efluente na irrigação proporcionou maior produção de grãos em relação à irrigação com a água de rio, quando foi utilizada uma dose de N-P-K equivalente a 75% daquela recomendada para a cultura do arroz. Após o cultivo, o solo irrigado com o efluente apresentou maiores níveis de Na+, CEes e PST em relação ao solo irrigado com a água do Rio Jaguaribe.

This study aimed to evaluate rice yield and soil chemical alterations in response to inland shrimp farm effluent use for irrigation, comparing the results to conventional irrigation, using the Jaguaribe River water. Rice yield obtained with the effluent was similar to that obtained with river water irrigation, when the N-P-K dose applied was equivalent to 100% of the crop recommended dose. Effluent irrigation produced higher grain yield as compared to river water irrigation when the N-P-K dose applied was equivalent to 75% of the rice recommended dose. After the crop was harvested the soil irrigated with the effluent presented higher levels of Na+, EC and ESR, as compared to the soil irrigated with the Jaguaribe River water.