Shrimp Immune Response Research Articles

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 μg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 μg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 μg/body weight) was administered than the group fed with a lower concentration of LPS (4 μg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

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.

Dietary cholesterol enhances osmoregulation, antioxidant defenses and immune response of Litopenaeus vannamei to alleviate the macromolecular damage induced by salinity stress

Cholesterol is an important essential nutrient for crustaceans. This experiment investigated the effects of dietary cholesterol on the osmoregulation, antioxidant defense, immune response and salinity stress resistance of Pacific white shrimp (Litopenaeus vannamei). Six diets (Chol 1.0, Chol 1.4, Chol 1.8, Chol 2.2, Chol 2.6, and Chol 3.0) with cholesterol levels of 0.1%, 0.14%, 0.18%, 0.22%, 0.26% and 0.30% were prepared and fed on shrimp (1.27 ± 0.18 g) for 56 d, followed by the low salinity stress experiment for 144 h. Sampling was conducted at 56 d and 24, 72, 144 h during salinity stress. At 56 d, cholesterol enhanced the growth performance of L. vannamei by promoting transport and absorption of endogenous lipid. Dietary 0.22%–0.30% cholesterol could enhance the antioxidant defense and immune response of shrimp. After salinity stress, Na+, Cl− and K+ ion concentrations and osmolality in hemolymph were decreased significantly (P < 0.05), and the Na+ concentration and osmolality of hemolymph in Chol 2.2–3.0 groups were significantly higher than those in Chol 1.0–1.4 groups (P < 0.05). After 24 h and 72 h of salinity stress, Na+/ K+- ATPase (NKA) activity in Chol 2.2–3.0 groups was significantly higher than that in Chol 1.0–1.4 groups (P < 0.05). The plasma dopamine (DA), norepinephrine (NE) and serotonin (5-HT) contents increased significantly at 24 h and recovered at 144 h in all groups (P > 0.05) with no significant differences in biogenic amines contents among all groups at the same time points (P > 0.05), indicating that cholesterol could improve the osmoregulation of L. vannamei by directly enhancing NKA activity independent of the regulation of biogenic amines. Dietary 0.26%–0.30% cholesterol could enhance the osmoregulation and salinity stress resistance of shrimp and protect against biomacromolecule damage induced by oxidative stress and ion toxicity. Insufficient cholesterol intake (below 0.1%) may presumably cause the death of shrimp by affecting the NKA conformation. Under physiological conditions, the optimum level of dietary cholesterol in shrimp feed was about 0.22%, while it was 0.26% - 0.3% under salinity stress.

Identification of a Yorkie homolog from Litopenaeus vannamei as a negative regulator in anti-WSSV immune response

Hippo signaling pathway is a serine threonine kinase cascade that is evolutionary conserved with well-established roles in organ size control, development, tumorigenesis and immunity. As its core molecule, Yorkie also plays an important role against pathogen. In this study, we cloned and characterized a Yorkie homolog from Litopenaeus vannamei, designed as LvYKI, which has a 1650 bp open reading frame. It has the characterized domains of Yokie family, and displayed to be close to the insects and crustacean. Quantitative Real-time PCR showed that LvYKI had different regulatory mechanisms in different tissues. The transcriptional level of Lvyki was down-regulated in gill, while up-regulated in hepatopancreas post white spot syndrome virus (WSSV) infection. Moreover, the expression and phosphorylation of LvYKI was reduced upon WSSV infection, which indicated that LvYKI was involved in WSSV infection. Furthermore, RNAi was performed to evaluate the role of LvYKI in shrimp immune responses. Knocking down of Lvyki resulted in inhibition of the transcription of WSSV gene ie1 and vp28, and delayed mortality of shrimp post WSSV infection. Meanwhile, the apoptosis of hemocyte was increased as well. All results suggested that shrimp can promote apoptosis to resist WSSV infection mediated by down-regulation of LvYKI. In addition, it was found that LvYKI could interact with Lvβ-catenin, which cross-linked the Wnt and Hippo signaling pathway in innate immunity. Conclusively, our study provided clues that LvYKI plays an important role in the interaction between shrimp and virus. It will promote our understanding of the molecular mechanism in innate immunity.

Effect of extracellular macromolecules from lactic acid bacteria on the immune response of black tiger shrimp (Penaeus monodon)

Extracellular polymeric substances (EPS) are known as prebiotic-like compounds that promote the growth of beneficial bacteria in the intestinal tract of humans and animals. To demonstrate the prebiotic potential of EPSs produced from lactic acid bacteria, Lactobacillus plantarumand Bifidobacterium bifidum, this study demonstrated the influence of these bacteria on the immune response in shrimp to infection with pathogenic bacteria. Vibrio parahaemolyticusdisease. The results showed that the diet supplemented with different types of EPS stimulated immunity of black tiger shrimp against acute hepatopancreatic necrosis disease, also known as early mortality syndrome in shrimp caused by Vibrio parahaemolyticus. The addition of EPS to shrimp feed significantly increased total white blood cell count, respiratory burst activity, phenoloxidase and superoxide dismutase activity. The study results show that EPS produced by lactic acid bacteria shows potential as a prebiotic, which can become an alternative to the use of antibiotics in shrimp farming to inhibit the growth of pathogens.

Dietary SYNSEA probiotic improves the growth of white shrimp, Litopenaeus vannamei and reduces the risk of Vibrio infection via improving immunity and intestinal microbiota of shrimp

The growth performance, immunological status, and intestinal microbiology of white shrimp, Litopenaeus vannamei, were evaluated after dietary administration of the commercial probiotic SYNSEA. Shrimp were fed a control diet (without probiotic supplement) and two levels of SYNSEA probiotic, a low concentration of SYNSEA (LSL) containing 105 CFU (g diet)−1Bacillus subtilis and 105 CFU (g diet)−1 lactic acid bacteria (LAB), and a high concentration of SYNSEA (LSH) containing 106 CFU (g diet)−1B. subtilis and 106 CFU (g diet)−1 LAB, for 12 weeks. Shrimp fed with the LSL diet significantly increased growth performance as well as final weight and feed efficiency compared to the control, but not the LSH diet. After being orally challenged with Vibrio parahaemolyticus, shrimp fed with LSL diet prior to the challenge or fed with LSL and pathogen simultaneously showed significantly lower mortality compared to the control. SYNSEA probiotic significantly improved shrimp immune response, including lysozyme activity in LSL and LSH groups, and phagocytic activity in the LSL group in comparison to the control. In addition, the gene expressions of anti-lipopolysaccharide factor 2 in LSL and LSH groups, and penaeidin 4 in LSL were also up-regulated. Although there was no significant difference among groups for hepatopancreas and intestinal morphology, the muscular layer thickness and villi height were slightly improved in the intestines of shrimp fed SYNSEA. The 16S rDNA gene amplicon sequence analysis using next-generation sequencing revealed a significant decrease in α-diversity (Margalef's species richness) after oral administration of SYNSEA due to an increase in the relative abundance of beneficial bacteria in the gut flora of shrimp, such as Lactobacillus, Shewanella, and Bradymonadales and a decrease in harmful bacteria, such as Vibrio, Candidatus_Berkiella, and Acinetobacter baumannii. Together the data suggest that the provision of SYNSEA probiotic at 105 CFU (g diet)−1B. subtilis and 105 CFU (g diet)−1 LAB can improve shrimp growth, enhance immunity, and disease resistance status of the host. In addition, these findings conclude that SYNSEA probiotic has great preventive and therapeutic potential for Vibrio infection in shrimp aquaculture.

The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei

SH2-domain-containing protein tyrosine phosphatases (PTPs), belonging to the class I PTP superfamily, are responsible for the dephosphorylation on the phosphorylated tyrosine residues in some proteins that are involved in multiple biological processes in eukaryotes. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway transduce signaling responding to interferons and initiate cellular antiviral responses. The activity of the JAK/STAT pathway is generally orchestrated by the de-/phosphorylation of the tyrosine and serine residues of JAKs and STATs, in which the dephosphorylation processes are mainly controlled by PTPs. In the present study, an SH2-domian-contianing PTP, temporally named as LvPTPN6, was identified in Litopenaeus vannamei. LvPTPN6 shares high similarity with PTPN6s from other organisms and was phylogenetically categorized into the clade of arthropods that differs from those of fishes and mammals. LvPTPN6 was constitutively expressed in all detected tissues, located mainly in the cytoplasm, and differentially induced in hemocyte and gill after the challenge of stimulants, indicating its complicated regulatory roles in shrimp immune responses. Intriguingly, the expression of LvPTPN6 was regulated by interferon regulatory factor (IRF), which could directly bind to the LvPTPN6 promoter. Surprisingly, unlike other PTPN6s, LvPTPN6 could promote the dimerization of STAT and facilitate its nuclear localization, which further elevated the expression of STAT-targeting immune effector genes and enhanced the antiviral immunity of shrimp. Therefore, this study suggests a PTPN6-mediated regulatory approach from IRF to the JAK/STAT signaling pathway in shrimp, which provides new insights into the regulatory roles of PTPs in the JAK/STAT signaling pathway and contributes to the further understanding of the mechanisms of antiviral immunity in invertebrates.

Dietary fish oil levels modulated lipid metabolism, immune response, intestinal health and salinity stress resistance of juvenile Penaeus monodon fed a low fish-meal diet

Dietary fish meal (FM) and fish oil (FO) replacement are crucial for the sustainable development of aquaculture. An eight weeks feeding trial was conducted to evaluate the potential replacing FM with soy protein concentrate, and the suitable FO level for a low FM diet of Penaeus monodon. A diet containing 25% FM was used as the control (C diet), and three other diets were formulated to contain 15% FM, and supplemented with 3%, 2%, 1% FO [high FO (HF), medium FO (MF) and low FO (LF), respectively]. The experiment was conducted in quadruplicates (30 shrimp per replicate, average weight 1.00 ± 0.01 g). The results indicated that besides the final body weight, there were no differences in weight gain, feed efficiency and survival rate observed among the four groups. The results of the acute salinity stress showed that shrimp fed the C and MF diets showed a similar survival rate, which was significantly higher than those fed a LF diet. Shrimp that were fed HF, MF and LF diets showed a lower level of fatty acid transport-related mRNA expression in the hepatopancreas, compared with those fed the C diet. Shrimp fed HF, MF and LF diets showed lower expression levels of superoxide dismutase, endoplasmic reticulum protein 57 and immune deficiency (IMD) in the hepatopancreas than those fed the C diet. Shrimp that were fed a LF diet showed the lowest expression levels of inhibitor of apoptosis proteins, Toll and tumor necrosis factor receptor associated factor 6 (TRAF6) in the hepatopancreas, as well as the highest expression levels of Toll, extracellular signal-regulated kinase, Relish and TRAF6 in the intestine among four groups. Intestinal microstructure damage was observed in shrimp that were fed a LF diet, and intestinal endoplasmic reticulum (ER) swelling was observed in shrimp fed MF and LF diets. In conclusion, these results indicated that the growth performance of shrimp was not affected when the FM and FO contents decreased, whereas a low content of FM negatively influenced the immune response in shrimp by modulating IMD and ER stress related gene expression. Under low FM conditions, a suitable dietary FO level would benefit to the lipid transport, hepatopancreas and intestine health, as well as the immune response of shrimp.

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.

Effect of bovine lactoferricin on the growth performance, digestive capacity, immune responses and disease resistance in Pacific white shrimp, Penaeus vannamei

The present study evaluated the growth performance, digestive enzyme activity, non-specific immunity, immunity and growth genes in Penaeus vannamei fed diets supplemented with Bovine lactoferricin (the basal diet without Bovine lactoferricin, the control; 1.0‰ Bovine lactoferricin，LCB1; 1.5‰ Bovine lactoferricin，LCB1.5; 2.0‰ Bovine lactoferricin, LCB2; 2.5‰ Bovine lactoferricin, LCB2.5) for 56 days. The feeding trial showed that the final weight, weight gain rate, and specific growth rate of the shrimp were improved significantly, while the feed conversion ratio was reduced significantly in the LCB1.5 group compared to the control (P < 0.05). The challenge test of Vibrio parahaemolyticus showed that the cumulative mortalities of shrimp in the LCB1.5, LCB2 and LCB2.5 groups were significantly lower than that in the control (P < 0.05). Compared with the control, Lipase and Trypsin activities in the hepatopancreas of LCB1.5 and LCB2 groups were significantly enhanced (P < 0.05). Compared with the control, alkaline phosphatase, acid phosphatase activities in the hepatopancreas and the relative expression levels of Relish, Toll, JAK, STAT, TOR, Raptor, 4E-BP, eIF4E1α, eIF4E2 genes in the hepatopancreas of LCB1.5, LCB2 and LCB2.5 groups were all significantly enhanced (P < 0.05). These results suggested that dietary Bovine lactoferricin could improve the growth performance, digestive capacity and immune responses of shrimp. When resistance against Vibrio parahaemolyticus in shrimp is considered, high dosage of Bovine lactoferricin showed a better effect than low dosage of Bovine lactoferricin. However, high dosage of Bovine lactoferricin can have a negative impact on the growth performance of shrimp. Considering collectively the above, Bovine lactoferricin could improve the growth performance, digestive enzymes activities, immune responses and disease resistance of P. vannamei.

Oral administration of white spot syndrome virus surface proteins VP24 and VP28 modulates immune gene expression in Penaeus monodon juveniles

Being primitive organisms, crustaceans such as shrimp and crab rely on innate immunity to defend pathogens. White spot syndrome virus is a deadly pathogen that causes total mortality in farmed shrimp. Although shrimps do not possess an adaptive immune system and immune memory, there are efforts to prevent mass mortality due to WSSV through the administration of antigenic proteins derived from the virus. In this paper, we describe the effectiveness of the two major WSSV antigenic proteins, VP24 and VP28, in eliciting an immune response in shrimp. Recombinant proteins rVP28 and rVP24 were mass-produced in E. coli and orally administered to Penaeus monodon juveniles. During the administration of recombinant antigens, differential expression analysis of shrimp biodefense genes was carried out. Among the genes analyzed, transcripts of TLR, Syntenin, PmRACK, Rab7, SOD and C-type Lectin exhibited upregulation during the administration of both rVP28 and rVP24 at least at a one-time point. Independently, rVP28 administration upregulated Lectin, PmRACK, Rab7, SOD, STAT, TLR and Syntenin genes at least at one-time point. Penaeidin and Crustin are either downregulated or similar to that of control. The rVP24 administration upregulated Crustin, Lectin, Penaeidin, PmRACK, Rab7, SOD, TLR and Syntenin. The STAT was either downregulated or similar to that of the control at least at one-time point. The results revealed the extent of the antigenic property and the ability of rVP28 and rVP24 to elicit an immune response after oral administration.

Integrated Analysis of mRNA-Seq and MiRNA-Seq Reveals the Molecular Mechanism of the Intestinal Immune Response in Marsupenaeus japonicus Under Decapod Iridescent Virus 1 Infection.

The intestine is not only an important digestive organ but also an important immune organ for shrimp; it plays a key role in maintaining homeostasis. Decapod iridescent virus 1 (DIV1) is a new type of shrimp-lethal virus that has received extensive attention in recent years. To date, most studies of the shrimp intestinal immune response under viral infections have relied on single omics analyses; there is a lack of systematic multi-omics research. In the current study, intestinal mRNA-seq and microRNA (miRNA)-seq analyses of Marsupenaeus japonicus under DIV1 infection were performed. A total of 1,976 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs (DEMs) were identified. Among them, 21 DEMs were negatively correlated with 194 DEGs from a total of 223 correlations. Functional annotation analysis revealed that M. japonicus can regulate glycosaminoglycan biosynthesis (chondroitin sulfate, dermatan sulfate, and keratan sulfate), vitamin metabolism (retinol metabolism and ascorbate and aldarate metabolism), immune pathway activation (Toll and IMD signaling pathways, Wnt signaling pathway, IL-17 signaling pathway, and Hippo signaling pathway), immunity enzyme activity promotion (triose-phosphate isomerase), antimicrobial peptide (AMP) expression, reactive oxygen species (ROS) production, and cell apoptosis through miRNAs to participate in the host’s antiviral immune response, while DIV1 can influence Warburg effect-related pathways (pyruvate metabolism, glycolysis/gluconeogenesis, and citrate cycle), glycosphingolipid biosynthesis-related pathways (glycosphingolipid biosynthesis—globo and isoglobo series and glycosphingolipid biosynthesis—lacto and neolacto series), and the tight junction and adhesion junction of the intestinal mucosal epithelium through the host’s miRNAs and mRNA to promote its own invasion and replication. These results indicate that intestinal miRNAs play important roles in the shrimp immune response against DIV1 infection. This study provides a basis for further study of the shrimp intestinal antiviral immune response and for the formulation of effective new strategies for the prevention and treatment of DIV1 infection.

A Glutathione Peroxidase Gene from Litopenaeus vannamei Is Involved in Oxidative Stress Responses and Pathogen Infection Resistance.

In shrimp, several glutathione peroxidase (GPX) genes have been cloned and functionally studied. Increasing evidence suggests the genes’ involvement in white spot syndrome virus (WSSV)- or Vibrio alginolyticus-infection resistance. In the present study, a novel GXP gene (LvGPX3) was cloned in Litopenaeus vannamei. Promoter of LvGPX3 was activated by NF-E2-related factor 2. Further study showed that LvGPX3 expression was evidently accelerated by oxidative stress or WSSV or V. alginolyticus infection. Consistently, downregulated expression of LvGPX3 increased the cumulative mortality of WSSV- or V. alginolyticus-infected shrimp. Similar results occurred in shrimp suffering from oxidative stress. Moreover, LvGPX3 was important for enhancing Antimicrobial peptide (AMP) gene expression in S2 cells with lipopolysaccharide treatment. Further, knockdown of LvGPX3 expression significantly suppressed expression of AMPs, such as Penaeidins 2a, Penaeidins 3a and anti-lipopolysaccharide factor 1 in shrimp. AMPs have been proven to be engaged in shrimp WSSV- or V. alginolyticus-infection resistance; it was inferred that LvGPX3 might enhance shrimp immune response under immune challenges, such as increasing expression of AMPs. The regulation mechanism remains to be further studied.

MiR-10c Facilitates White Spot Syndrome Virus Infection by Targeting Toll3 in Litopenaeus vannemei.

Toll-like receptors (TLRs) are canonical cell membrane receptors functioning to recognize pathogens and transduce signals to activate immune responses. It has been known that Toll3 in Pacific white shrimp Litopenaeus vannamei (LvToll3) plays a critical role in antiviral immunity by inducing the transcription of interferon regulatory factor (IRF), which mediates a signaling axis that is similar to the interferon system of vertebrates. However, the regulatory mechanism of the Toll3-IRF signaling is still unclear. In this study, a novel microRNA (miRNA) of miR-10 family, temporarily named as miR-10c, was identified from L. vannamei. miR-10c may play a nonnegligible regulatory role in shrimp immune responses since it was constitutively expressed in all detected tissues and transcriptionally induced by immune stimulation. Functional analysis validated that miR-10c could target LvToll3 to inhibit its expression, through which miR-10c blocked the nuclear translocation of IRF and facilitated white spot syndrome virus (WSSV) infection. To our knowledge, the present study revealed the first report of a Toll targeted by miRNA in crustaceans and provided a solid evidence base for supporting the role of LvToll3 in antiviral defense by activating IRF signaling in L. vannamei. Identification of the miR-10c/Toll3/IRF regulatory axis in shrimp provides new insights into the participation of miRNA in the regulation of immune responses and contributes to in-depth understanding of the mechanisms of Toll-induced immune responses in L. vannamei.

Shrimp immune response: A transcriptomic perspective

AbstractDuring the last 15 years, a significant amount of data related to the crustacean immune system has been generated, mainly led by the advances made in the field of transcriptomic analysis through massive mRNA sequencing technology. In order to shed new light on the genes and metabolic pathways associated with the immune system in shrimp species, we have carried out a meta‐analysis using the current literature and the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI), which has 1195 published transcriptomic data sets until 31 December 2020, from different tissues and 14 Penaeus species. The transcriptomic analyses published so far have explored the effects of the challenge from viral and bacterial pathogens, as well as abiotic stressors, such as ammonia, temperature, salinity, oxygen deprivation and pH. They have also explored the effect of dietary factors and beneficial microbes (pre‐ and probiotics) in the immune response. In order to harness the vast amount of information available, multidisciplinary efforts have to generate better genomic tools to overcome the complexity of the genome and to implement the knowledge gained into commercial applications for the improvement of the culture conditions, adaptation of more sustainable feeds and producing strains showing more resilience/tolerance to diseases.

Rearing of White Leg Shrimp Litopenaeus vannamei (Boone, 1931) in Biofloc and Substrate Systems: Microbial Community of Water, Growth and Immune Response of Shrimp

The microbial composition of rearing water, growth and immune status of Litopenaeus vannamei juveniles in biofloc (T1), substrate-integrated biofloc (T2), substrate (T3) systems and a control with four replicates each were evaluated in a 49-day indoor trail. In each HDPE tank of 70 L capacity filled with 10 g L-1 salinity water, ten shrimp (4.56±0.13 g) were stocked. The C: N ratio of 15:1 was maintained in T1 and T2 using wheat flour as carbon source for production of biofloc. The TAN, NO2 and NO3 were lower (P&lt;0.05) in treatment tanks than that in control. It was also observed that the counts of Bacillus, Lactobacillus, Vibrio and zooplankton were high in T2 than T1, T3 and control. There was higher net weight gain (10.38±0.14 g) and lowest FCR (1.27±0.12) with T2 when compared to T1, T3 and control tanks. Moreover, the survival rate is significantly higher in treatments than control. Significant increase in THC (47.24±4.49 x 106 cells ml-1), serum protein (82.67±0.01 mg/ml), Phenoloxidase (0.73±0.03, OD 490 nm) and Lysozyme activity (56.32±0.03%) was observed in T2 than T1, T3 and control. The result shows that substrate-integrated biofloc system assures higher growth, survival and better immune response in L. vannamei.

Non-specific immune response of Pacific white shrimp Litopenaeus vannamei by supplementation of sodium alginate of Sargassum collected from Lampung Indonesia

This study aims to determine the effectiveness of supplementation of sodium (Na) alginate Sargassum sp. from Lampung waters to enhance the non-specific immune response and the resistence of Pacific white shrimp (Litopenaeus vannamei) against white spot disease. This study used a completely randomized design with 3 treatments, namely feeding without alginate supplementation (A) or control, dietary Na alginate Sargassum supplementation at a dose of 2.0 (B), and 4.0 g kg−1 feed (C), each with four replications, for 14 days. Hemolymph sampling was performed on days 0, 7 and 14 to observe the parameter of shrimp hematology included total haemocyte count (THC), phagocytocyte activity (PA), phagocytic index (PI), and total plasma protein (TPP). The hepatopancreas histology profile and water quality were observed at the end of treatment. Data were analyzed by Anova at 95% of confidence interval and continued with Duncan test. Result showedthat the supplementation of Na alginat from Sargassum sp. significantly enhance several shrimp immune response namely THC, PA, and TPP. Meanwhile, the histology of the hepatopancreas of both shrimp treatment and control showed not significantly effect on tissue damage. The water quality was still in normal condition during the treatment. The results of this study indicate that the application of Sargassum alginate supplementation at a dose of 2gr/kg of feed is the best treatment to enhance the immune response of Pacific white shrimp.

Co-culture of Indian white shrimp, Penaeus indicus and seaweed, Gracilaria tenuistipitata in amended biofloc and recirculating aquaculture system (RAS)

A 60 days nursery-based co-culture of Indian white shrimp, Penaeus indicus along with seaweed, Gracilaria tenuistipitata trial was carried out in Recirculatory Aquaculture System (RAS) of 6000 Liters capacity tank (FRP, reinforced fibre plastics) by using biofloc technology (BFT) to evaluate the growth, survival performance and immune potential. Accordingly a completely randomized experiment was designed with six treatments. These treatments were: T1- Static system (Control), T2 - RAS with BFT, T3 - RAS with Gracilaria, T4 - RAS and BFT customized with bacterial consortium, T5 - RAS with BFT + Gracilaria + bacterial consortium, T6 - Static + Gracilaria + BFT (without consortium and without RAS). Shrimp post larvae (0.08 ± 0.03 g) were stocked at a density of 3000 nos./ton. RAS with BFT along with the seaweed and bacterial consortium showed significantly higher growth and survival compared to control (T1) and treatments without BFT (T3). The control (T1) exhibited significantly lower growth, weight gain, specific growth rate, yield and higher FCR value than that of the RAS + BFT (T2), RAS+ seaweed (T3) and RAS + BFT or their combinations with bacterial consortium (T4 and T5). The treatment having seaweed without BFT (T3) showed higher survival than the static control (T1), but no significant difference in growth. The highest ABW of 4.23 ± 0.39 was obtained in T4 (BFT customized with consortium) followed by T5 (BFT + seaweed + bacterial consortium) and lowest was observed in T1 (2.14 ± 0.4). Similarly, the highest survival (92.33 ± 1.52) and production (10.39 ± 0.24 kg/ton) was observed in T5. Significantly higher NH4-N and NO2-N was accumulated in the control (T1) in comparison with other treatment like T4 and T5. Immune parameters viz., PPO activity, Total Serum Protein and Total Hemocyte Count (THC) showed significantly higher values in all the treatments compared to control. This study establishes the novel concept of incorporation of macroalgae in biofloc based RAS improving the production performance and immune response of shrimp during nursery as well as grow-out phase.

KLF13 induces apoptotic cell clearance in Penaeus vannamei as an essential part of shrimp innate immune response to pathogens

Although, in mammals, the Krüppel-like transcription factor 13 (KLF13) plays an essential role in cell proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation, and inflammation, its role in penaeid shrimp is unclear. In the current study, we characterized a KLF13 homolog in Penaeus vannamei (PvKLF13), with full-length cDNA of 1677 bp and 1068 bp open reading frame (ORF) encoding a putative protein of 355 amino acids, which contains three ZnF_C2H2 domains. Sequence and phylogenetic analysis revealed that PvKLF13 shares a close evolutionary relationship with KLF13 from invertebrates. Transcript levels of PvKLF13 were ubiquitously expressed in shrimp and induced in hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, and white spot syndrome virus (WSSV), suggesting the involvement of PvKLF13 in shrimp immune response to pathogens. Besides, knockdown of PvKLF13 decreased hemocytes apoptosis in terms of increased expression of pro-survival PvBcl-2, but decreased expression of pro-apoptotic PvBax and PvCytochrome C, coupled with high PvCaspase3/7 activity, especially upon V. parahaemolyticus challenge. The findings here indicate the involvement of PvKLF13 in apoptotic cell clearance as an essential part of shrimp innate immune response to pathogens.

NADPH oxidase involved in immune response via regulating the expression of antioxidant genes in Litopenaeus vannamei

Free radicals such as reactive oxygen species (ROS) play a significant role in the innate immune system. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) are found to be involved in ROS production and played crucial roles in host biological processes of humans, insects, and plants. However, there are relatively few studies on the function of Noxes in crustaceans. In this study, the full-length cDNA of Nox was cloned from Litopenaeus vannamei by rapid amplification of cDNA ends (RACE) to obtain a 3790 base pairs (bp) cDNA sequence. LvNox cDNA comprised a 5’-untranslated region (UTR) of 454 bp, a 3’-UTR of 333 bp, and a putative open reading frame (ORF) of 3006 bp. Structural analysis showed that LvNox had a transmembrane helical segment (THS) at positions 233-255, a flavin adenine dinucleotide (FAD) binding domain (at positions 447-579), and a nicotinamide adenine dinucleotide (NAD) binding domain (at positions 815-990). Tissue distribution analysis revealed that LvNox mRNA was expressed in heart, intestine, muscle, gill, hepatopancreas, stomach, epidermis, hemocyte, and eyestalk. High expression levels of LvNox were observed in epidermis, hemocyte and eyestalk. The expression levels of LvNox were significantly induced after Vibrio alginolyticus injection for 6, 12, 24 and 48 h, and after Cu exposure for 24 and 48 h. Furthermore, the RNA interference of LvNox significantly reduced the production of ROS and the expression levels of copper/zinc superoxide dismutase (Cu-Zn SOD), catalase (CAT) and nitric oxide synthase (NOS). These results indicated that LvNox could be induced by V. alginolyticus infection and Cu exposure, which might be involved in immune response of shrimp via regulating the expression of antioxidant genes. These findings provide a solid foundation for further study of the function of Noxes in crustaceans.