Crayfish Mortality Research Articles

PHB2 inhibits WSSV replication by promoting the nuclear translocation of STAT

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses and cell proliferation. However, the function of the PHBs in immune regulation has largely not been determined. In the present study, we identified PHB2 in the red swamp crayfish Procambarus clarkii. PHB2 was found to be widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge. PHB2 significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. Here, we observed that PHB2 promotes the nuclear translocation of STAT by binding to STAT. After blocking PHB2 or STAT with antibodies or interfering with PHB2 or STAT, the expression levels of the antiviral genes β-thymosin (PcThy-4) and crustin2 (Cru2) decreased. The gene sequence of PHB2 was analyzed and found to contain a nuclear introgression sequence (NIS). After in vivo injection of PHB2 with deletion of NIS (rΔNIS-PHB2), the nuclear translocation of STAT did not change significantly compared to that in the control group. These results suggest that PHB2 promoted the nuclear translocation of STAT through NIS and mediated the expression of antiviral proteins to inhibit WSSV infection.

The smaller, the better? First evaluation of growth and mortality in crayfish internally tagged with p-Chips

Abstract Small-bodied aquatic animals present a challenge to researchers seeking to uniquely mark individuals for scientific study. Microtransponder tags, such as p-Chips, represent the smallest electronic animal tags available to meet this need. The use of p-Chips to tag freshwater crayfishes, however, has not been explored. The goal of this study, therefore, was to determine the effects of p-Chip tagging on growth and survival of crayfishes in a controlled laboratory environment. We also investigated potential influences from variables such as sex, reproductive form, number of molt events, and crayfish size on mortality. We internally tagged individuals of the woodland crayfish Faxonius hylas (Faxon, 1890) (12.2–26.6 mm carapace length; CL) with either one p-Chip (single-tag) or one p-Chip and one visual implant elastomer tag (double-tagged) and observed the effects over a period of 90 days. Survival probability over time was not statistically different (P > 0.05) among tagging groups, sex, and reproductive form. Survival rates were similar across all tagging groups, with 75% in the control group, 77% in the double-tagged group, and 78% in the single-tag group. A strong correlation, however, was observed between survival and the number of molt events. Additionally, there was a negative correlation between survival and crayfish size (CL), indicating a higher risk of mortality for larger individuals. There were no statistically significant differences in growth in CL (P = 0.30) or mass (P = 0.19) among the tagging groups. We conclude that p-Chips are a viable tagging option for the study of crayfishes given their size, readability, and retention through molting cycles. We recommend that future studies repeat this experiment using smaller individuals to determine the minimum crayfish size compatible with p-Chip tagging. It is also important to test p-Chips with other crayfish species and compare the growth and mortality of crayfish tagged with p-Chips in natural habitats.

Mannose-binding C-type lectin from Procambarus clarkii exhibited antimicrobial activity to mediate crayfish innate immunity

Mannose-binding lectin (MBL), which is a member of the C-type lectin (CTL) family, plays crucial roles in non-self recognition and the clearance of invading microorganisms. In this study, an MBL (designated as PcMBL) was cloned and characterized from the crayfish Procambarus clarkii. PcMBL cDNA consisted of 816 bp encoding a protein with 271 amino acid residues. The deduced PcMBL contained a signal peptide, three low-complexity regions, and a carbohydrate-recognition domain, as found in most CTLs. The mRNA encoding PcMBL was detected in all the examined tissues. PcMBL expression in hemocytes and intestines was significantly upregulated after stimulation with Vibrio parahaemolyticus and Staphylococcus aureus. RNA interference studies revealed that PcMBL silencing remarkably downregulated the transcription of five antimicrobial peptide genes, namely, Anti-lipopolysaccharide factor 1 (ALF-1), ALF-4, ALF-10, Crustin 1 (Crus-1), and Crus-4. Recombinant PcMBL protein was used for the functional analysis. The results showed that rPcMBL had the capacity to bind to all seven tested microorganisms and three carbohydrates. rPcMBL facilitated the clearance of V. parahaemolyticus and S. aureus and inhibited the formation of biofilms and growth of the two bacteria. In addition, rPcMBL could reduce the mortality of crayfish infected with V. parahaemolyticus or S. aureus. All these results suggest that PcMBL may be involved in immune recognition and pattern elimination in the innate immunity of P. clarkii.

Dietary quercetin protects Cherax quadricarinatus against white spot syndrome virus infection

Quercetin is a plant flavonoid with a molecular formula C15H10O7. It has antioxidant, anti-inflammatory, antibacterial, and anti-apoptotic effects in animals. We used red claw crayfish (Cherax quadricarinatus) infected with white spot syndrome virus (WSSV) to investigate quercetin's effects on innate immunity of crustaceans. Quercetin supplementation significantly reduced the mortality of crayfish caused by WSSV infection and the number of VP28 copies in WSSV-infected crayfish. Quantitative real-time PCR analysis showed that dietary quercetin supplementation increased the expression of immune-related genes, like JAK, STAT and ALF. Quercetin supplementation affected the activity of six immune-related enzymes and increased the total number of hemocytes in crayfish. It also significantly reduced the rate of hemocyte apoptosis in both WSSV-infected and uninfected crayfish. These results demonstrate the potential for commercial use of quercetin for the prevention of WSSV disease in crustaceans.

Enhance the biocontrol efficiency of Bacillus velezensis Bs916 for white spot syndrome virus in crayfish by overproduction of cyclic lipopeptide locillomycin

White spot disease, caused by white spot syndrome virus (WSSV), is a serious crustacean disease in China, and currently, there is no specific treatment for it. In this study, the addition of locillomycin could help to improve the cumulative survival rate of WSSV-infected crayfish. Then further analysis showed that Spo0A and SpoIIID could negatively regulate the production of locillomycin in Bacillus velezensis Bs916, and the disruption of them caused a significant increase in the production of locillomycin, which was 8.9 times higher than that of wild-type. Subsequently, we investigated the protective effects of B. velezensis Bs916 and ΔSpo0A + SpoIIID as probiotics, and the results showed that addition of their cultures to the basal control diet significantly reduced the mortality of WSSV-challenged crayfish. Further results indicated that dietary B. velezensis Bs916 or ΔSpo0A + SpoIIID supplement reduced the copy number of WSSV and improved the immune activities of acid phosphatase (ACP), alkaline phosphatase (AKP), nitric oxide synthase (NOS) and lysozyme (LZM) in crayfish. Moreover, compared with B. velezensis Bs916, all above performances were further enhanced when ΔSpo0A + SpoIIID was provided, so we speculated that overproduction of locillomycin might contribute to the enhancement of biocontrol efficiency of B. velezensis Bs916 for WSSV in crayfish. In summary, this study provides some insight into the therapeutic or prophylactic intervention with B. velezensis Bs916 or its metabolic product, locillomycin, to protect crayfish against WSSV infection, and also provides a theoretical basis for the construction of high yielding locillomycin strains.

Identification and characterization of a Reeler domain containing protein in Procambarus clarkii provides new insights into antibacterial immunity in crustacean

Crayfish, as an invertebrate, relies only on the innate immune system to resist external pathogens. In this study, a molecule containing a single Reeler domain was identified from red swamp crayfish Procambarus clarkii (named as PcReeler). Tissue distribution analysis showed that PcReeler was highly expressed in gills and its expression was induced by bacterial stimulation. Inhibiting the expression of PcReeler by RNA interference led to a significant increase in the bacterial abundance in the gills of crayfish, and a significant increase in the crayfish mortality. Silencing of PcReeler influenced the stability of the microbiota in the gills revealed by 16S rDNA high-throughput sequencing. Recombinant PcReeler showed the ability to bind microbial polysaccharide and bacteria and to inhibit the formation of bacterial biofilms. These results provided direct evidence for the involvement of PcReeler in the antibacterial immune mechanism of P. clarkii.

Dietary glycerol monolaurate protects Cherax quadricarinatus against white spot syndrome virus infection

Glycerol monolaurate (GML), one of the medium-chain fatty acid esters, is often used as an emulsifier or preservative. Its biological functions include antibacterial and antiviral activities. In this study, we examined the effects of dietary GML on the resistance of the red claw crayfish to WSSV infection. Crayfish fed with 4 g/kg GML showed higher survival rate and lower WSSV copy numbers than the control after WSSV infection. A RT–qPCR analysis showed that GML supplementation enhanced the expression of immune-related genes, especially JAK and caspase. Our data indicate that GML affects the immune parameters of crayfish, including the total hemocyte counts and phenoloxidase, acid phosphatase, superoxide dismutase, lysozyme, and peroxidase activities. After treatment with GML, the apoptosis of hemocytes increased significantly in both WSSV-infected and uninfected crayfish. In summary, GML reduced the mortality of WSSV-infected crayfish, perhaps by modulating the innate immunity of the crayfish. Our study shows that GML can be used to induce the innate immunity and enhance the immune protection of the red claw crayfish against WSSV infection, either therapeutically or as a preventive measure.

Impact of aluminum exposure on oxidative stress, intestinal changes and immune responses in red swamp crayfish (Procambarus clarkii)

Aluminum (Al) is an abundant metal that has been classified as a threatening pollutant due to indiscriminate use and anthropogenic activities. This study aimed to evaluate the impacts of Al on crayfish (Procambarus clarkii), including biochemical change, histological alteration, gut microbial community diversification, and immune changes. The bioaccumulation of Al was detected in the hemolymph and intestine of crayfish after Al exposure at different time points. Results showed that Al exposure significantly induced oxidative stress and caused pathohistological changes on intestinal barrier structures in crayfish. It was found that the intestinal microbiota was affected by retained Al and the intestinal community diversity was changed after Al treated in the crayfish. Furthermore, Al exposure affected the immunity in crayfish, by altering the expression of a set of immune-related genes, as well as reducing the phenoloxidase and lysozyme activities. Moreover, Al exposure promoted hemocytes apoptosis and impaired hemophagocytic capacity against Vibro parahamolyticus, resulting in higher mortality of crayfish upon bacterial infection. Taken these results together, we conclude that excessive Al exposure caused adverse effects on multiple biological processes of crayfish and Al pollution is a potential threat to crayfish culture.

Field estimates of crayfish mortality associated with passive integrated transponder tagging

Abstract Mark–recapture studies of crayfish using internally implanted passive integrated transponder (PIT) tags is a promising method for understanding details of crayfish ecology. The size of crayfish that can be marked is dependent on the size of the PIT tag. Several studies have examined mortality rates associated with the insertion of the smallest currently available PIT tags (Biomark MiniHPT8, 8.4 × 1.4 mm) using different insertion locations and husbandry conditions. Here, carapace length‐ and sex‐dependent mortality rates were evaluated for three ventral PIT tag insertion locations (central, pleopod and lateral) in a field experiment focusing on the northern crayfish (Faxonius virilis) in the Gila River Basin where it was introduced several decades ago. The central insertion method was the only treatment group that did not exceed background mortality in two trials for all individuals combined. When evaluated by sex, no insertion method exceeded control mortality in males, and only the lateral insertion method exceeded control mortality in females. The relationship between carapace length and mortality was different depending on the PIT tag insertion location and sex. Regardless of sex or size, F. virilis responded with lower mortality rates when the PIT tag was inserted using the central insertion method, and this method is preferred for tagging F. virilis of ≥20 mm carapace length. Crayfish are large‐bodied macroinvertebrate omnivores with important roles in freshwater ecosystems. This article identifies best practices for internal PIT tag placement, provides estimates of mortality associated with tagging, and thereby advances ecological research focused on the conservation of imperilled or sensitive crayfishes. It also provides useful guidance for studies of non‐native crayfish in communities where they have become invasive.

ScFv antibody-mediated targeted drug delivery system improves the antiviral activity of geniposidic acid against WSSV

White spot disease (WSD) caused by white spot syndrome virus (WSSV) is one of the most dangerous diseases in the shrimp production industry, but there is no effective drug to control WSSV infection to date. The main challenges are the existence of biological barriers and untargeted drug delivery. In this study, a single-chain fragment variable (scFv) antibody-mediated WSSV-targeting drug delivery system was constructed for WSD targeting therapy. Natural compound geniposidic acid (GPA) could directly inhibit the expression of WSSV structural proteins VP24, VP26, and VP28 in Drosophila S2 cells. Yeast two-hybrid and intracellular co-localization showed that scFv antibody P75E8 could recognize and bind to VP28. Indirect ELISA showed that P75E8 had a good affinity to WSSV. Moreover, single-walled carbon nanotubes, GPA, and P75E8 were respectively selected as the nanocarrier, anti-WSSV drug, and targeting ligand to construct targeted drug delivery system SWCNTs-GPA-P75E8. Compared with GPA, SWCNTs-GPA-P75E8 showed a stronger anti-WSSV activity by inhibiting WSSV replication in crayfish and reducing the mortality of WSSV-infected crayfish. The mortality of WSSV-infected crayfish decreased by 23.33% and 46.67% after treatment with GPA and SWCNTs-GPA-P75E8 for 240 h, respectively. The results of GPA metabolism showed that SWCNTs-GPA-P75E8 could transport more GPA to target tissues of WSSV. Taken together, SWCNTs-GPA-P75E8 showed stronger WSSV-targeting ability and anti-WSSV activity. WSSV-specific antibody-mediated targeted drug delivery system is an effective strategy for WSD targeting therapy.

Natural component geniposide enhances survival rate of crayfish Procambarus clarkii infected with white spot syndrome virus

White Spot Disease (WSD), caused by white spot syndrome virus (WSSV), is an acute and highly lethal viral disease of shrimp. Currently, there are no commercially available drugs to control WSD. It is urgent and necessary to find anti-WSSV drugs. Natural compounds are an important source of antiviral drug discovery. In this study, the anti-WSSV activity of natural compound geniposide (GP) was investigated in crayfish Procambarus clarkii. Results showed that GP had a concentration-dependent inhibitory effect on WSSV replication in crayfish at 24 h, and highest inhibition was more than 98%. In addition, GP significantly inhibited the expression of WSSV immediate-early gene ie1, early gene DNApol, late gene VP28. The mortality of WSSV-infected crayfish in control groups was 100%, while it reduced by 70.0% when treated with 50 mg/kg GP. Co-incubation, pre-treatment and post-treatment experiments showed that GP could prevent and treat WSSV infection in crayfish by significantly inhibiting WSSV multiplication. Mechanistically, the syntheses of WSSV structural proteins VP19, VP24, VP26 and VP28 were significantly inhibited by GP in S2 cells. Furthermore, GP could also suppress WSSV replication by blocking the expression of antiviral immunity-related factor STAT to reduce ie1 transcription. Moreover, GP possessed anti-inflammatory and anti-oxidative activity in crayfish. Overall, GP has the potential to be developed as a preventive or therapeutic agent against WSSV infection.

The effects of Agaricus bisporus polysaccharides enriched diet on growth, nonspecific immunity and disease resistance in crayfish (Procambarus clarkii)

In order to accelerate the application of ABPs in shrimp and crab feed, we evaluated the effect of ABPs on the growth, non-specific immunity and disease resistance of crayfish. Crayfish (Procambarus clarkii) were randomly divided into six groups, the fed diets supplemented with different levels of ABPs: 0, 0.05, 0.15, 0.3, 0.45 and 0.6 g/kg. After 28 days of feeding, ABPs were found to significantly increase FBW and WG of crayfish. The addition of 0.15–0.6 g/kg of ABPs to the feed significantly increased the activities of ACP, AKP and T-SOD, decreased the activity of MDA enzymes in the hepatopancreas of crayfish. In the hemolymph, ABPs at 0.3–0.45 g/kg can significantly increase the activities of ACP, AKP, T-SOD and LZM enzymes, while the addition of 0.15 or 0.6 g/kg has no significant difference with the control group. The results of qPCR showed the addition of 0.15–0.6 g/kg ABPs to the feed could significantly up-regulate the expression levels of gpx, lec and hsp70 mRNA in the hepatopancreas (P < 0.05). Adding 0.15–0.6 g/kg of ABPs could up-regulate the expression of ctsb and lec gene in gills. In addition, the expression of alf was up-regulated in gills only when the addition amount of ABPs was 0.3 g/kg. While the expression of mtMnsod in gills was up-regulated when the addition amount was 0.3–0.6 g/kg. Addition of 0.05–0.6 g/kg of ABPs reduced the mortality of crayfish infected by A.hydrophila. The above results indicated that the addition of ABPs could improve the growth, non-specific immunity and disease resistance of crayfish, and the addition of 0.3 g/kg and 0.45 g/kg of ABPs might be beneficial to the culture of crayfish.

Crayfish (Procambarus clarkii) TRPA1 is required for the defense against Aeromonas hydrophila infection under high temperature conditions and contributes to heat sensing.

Temperature is an important environmental factor influencing immune responses of crayfish. However, the mechanism underlying how temperature affects immune responses remains unclear. Here, we identified an ortholog of the transient receptor potential ankyrin subtype 1 (TRPA1), a temperature sensor of Drosophila, from Procambarus clarkii (PcTRPA1-1). Its expression was induced by high temperature and challenge with heat-killed A. hydrophila at high temperature, but not at lower temperature. PcTRPA1-1 silencing led to increased mortality of crayfish challenged with live A. hydrophila at high temperature (32°C), but had no statistically significant effect on crayfish mortality at 24°C. This suggests that PcTRPA1-1 is involved in the immune responses of crayfish at high temperature as a potential temperature sensor. Further assay exhibited that PcTRPA1-1 silencing affected immune responses of crayfish, including increase of lipid peroxidation, reduction of total antioxidant capacity, decreased phenoloxidase activity and disruption of circadian rhythm of total hemocyte count entrained by temperature cycles. PcTRPA1-1 silencing also decreased the expression of PcHSP70 and PcHSP90 which are responsive to heat stimuli and bacterial challenge. The results collectively indicate that TRPA1 contributes to heat sensing of crayfish and is required for crayfish defense against bacterial infection.

Effects of dietary quercetin on the innate immune response and resistance to white spot syndrome virus in Procambarusclarkii

In recent years, the use of natural products with immune-stimulating and antimicrobial properties has attracted increasing attention in aquaculture researches. In our study, the effect of diet supplemented with quercetin, a flavonoid commonly found in some types of plants substance on the innate immune response and disease resistance in crayfish (Procambarus clarkii) against white spot syndrome virus (WSSV) is reported. It was found that dietary 40 mg/kg quercetin significantly reduced the mortality of crayfish and WSSV copy number after WSSV challenge. Dietary quercetin increased catalase (CAT), and lysozyme (LZM) activity in crayfish. Dietary quercetin increased the expression of NF-κB, anti-lipopolysaccharide factor (ALF) and toll-like receptor (TLR) genes in crayfish. The apoptosis rate of hemocyte was increased by quercetin supplement in crayfish. Our results suggest that dietary quercetin may affect the innate immunity of crayfish and protect crayfish from WSSV infection.

Use of glycerol monolaurate as a treatment against white spot syndrome virus in crayfish (Procambarus clarkii)

Glycerol monolaurate (GML), a monoester formed from glycerol and lauric acid, is widely used in food, cosmetics, and dietary supplements. GML also has antiviral activity against enveloped viruses. In this study, we conducted a challenge experiment to assess the effects of GML on the survival of crayfish (Procambarus clarkii) infected with white spot syndrome virus (WSSV). Dietary GML treatment reduced the mortality of WSSV-infected crayfish and reduced the WSSV copy number. Quantitative reverse transcriptase polymerase chain reaction analysis showed that GML enhanced the expression of immune-related genes, such as nuclear factor-κB and C-type lectin. GML increased three immune parameters (total hemocyte count, phenoloxidase activity, and superoxide dismutase activity) in crayfish with or without WSSV infection. GML also increased the hemocyte apoptosis rate in crayfish with or without WSSV infection. These data indicated that GML increased hemocyte apoptosis, THC, PO and SOD activity and reduced the mortality in crayfish after oral WSSV challenge, which suggested that GML can be used to keep the crayfish healthy.

Effect of dietary sodium butyrate on the innate immune response of Procambarus clarkii and disease resistance against white spot syndrome virus

Sodium butyrate is a compound used as a feed additive in animal production. Sodium butyrate regulates electrolyte balance, gastrointestinal micro-ecology, intestinal mucosal nutrients, and also acts as an immunostimulant. It has great potential research value. The effect of sodium butyrate on the innate immunity of crustaceans was investigated in this study. Procambarus clarkii (crayfish) was used as a model organism and healthy crayfish were challenged with White spot syndrome virus (WSSV). The results showed that the group treated with sodium butyrate had significantly lower levels of mortality caused by WSSV, and the replication of WSSV was also reduced to a certain extent. Fluorescence quantitative PCR showed that sodium butyrate could up-regulate the expression of two immune-related genes, including C-type-lectin and prophenoloxidase. It was found that sodium butyrate treatment had positive effects on the total number of hemocyte (THC), phenoloxidase activity (PO), superoxide dismutase activity (SOD), and CAT activity of crayfish. In addition, we found that whether infected with WSSV or not, sodium butyrate treatment can significantly reduce the hemocyte apoptosis rate of crayfish. The results show that sodium butyrate can regulate the innate immunity of crayfish and reduce crayfish mortality following WSSV infection. This study provides evidence for the novel use of sodium butyrate to regulate the immunity of crayfish, treat WSSV infection, and provide a new theoretical basis for the research of sodium butyrate on crustaceans as a new additive for aquatic feed.

The SNP polymorphisms associated with WSSV-resistance of prophenoloxidase in red swamp crayfish (Procambarus clarkii) and its immune response against white spot syndrome virus (WSSV)

Prophenoloxidase (proPO), a critical enzyme in innate immune, has been extensively studied in various crustacean species including red swamp crayfish Procambarus clarkii. However, little is known about the genetic structure of proPO from P. clarkii and its role in antivirals. In this study, the expression pattern of proPO upon white spot syndrome virus (WSSV) infection was investigated, and the proPO genomic DNA sequence was obtained from P. clarkii for the first time and analyzed. The results indicated that the mRNA level of proPO could be significantly upregulated in haemocytes, hepatopancreas, gill and midgut. The proPO genomic DNA sequence is 9085 bp in length, which includes five exons interrupted by four introns, an 82 bp 5′-flanking region, and a 1671 bp 3′-untranslated region. Two TATA boxes as well as three repeat sequences were predicted in the intron region of proPO. Subsequently, the single nucleotide polymorphisms (SNPs) of proPO were identified and genotyped in the crayfish population and the association between SNPs and anti-WSSV traits was investigated. Three SNPs in intron regions were identified, and the SNP 7081 T > C showed a significant association (P < 0.05) with anti-WSSV traits. In addition, the role of purified recombinant protein of proPO in anti-WSSV infection was explored. The results showed that recombinant proPO significantly reduced the amount of WSSV copy number in hepatopancreas and gill compared to the control group injected with bovine serum albumin (BSA). It was found that recombinant proPO could enhance the expression of proPO in hepatopancreas, peroxinectin in midgut, and the C-type lectin (ctl) in gill and midgut. Intramuscularly injection of juvenile crayfish with recombinant proPO could significantly decrease 65% mortality of WSSV-infected crayfish at 10 days as compared to the control group with BSA. The findings of this study suggested that the proPO is an important molecule in the antiviral immune system of crustacean and could provide theoretical for molecular marker-assisted WSSV resistance breeding of P. clarkii.

Antiviral activity of geniposidic acid against white spot syndrome virus replication in red swamp crayfish Procambarus clarkii

White spot syndrome virus (WSSV) causes high mortality in crustacean, but there are no effective therapies up to now. Medicinal plants and their bioactive ingredients are important sources of therapeutic agents. In the study, the anti-WSSV activities of 10 plant extracts were investigated in crayfish Procambarus clarkii. The results showed that the ethanol extracts of Eucommia ulmoides Oliver had the highest inhibition on WSSV replication among these tested extracts, with 84.12%. Geniposidic acid (GPA) is a major active ingredient of E. ulmoides. The contents of GPA in ethanol extracts of E. ulmoides were 32.148 mg/g. GPA could also suppress WSSV infection in vitro and in vivo. The inhibition rate was over 97% after treatment with 50 mg/kg GPA for 24 h. In addition, the mortality of WSSV-infected crayfish treated with GPA was 56.67%, while it was 100% for the controls. GPA possessed anti-inflammatory and anti-oxidative activities in crayfish. Mechanistically, GPA inhibited WSSV replication at least by reducing STAT expression to block the transcription of WSSV IE gene ie1. GPA also inhibited the expression of apoptosis related factor Bax inhibitor-1 which is beneficial for WSSV replication. In conclusion, GPA can be used as a potential agent to treat and prevent WSSV infection.

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish

Bacillus amyloliquefaciens, which is closely related to Bacillus subtilis, produces a series of metabolites that can inhibit the growth of fungi and bacteria. Here, we investigated the effect of B. amyloliquefaciens used as a probiotic on the innate immunity of the crayfish Procambarus clarkii when challenged with white spot syndrome virus (WSSV). Dietary B. amyloliquefaciens supplement significantly reduced the mortality of WSSV-challenged crayfish and reduced copy numbers of WSSV. The quantitative reverse transcription-polymerase chain reaction results showed that B. amyloliquefaciens supplement increased the expression of several immune-related genes, including Toll-like receptor, NF-κB and C-type-lectin. Further analysis showed that B. amyloliquefaciens supplement also had an effect on three immune parameters, including total hemocyte count, phenoloxidase activity and superoxide dismutase activity. In both infected and uninfected crayfish, B. amyloliquefaciens supplement significantly decreased hemocyte apoptosis. Our results showed that B. amyloliquefaciens can regulate innate immunity of crayfish and reduce the mortality following WSSV challenge. This study provides a novel insight into the potential for therapeutic or prophylactic intervention with B. amyloliquefaciens to regulate crayfish immunity and protect against WSSV infection, and also provides a theoretical basis for the use of probiotics as aquatic feed additives.

Bursicon homodimers induce the innate immunity via Relish in Procambarus clarkii

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs β), and burs α-β is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs β are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.