White Spot Syndrome Research Articles

Effect of the Flagellar Gene fliL on the Virulence of Pseudomonas plecoglossicida to Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Pseudomonas plecoglossicida is the pathogen of visceral white spot disease in marine fish, which usually occurs at 16–19 °C and has resulted in heavy economic losses. Our previous RNA sequencing revealed that the expression of the fliL gene in P. plecoglossicida was significantly up-regulated during infection of the host. In order to study the influence of the fliL gene on the virulence of P. plecoglossicida, the fliL gene of the NZBD9 strain was knocked out by the homologous recombination method, the fliL gene-deleted strain (ΔfliL strain) constructed, and complemented the fliL gene to the ΔfliL strain to obtain the C-ΔfliL strain. The growth curves of the NZBD9 strain, ΔfliL strain, and C-ΔfliL strain did not show significant differences. Compared with the NZBD9 strain, the motility, adhesion, and biofilm formation ability were tendered in the ΔfliL strain (p < 0.05); the complement of the fliL gene enhanced these abilities to the level of the NZBD9 strain. The results of artificial infection experiments showed that the LD50 of NZBD9 strain, ΔfliL strain, and C-ΔfliL strain in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) were 5.0 × 103 CFU/fish, 6.3 × 104 CFU/fish, and 1.3 × 103 CFU/fish, respectively. RNA sequencing was performed on wild-type strains and ΔfliL strains. A total of 126 differentially expressed genes (DEGs) were screened (p < 0.05), of which 114 were downregulated and 12 were upcontrolled, among which several genes related to the six-type secretion system and transport activity were significantly downregulated. The DEGs were aligned to the GO and KEGG databases and enriched to 44 GO pathways and 39 KEGG pathways, respectively. The active pathways of ABC transporters were significantly enriched in both databases. These results indicate that the fliL gene is related to the movement, biofilm formation, and adhesion ability of P. plecoglossicida, and may reduce virulence by affecting substance transport and bacterial secretion.

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

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

The MEF2 homolog of Penaeus vannamei is essential for maintaining the WSSV latent infection

White spot syndrome virus (WSSV) is a lethal shrimp pathogen that has a latent infection cycle. The latent virus can easily turn into an acute infection when the culture environment changes, leading to widespread shrimp mortality. However, the mechanism of WSSV latent infection is poorly understood. Bioinformatic analysis revealed that the promoters of WSSV latency-related genes (i.e., wsv151, wsv366, wsv403, and wsv427) contained putative myocyte enhancer factor 2 (MEF2) binding sites. This suggested that the transcription factor MEF2 may be involved in WSSV latent infection. To further investigate this, a MEF2 homolog (PvMEF2) was cloned from Penaeus vannamei and its role in WSSV latent infection was explored. The results showed that knockdown of PvMEF2 led to an increase in the copy number of WSSV, indicating reactivation of WSSV from a latent infection. It was further demonstrated that suppression of PvMEF2 significantly decreased expression of the viral latency-related genes in WSSV-latent shrimp, while overexpression of PvMEF2 in Drosophila S2 cells activated the promoter activity of the viral latency-related gene. Additionally, we demonstrated that silencing of PvMEF2 was able to upregulate the expression of pro-apoptosis genes, thereby promoting cell apoptosis during latent infection. Collectively, the present data suggest that PvMEF2 could promote the expression of virus latency-related genes and enhance cell survival to maintain WSSV latent infection. This finding would contribute to a better understanding of the maintenance mechanism of WSSV latent infection.

Comparative analysis of the effect of nano‑copper and Cu2O treatment on Cryptocaryon irritans tomonts in large yellow croaker (Larimichthys crocea) farming

Cryptocarya irritans is an obligate parasite causing the “white spot disease.” This disease often leads to the death of a large number of cultured marine fish. This study aimed to compare the effects of cuprous oxide (Cu2O) and nano‑copper on the morphology and hatchability of tomonts to investigate the effects of copper derivatives on C. irritans tomonts. The potential mechanistic difference was also explored using the transcriptome. The results showed that Cu2O seriously damaged the internal cell structure of tomonts, and the hatching rate of the tomonts in the Cu2O-treated group was lower than that in the control group within 10 days. On the contrary, the effect of nano‑copper on tomonts was not significant. The cytoplasm of tomonts treated with nano‑copper dissolved, the internal structure was slightly loose, and the hatching rate of tomonts was even slightly higher than control group within 5 days. In the transcriptome analysis, 850 differentially expressed genes (DEGs), including 332 upregulated genes and 518 downregulated genes, were identified in the Cu2O-treated groups. However, only 56 DEGs, including 34 upregulated genes and 22 downregulated genes, were identified in the nano‑copper-treated groups. This indicated that the effect of Cu2O on tomonts was greater than nano‑copper. In the enrichment pathway analysis, DEGs in the Cu2O-treated group were mostly enriched in the ribosome and apoptosis pathways. Further analysis showed that genes (RPL18e, RPLP0, RPL10, RPL17, RPL26, RPL35, and RPL37) related to cell stress and defense were significantly upregulated and those (P53 and PKA) related to cell proliferation and development were downregulated, indicating that the killing effect was mainly mediated by different ribosomal and structural proteins. However, the DEGs (KCNMA1, CACNA1C, CNGA1) in the nano‑copper-treated group were enriched in the insulin secretion and cAMP signaling pathways, which were activate part of tomonts' innate immune response, completely different from those in the Cu2O-treated group. The results provided more information on the mechanism of killing parasites by copper derivatives.

Effects of dietary peptidoglycan on Litopenaeus vannamei: Growth performance, disease resistance, non-specific immunity and transcriptome analysis of immune response

At present, peptidoglycan (PGN) is commonly used as an immunostimulant in shrimp. However, little has been known about its mechanism. In this study, comparative transcriptome analysis was employed to investigate the impact of dietary PGN on the immune response in Litopenaeus vannamei. A total of 720 healthy juvenile L. vannamei (initial weight 0.31 ± 0.05 g) were randomly evenly allocated to 18 fibreglass buckets as 6 groups, fed with six isonitrogen (41.05%) and isolipid diets (8.16%) containing PGN at the doses of 0 (control), 0.2, 1, 5, 25, and 125 mg/kg, respectively. After 8 weeks feeding trail, the growth performance, immunity, and transcriptome response of L. vannamei were analyzed. The results showed that the growth performance of L. vannamei was not significantly influenced by dietary PGN (P > 0.05). According to the analysis of weight gain rate (WGR), the optimum dietary PGN level was 71.46 mg/kg. Compared with the control group, the survival rate (SR) after Vibrio parahaemolyticus infection of L. vannamei were significantly increased by dietary PGN at 5 mg/kg and the SR after white spot syndrome virus (WSSV) infection was improved by dietary PGN at 25 mg/kg (P < 0.05). The superoxide dismutase (SOD), phenoloxidase (PO), catalase (CAT), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities could be enhanced remarkably while the maleic dialdehyde (MDA) levels were significantly decreased by dietary PGN compared with the control group (P < 0.05). Transcriptome analysis showed that there were 1350 (902 down, 448 up) and 1697 (1183 down, 514 up) DEGs in the PGN5 and PGN25 when compared with the PGN0 group, respectively. Most DEGs were significantly enriched in immune and metabolism related signalling pathways, such as antigen processing and presentation, autophagy-animal, apoptosis-multiple species, carbohydrate digestion and absorption, and amino sugar and nucleotide sugar metabolism signalling pathway. Besides, the metabolism related signalling pathways occupied top significantly enriched placement. In conclusion, moderate dietary PGN could significantly improve the non-specific immunity of L. vannamei, presumably by modulating its metabolism.

Stationary metal sheets (copper, zinc or brass) in fish tanks prevent Ichthyophthirius multifiliis Fouquet, 1876 infection of rainbow trout: In vivo and in vitro effects

Infections with the parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 challenge health of freshwater fishes worldwide by eliciting white spot disease (ichthyophthiriosis). A series of chemicals, pharmaceuticals and herbal extracts are currently in use to control the infections by regular or continuous administration to aquaculture systems. Recently application of brass (copper/zinc alloys) metal sheets in marine fish tanks was shown to eliminate tomonts of a related ciliate, Cryptocaryon irritans Brown, 1951 causing cryptocaryonosis (marine white spot disease). The present work documents that brass, and each of its pure constituents (copper and zinc), have in vivo and in vitro antiparasitic effects in freshwater. We used an experimental model with I. multifiliis infected rainbow trout (Oncorhynchus mykiss Walbaum, 1792). In the in vivo study metal sheets of either brass (alloy of copper and zinc), pure copper or pure zinc, were placed in fish tanks with trout experimentally infected with I. multifiliis (duplicated trials). All three metals (pure or combined) effectively blocked the life cycle of I. multifiliis and inhibited reinfection and morbidity. In control tanks with infected trout, but no metal sheets, we observed significantly elevated infection levels and development of disease. In vitro assays, exposing three life cycle stages (tomont, tomocyst and theront) to the same metal sheets in Petri dishes, confirmed the antiparasitic effects. Tomonts were sensitive to pure copper and brass (100% and 66% mortality within 15 min, respectively) and zinc (100% mortality within 30 min). Tomocysts, with enclosed tomites, were less sensitive, as even 12 h exposure to brass, pure copper and zinc merely led to mortality rates of 44%, 50% and 60%, respectively. Theronts were killed within 75 min when exposed to brass and pure copper plates, whereas zinc plate exposure eliminated all theronts within 60 min. Microscopical observations suggested that direct contact between the parasite and the metal sheet increased the parasiticidal effect, although the released metal ions may contribute may play a role. The use of stationary metal sheets in aquaculture settings may be a possible way to control ichthyophthiriosis. However, we measured the concentration of free metal ions in the fish tanks with metal sheets, which suggested release of metal to the water. This calls for additional studies on toxicology and environmental effects of metal sheet use before implementation at fish farms level.

A simple sequence repeats marker of disease resistance in shrimp Litopenaeus vannamei and its application in selective breeding.

The polymorphism of the simple sequence repeat (SSR) in the 5' untranslated coding region (5'-UTR) of the antiviral gene IRF (LvIRF) has been shown to be implicated in the resistance to viral pathogens in shrimp Litopenaeus vannamei (L. vannamei). In this study, we explored the potential of this (CT)n-SSR marker in disease resistance breeding and the hereditary property of disease resistance traits in offspring. From 2018 to 2021, eight populations were generated through crossbreeding by selecting individuals according to microsatellite genotyping. Our results demonstrated that shrimp with the shorter (CT)n repeat exhibited higher resistance to white spot syndrome virus (WSSV) or Decapod iridescent virus 1 (DIV1); meanwhile, these resistance traits could be inherited in offspring. Interestingly, we observed that the longer (CT)n repeats were associated with bacterial resistance traits. Accordingly, shrimp with longer (CT)n repeats exhibited higher tolerance to Vibrio parahaemolyticus infection. Taken together, these results indicate that the single (CT)n-SSR marker could be used to selective breeding for both resistance to virus and bacteria in shrimps.

Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture.

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.

Identifikasi Infeksi WSSV (White Spot Syndrome Virus) Pada Udang Vaname (Litopaneus vannamei) Dengan Metode PCR (Polimarace Chain Reaction) Di Kecamatan Wanggarasi

This study aims to identify White Spot Syndrome Virus (WSSV) infection in vannamei shrimp ponds cultivated in Wanggarasi Sub-district, Pohuwato Regency, using the Polymerase Chain Reaction (PCR) test method. The method applies descriptive analysis method, observation method, and laboratory analysis. The results showed that after being detected by PCR from 4 sampling stations, stations 3 was detected positive (+) for moderate virus infection at stations 1, 2, and 4. The results of identification using the PCR (Polymerase Chain Reaction) method at the Fish Quarantine Station and Quality Assurance of Gorontalo Province, the white spot syndrome virus (WSSV) was not detected in vannamei shrimp, or negative (-) with clinical symtomps, namely; pale color, slow movement, leaning against the embankment, there is a light spot on the head, swimming on the surface.

Replacement of dietary fish meal by fermented soybean meal improves growth, energy metabolism, and innate immune response in Penaeus vannamei Boone, 1931 (Decapoda: Dendrobranchiata: Penaeidae)

Abstract Fishmeal (FM) is considered a preferred source of protein for aquatic feed due to its good palatability and digestibility, a balanced amino acid profile, and a low level of anti-nutritional factors (ANFs). Shortage of FM supply and increases in price, however, are major challenges faced by feed producers and aquaculture farmers. Fermented soybean meal (FSBM) has been proposed to partially replace FM because of its high nutritional value and low cost. The optimal FSBM concentration for substituting FM and effects of FSBM substitution on growth performance, energy metabolism, and immune response nevertheless remain unclear. We evaluated the effects of shrimp diets containing FSBM at 0% (FM), 25% (FSBM25), 50% (FSBM50), 75% (FSBM75), and 100% (FSBM100) on the shrimp Penaeus vannameiBoone, 1931. FSBM50 led to a significantly higher weight gain (postlarvae 1927.36 ± 46.64%, young juveniles 237.00 ± 8.25%, and juveniles 153.96 ± 2.18%) than the other experimental and control diets in different stages of the shrimp. The FM group consumed less oxygen than the experimental diet groups. FSBM75 and FSBM100 led to a significantly higher ammonia-N excretion than FM, FSBM25, and FBSM50. All experimental diet groups exhibited a significantly higher O:N ratio (8.3 ± 0.8–9.4 ± 1.7) than the FM group (7.1 ± 0.2). Shrimp fed with FSBM25- and FSBM50-containing diets exhibited an increased total hemocyte count, and FSBM25, FBSM50, and FSBM100 led to a significantly higher superoxide anion production than FSBM75 and FM. FSBM substitution in shrimp diets resulted in a significantly higher phagocytic activity than the FM diet, suggesting that FSBM substitution improves the cell-mediated immune response in shrimps. In challenge trials with white spot syndrome virus (WSSV), all experimental diets led to a higher survival rate than the FM diet, and in Vibrio parahaemolyticus challenge trials, FSBM50 led to a higher survival rate than the other experimental and control diets, indicating that FSBM substitution in shrimp diets could improve the defense capacity of shrimps against WSSV and V. parahaemolyticus.

Experimental infection reveals mud crab, Scylla serrata is less susceptible than Scylla olivacea and shrimp, Penaeus vannamei to white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) causes severe mortality in farmed shrimps, besides causing infection in a wide range of crustaceans. Among the hosts, mud crabs are reported to be relatively resistant to the virus. To validate this, the present study investigated the susceptibility of two species of mud crabs, Scylla olivacea and S. serrata compared with shrimp, Penaeus vannamei to WSSV through experimental infection. Initially, to find out the pathogenicity of the virus, a quantified, 10-fold serially diluted WSSV inoculum was injected intramuscularly into shrimp and mud crabs. Cumulative mortality of 100% was observed in shrimp injected with 10−1 and 10−2 WSSV dilution at 5 and 6 day post-injection (dpi), respectively. However, injection of the virus inoculum of 10−1 dilution resulted in 100% and 85% cumulative mortality at 7 dpi in S. olivacea and S. serrata, respectively. The LD50 (lethal dose 50% endpoint) value of WSSV in P. vannamei, S. olivacea and S. serrata was determined to be 1.74 × 105, 3.80 × 106 and 8.51 × 106 viral copies per animal, respectively. PCR amplification of DNA extracted from different tissues of the experimental animals at 24, 48 and 72 h post-injection (hpi) confirmed the infection. A SYBR green-based real-time PCR assay revealed comparatively higher viral load in shrimp tissues (4.1 × 103, 2.8 × 106 and 4.3 × 107 copies/ 100 ng DNA in stomach, 1.0 × 103, 2.4 × 106 and 3.5 × 107 copies/ 100 ng DNA in gill and 3.5 × 102, 1.1 × 104 and 1.1 × 105 copies/ 100 ng DNA in hepatopancreas at 24, 48 and 72 h, respectively). The viral load in S. olivacea was estimated to be 2.9 × 103, 2.2 × 106 and 3.0 × 107 copies/ 100 ng DNA in the stomach, 7.1 × 102, 1.6 × 106 and 2.4 × 107 copies/ 100 ng DNA in gill and 2.0 × 102, 8.6 × 103& 7.8 × 104 copies/ 100 ng DNA and in S. serrata the viral copy numbers detected were 1.1 × 103, 1.7 × 106 and 1.8 × 107 copies/ 100 ng DNA in stomach, 6.6 × 102, 1.3 × 106 and 1.4 × 107 copies/ 100 ng DNA in gill and 5.8 × 101, 7.7 × 102 & 1.1 × 104 copies/ 100 ng DNA at 24, 48 and 72 h, respectively. A higher viral load was detected in the stomach tissue compared to the gill and hepatopancreas of all the experimental animals. Histological examination of the stomach and gill of P. vannamei, S. olivacea and S. serrata at different time points post-infection revealed characteristic WSSV pathology. Hepatopancreatic tubular epithelium showed no viral inclusions, though pathological changes could be noticed in the intertubular connective tissue. The most pronounced changes were found in the stomach epithelium of P. vannamei and S. olivacea, which revealed significant nuclear hypertrophy and large-sized inclusions, compared to S. serrata which showed relatively smaller-sized inclusion bodies.

Expression of Hsp70 in the red swamp crayfish, Procambarus clarkii in response to immune challenge

A heat shock protein 70 (Hsp70) was identified from Procambarus clarkii (named as PcHsp70). The open reading frame of the PcHsp70 cDNA was 1917 bp, encoding a 638 aa protein with signature sequences of the Hsp70 family. Tissue distribution results showed that PcHsp70 has relatively high expression in immune-related tissues such as hepatopancreas, gill, and hemocytes. The expressions of PcHsp70 in hepatopancreas and gill were significantly induced by Gram-positive bacterium, Gram-negative bacterium, and white spot syndrome virus stimulation, indicating that PcHsp70 may be involved in the immune response to pathogen invasion. The research will provide a foundation for understanding the resistance and stress mechanism of Hsp70 in P. clarkii.

Flagellar gene fliP contributes to the virulence of Pseudomonas plecoglossicida by regulating its motility

As an opportunistic causative pathogen of “Visceral White Spot Disease” in several kinds of teleost, Pseudomonas plecoglossicida has caused huge economic losses to aquaculture. Flagella is the motility organ of bacteria as well as a vital virulence factor. The protein encoded by the fliP gene is involved in the formation of the flagellar type III secretion export apparatus, which plays a crucial role in flagellar assembly. In this study, the ΔfliP and C-ΔfliP strains of P. plecoglossicida were constructed to explore the biological profiles mediated by the fliP gene and its effects on the virulence of this organism. Compared to the wild type strain (NZBD9) of P. plecoglossicida, the deletion strain ΔfliP exhibited a significant impairment in swimming motility, chemotaxis, adhesion, and biofilm formation abilities. The transcriptomic analysis of the NZBD9, ΔfliP and C-ΔfliP strains revealed that the fliP gene may affect the biological profiles of P. plecoglossicida by down-regulating the expression of flagellar assembly, type 6 secretion system and bacterial chemotaxis related genes. Furthermore, the results of artificial infection showed that the virulence of ΔfliP strain to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was attenuated, with a LD50 value of 2.5 × 104 CFU/fish. These findings demonstrate that the fliP gene contributes to the virulence of P. plecoglossicida by regulating its motility.

Correlation of Atmospheric Temperature with the Prevalence of White Spot Disease (WSD) and Acute Hepatopancreatic Necrosis Disease (AHPND) in the Philippines

The growing crustacean aquaculture industry in the Philippines is a significant contributor to the seafood production sector. However, several problems hinder the growth and overall market production, the main factor being diseases. Previous studies observed that temperature changes affect shrimp’s physiological and immunological responses to different pathogens and parasites; hence, this meta-analysis examined the correlation of white spot disease (WSD) and acute hepatopancreatic necrosis disease (AHPND) with atmospheric temperature changes. The analysis was conducted using the available data from the Quarterly Aquatic Animal Disease report of the Network of Aquaculture Centres in Asia Pacific, the Bureau of Fisheries and Aquatic Resources, and the Philippine Atmospheric, Geophysical, and Astronomical Services Administration. Results indicated that temperatures ranging from 27–28 °C generally cause higher disease occurrences in the Philippines, as it facilitates optimum conditions for the infectivity of the causative agents of both diseases. Statistical analysis also has shown a positive correlation of temperature to the disease occurrence, indicating that temperature monitoring is crucial, especially in the culture of shrimps in a controlled environment. This information will serve as a guide to the fish health and aquaculture industry in mitigating possible disease outbreaks.

The involvement of the T6SS vgrG gene in the pathogenicity of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida, the causative agent of white spot disease of large yellow croaker, has caused serious economic losses to the aquaculture industry. The type VI secretion system (T6SS) is a significant virulence system widely distributed among Gram-negative bacteria. VgrG, a structural and core component of T6SS, is crucial to the function of T6SS. To explore the biological profiles mediated by vgrG gene and its effects on the pathogenicity of P. plecoglossicida, the vgrG gene deletion (ΔvgrG) strain and complementary (C-ΔvgrG) strain were constructed and the differences in pathogenicity and virulence-related characteristics between different strains were analysed. The results showed that vgrG gene deletion significantly affected the virulence-related characteristics of P. plecoglossicida, including chemotaxis, adhesion, and biofilm formation. In addition, the LD50 of ΔvgrG strain was nearly 50-fold higher than that of the NZBD9 strain. Transcriptome data analysis suggested that the vgrG gene may affect the virulence of P. plecoglossicida by regulating the quorum sensing pathway to inhibit the secretion of virulence factors and affect biofilm formation. Besides, deletion of the vgrG gene may reduce bacterial pathogenicity by affecting bacterial signal transduction processes and the ability to adapt to chemotactic substances.

SIMULTANEOUS PATHOGEN DETECTION OF SHRIMP VIRUSES ON CULTURED TIGER SHRIMPS (Penaeus monodon) IN INDONESIA

The multiple-pathogen infection causes severe economic impact to shrimp industry in Indonesia and worldwide due to mass mortality and multiple abnormalities of the survived infected shrimps. However, multiple-pathogen detection tools in shrimp diseases have not yet widely used. The purpose in this study was to develop and applied simultaneous detection system using multiplex polymerase chain reaction (PCR) assay from natural infections caused by white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV) in Black tiger shrimp culture. To analyze multiple-pathogen infections in the shrimp, the study designed and used three pairs of specific primers targeting DNA virus from the shrimp diseases. All amplifications used a specific master mix for multiplex PCR assay and standardized extracted nucleic acid from the samples. This mPCR assay successfully amplified the DNA of three viruses in a single tube-run by multiplex PCR for each virus. Based on the results, the study confirms that multiple-pathogen infection contributes the highest mass mortality rather than from single infection by either WSSV, IHHNV or MBV. This study also confirms that the mPCR assay is a faster, cheaper, and efficient method to detect and subsequently prevent the spreading of multi-pathogen shrimp diseases.

Molecular characterization, adaptive evolution, and expression analysis of the Toll-like receptor gene family in Fenneropenaeus chinensis

Global warming is a challenge to animal health because of the increased environmental temperature, with subsequent induction of immune suppression and increased susceptibility to disease during summer. The Toll-like receptor (TLR) family is an essential pattern recognition receptor (PRR) that initiates the innate immune response by sensing conserved molecular patterns of pathogens. However, research on the TLR gene family in decapod crustaceans has been conducted sporadically, without systematic naming, and the relationship between pathogen immunity adaptation and adaptive evolution of immune-related genes is unclear. In this study, various TLR gene sequences in decapod crustaceans were collected, and the unified name of Fenneropenaeus chinensis was confirmed using sequence alignment. Structural characteristics and evolutionary analyses of TLR genes in decapod crustaceans were performed, and ten FcTLR genes were identified in F. chinensis. Protein domain analysis revealed that FcTLR proteins contain 4–25 LRR domains used to recognize different pathogens. Selection pressure analysis revealed that TLR1 and TLR9 were subjected to positive selection pressure in decapod crustaceans, which may be related to their resistance to environmental changes. Furthermore, the expression of ten TLR genes was detected in F. chinensis following white spot syndrome virus (WSSV) infection. The results demonstrated that FcTLR1, FcTLR7, and FcTLR9 responded positively, which was also consistent with the results of the protein domain and selection pressure analyses. This study provides new insights into the immune response and adaptive evolution of TLRs in decapod crustaceans to prevent environmental damage, such as pathogens and high temperature.

Detection and diagnosis of Panulirus argus virus 1 in captive spiny lobsters using qPCR in conjunction with histopathology and transmission electron microscopy.

Panulirus argus virus 1 (PaV1) is the first and only naturally occurring pathogenic virus described in the Caribbean spiny lobster, Panulirus argus. PaV1 infection in decapod species that commonly co-occur with P. argus, including the spotted spiny lobster Panulirus guttatus, has not been previously described. In 2016, 14 Caribbean and 5 spotted spiny lobsters were collected near Summerland Key, Florida, to supplement the resident population of the Audubon Aquarium of the Americas in New Orleans, Louisiana. After 5 months in quarantine, Caribbean and spotted spiny lobsters began to exhibit clinical signs of lethargy and dying in the molt. Initial histologic evaluation revealed intranuclear inclusion bodies in circulating hemocytes in the spongy connective tissue of the epidermis, suggesting a viral infection. Samples of hepatopancreas and hemolymph from deceased Caribbean and spotted spiny lobsters tested negative for white spot syndrome virus and positive for PaV1 using real-time quantitative polymerase chain reaction (qPCR). Intranuclear, eosinophilic to amphophilic, Cowdry type A inclusion bodies observed primarily within fixed phagocytes and circulating hemocytes in the hepatopancreas of freshly euthanized Caribbean spiny lobsters were consistent with PaV1 infection. Transmission electron microscopy revealed that hemocytes associated with hepatopancreatic tubules contained viral inclusions with location, size, and morphology consistent with previously described PaV1 infection. These findings highlight the significance of using molecular diagnostics in conjunction with histopathology and electron microscopy in the investigation and diagnosis of PaV1 in spiny lobsters. Further study is required to investigate the relationship of PaV1-associated mortality events and microscopic lesions in the spotted spiny lobster.

The histidine phosphatase LHPP of Penaeus vannamei is involved in shrimp hemocytes apoptosis

LHPP (Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase) is a protein histidine phosphatase that modulates a hidden posttranslational modification called histidine phosphorylation. LHPP also acts as a tumor suppressor, which plays a pivotal role in various cellular processes. However, whether LHPP participates in the regulation of invertebrate's immunity is still unknown. Here we characterized a LHPP homolog in P. vannamei (designated PvLHPP), with a 807 bp length of open reading frame (ORF) encoding a putative protein of 268 amino acids. Sequence analysis revealed that PvLHPP contains a typical hydrolase 6 and hydrolase-like domain, which was conserved from invertebrate to vertebrate. PvLHPP was ubiquitously expressed in tissues and induced in hemocyte and hepatopancreas by Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, indicating that PvLHPP participated in the immune responses. Moreover, silencing of PvLHPP followed by V. parahaemolyticus inhibited hemocyte apoptosis. This study enriches our current insight on shrimp immunity, and provides novel perspective to understand immune-regulatory role of PvLHPP.

Characterization and immune functional analysis of two new type I crustins in the oriental river prawn Macrobrachium nipponense

Crustins are cationic antimicrobial peptides that are categorized into five types and play a key role in the nonspecific immunity of crustaceans. In the present study, full-length cDNA sequences of two Macrobrachium nipponense crustin genes (MnCrustin1 and MnCrustin2) were cloned and identified. The full-length cDNAs of MnCrustin1 and MnCrustin2 were 715 and 881 bp, including 351 and 330 bp open reading frames (ORFs) encoding 117 and 110 amino acids, respectively. A signal peptide, a cysteine (Cys)-rich region and a whey acidic protein (WAP) domain existed in both MnCrustin1 and MnCrustin2. Phylogenetic analysis and multiple sequence alignment indicated that MnCrustin1 and MnCrustin2 belonged to the type I crustin family. The MnCrustin1 and MnCrustin2 mRNAs were expressed in various tissues of M. nipponense and were highly expressed in hemocytes and gills. Recombinant MnCrustin1 and MnCrustin2 inhibited the growth of both gram-positive and gram-negative bacteria in vitro. After Aeromonas hydrophila and white spot syndrome virus (WSSV) infection, MnCrustin1 and MnCrustin2 expression levels were elevated in the hepatopancreas, hemocytes and gills of M. nipponense. Tests of RNA interference (RNAi) and recombinant protein injection were employed to further study the roles of MnCrustin1 and MnCrustin2 in pathogen infection. The results indicated that silencing MnCrustin1 and MnCrustin2 accelerated the mortality rate of M. nipponense after challenge with A. hydrophila or WSSV, while recombinant MnCrustin1 and MnCrustin2 decreased the mortality rate under these conditions. These results suggest that MnCrustin1 and MnCrustin2 both play prominent roles in the immune response of M. nipponense to bacterial and viral infection and are important immune effectors of this prawn against pathogen infection.