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Microsporidium Ecytonucleospora hepatopenaei (EHP) spores were purified from the hepatopancreas of Penaeus vannamei infected with EHP by percoll density gradient centrifugation and differential centrifugation. The EHP spores contain a thick chitin wall and might not rupture using the routine DNA extraction protocol. In this study, three enzymes were used, including chitinase, proteinase K, and DNase I. Chitinase or proteinase K digestions caused weakened fluorescence of chitin showing by a blurred edge of EHP spores stained with calcofluor white under a fluorescence microscope. Different combinations of these enzymes followed by DNA extraction with phenol-chloroform from EHP spores showed significant increases in the copy number of the EHP SSU gene per spore. The combination of the chitinase and proteinase K treatments resulted 4.46 ± 1.07 copies/spore detected, which is 31.6 ± 20.7 folds of no treatment groups, accounting to (55.7 ± 13.4)% of the total copies of the gene in the spore. The additional treatment with chitinase to the conventional extraction protocol with a proteinase K digestion step for feces and hepatopancreas samples of P. vannamei resulted in a significant difference in EHP copies in the DNA of (83.8 ± 64.1)% and (55.3 ± 88.0)% increases. The study proved that chitinase and proteinase K treatment enhance the DNA extraction from microsporidian spores resulting in high yield., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

A recurring trend in evidence scrutinized over the past few decades is that disease outbreaks will become more frequent, intense, and widespread on land and in water, due to climate change. Pathogens and the diseases they inflict represent a major constraint on seafood production and yield, and by extension, food security. The risk(s) for fish and shellfish from disease is a function of pathogen characteristics, biological species identity, and the ambient environmental conditions. A changing climate can adversely influence the host and environment, while augmenting pathogen characteristics simultaneously, thereby favoring disease outbreaks. Herein, we use a series of case studies covering some of the world's most cultured aquatic species (e.g., salmonids, penaeid shrimp, and oysters), and the pathogens (viral, fungal, bacterial, and parasitic) that afflict them, to illustrate the magnitude of disease-related problems linked to climate change., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Introduction The development of diseases associated with Helicobacter pylori  ( H. pylori ) infection is closely linked to its virulence genes, which vary by geographic region. This study aimed to determine the prevalence of H. pylori cytotoxin-associated gene A ( cagA ) and vacuolating cytotoxin gene A ( vacA ) genes and their genotypes in patients with gastrointestinal diseases. Methods Patients diagnosed with gastrointestinal disease based on endoscopic findings were recruited for the study. Gastric biopsies were collected to screen for H. pylori infection using polymerase chain reaction (PCR). Subsequently, infected samples were tested for  cagA  and vacA genes, and their genotypes were analyzed by sequencing. Results Among 250 cases, 56% (140/250) exhibited gastrointestinal diseases. Of these cases, 32.1% (45/140) were infected with H. pylori . Regarding gene detection, 40 (88.9%) samples were positive for cagA , while all samples were positive for vacA . For cagA , the Western type with the ABC pattern was the most prominent. There was a statistically significant association between cagA genotypes and clinical outcomes, with the Western type being more prevalent in gastritis patients. For vacA , there was a high prevalence of the s1 and i1, while the m1 and m2 showed similar prevalence. In our combined analysis, the dominant vacA genotype combinations were s1m1i1 (46.7%). There were no statistical differences between the vacA genotypes and clinical outcomes (P > 0.05). Conclusion This study revealed a high prevalence of H. pylori cagA and vacA genes, but there were variations in their genotypes. A correlation was observed between the Western-type cagA and gastritis; however, no association was found between vacA genotypes and clinical outcomes., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. The Institutional Review Board of the National Cancer Institute, Thailand, issued approval EC 65001. The study has been reviewed and approved by the Ethics Committee, National Cancer Institute, Thailand, based on the Declaration of Helsinki and Good Clinical Practice. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Sukthaworn et al.)

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat to shrimp health worldwide. Severe EHP infections in shrimp cause growth retardation and increase susceptibility to opportunistic infections. EHP produces spores with a chitin wall that enables them to survive prolonged environmental exposure. Previous studies showed that polar tube extrusion is a prerequisite for EHP infection, such that inhibiting extrusion should prevent infection. Using a proteomic approach, polar tube protein 2 of EHP (EhPTP2) was found abundantly in protein extracts obtained from extruded spores. Using an immunofluorescent antibody against EhPTP2 for immunohistochemistry, extruded spores were found in the shrimp hepatopancreas (HP) and intestine, but not in the stomach. We hypothesized that presence of EhPTP2 might be required for successful EHP spore extrusion. To test this hypothesis, we injected EhPTP2-specific double-stranded RNA (dsRNA) and found that it significantly diminished EHP copy numbers in infected shrimp. This indicated reduced amplification of EHP-infected cells in the HP by spores released from previously infected cells. In addition, injection of the dsRNA into EHP-infected shrimp prior to their use in cohabitation with naïve shrimp significantly (p < 0.05) reduced the rate of EHP transmission to naïve shrimp. The results revealed that EhPTP2 plays a crucial role in the life cycle of EHP and that dsRNA targeting EHP mRNA can effectively reach the parasite developing in host cells. This approach is a model for future investigations to identify critical genes for EHP survival and spread as potential targets for preventative and therapeutic measures in shrimp., (© 2024. The Author(s).)

Background: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV)., Results: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV., Conclusions: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them., (© 2022. The Author(s).)

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp ( Penaeus monodon ) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp ( Penaeus vannamei ) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Taengchaiyaphum, Buathongkam, Sukthaworn, Wongkhaluang, Sritunyalucksana and Flegel.)

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP AHPND ), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb-protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10 7  cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria., (© 2019 John Wiley & Sons Ltd.)

Previous work has shown that non-retroviral endogenous viral elements (EVE) are common in crustaceans, including penaeid shrimp. So far, they have been reported for infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). For the latter, it was shown that shrimp sperm were positive for an EVE of WSSV called EVE 366 , suggesting that it was heritable, since shrimp sperm (non-motile) do not contain mitochondria. However, to prove this hypothesis that EVE 366 was heritable and located in chromosomal DNA, it was necessary to carry out mating tests to show that EVE 366 could be detected in parental shrimp and distributed in their offspring in a Mendelian fashion. To do this, we analyzed two shrimp crosses using polyacrylamide gels with a multiple-allele, microsatellite marker Pmo11 as a quality control for single allele detection. In both crosses, all of the shrimp (parents and siblings) were positive for 2 Pmo11 alleles as expected. In Cross 1, the female was PCR-positive for EVE 366 while the male was negative, and in Cross 2, both the female and male were PCR-positive for EVE 366 . Individual analysis of the offspring of Cross 1 revealed a distribution of 1:1 for EVE 366 , indicating that the EVE 366 -positive female parent was heterozygous for EVE 366 . In the second cross, the distribution of EVE 366 in the offspring was 3:1, indicating that both PCR-positive parents were heterozygous for EVE 366 . These results supported the hypothesis that EVE 366 was present in shrimp chromosomal DNA and was heritable in a Mendelian fashion. This work provides a model to screen for heritable EVE in shrimp and shows that selection of one parent heterozygous for an EVE and the other negative for it can result in approximately half of the siblings positive and half negative for that EVE as expected. Dividing the siblings of such a cross into an EVE positive group and an EVE negative group followed by challenge with the originating lethal virus should reveal whether or not possession of that specific EVE results in any significant protection against disease caused by the homologous virus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To achieve in creating permanent shrimp cell lines, cellular arrest of primary cells in the culture is needed to be firstly solved. Considering the insertion of some markers affecting cellular proliferation into primary haemocytes in order to produce the black tiger shrimp cell line and the very low percent of transduced cells previously reported in penaeid shrimps, these paved us the way to set up suitable gene delivery protocols to increase percent of transduced cells in the shrimp as our primary aim. In this study, electroporation and lipofection were used to transfer construct plasmids (pLL3.7 plasmids containing CMV promoters and pGL-IE1-126(A)-EGFP plasmids carrying WSSV IE1 promoters) into primary haemocytes. As it was difficult to distinguish between cells expressing EGFP signal and auto-fluorescence of many dead cells occurred by electroporation during the first 72 h of experiment; so, only lipofection was managed to deliver plasmids into primary cells. Surprisingly, numbers of suspected proliferative cells were derived after electroporation with no insertion of immortalising markers. These cells survived in vitro for up to 45 days with high rate of cell viability, but the number of viable cells decreased throughout the experiment. In addition, these cells expressed genes and proteins closely related to hyaline cells determined using RT-PCR and western blot. For the lipofection experiment, no green fluorescence signal was detected in any primary cell introduced with these plasmids, suggesting that plasmids were not successfully inserted into cells. Also, a number of primary haemocytes had the apoptotic cell death characteristic within 5 days after lipofection. These possibly result from using inappropriate lipofection protocol and chemical substances. In summary, finding out suitable protocols to elevate the percent of transduced cells is still necessary. Additionally, continuous shrimp cell lines would be possibly established by transforming suspected proliferative cells derived from electroporation in this study., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VP AHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VP AHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VP AHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions. IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VP AHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VP AHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VP AHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid., (Copyright © 2017 Phiwsaiya et al.)

Yellow head virus (YHV) causes acute infections and mass mortality in black tiger shrimp culture. Our study aims to investigate molecular interaction between YHV and circulating hemocytes of Penaeus monodon at early infection. Total shrimp hemocytes were isolated by Percoll gradient centrifugation and identified by flow cytometric analysis. At least three types of hemocyte cells were identified as hyaline, semi-granular, and granular hemocytes. Experimental infection of YHV in shrimp culture demonstrated drastic changes in total and each hemocyte cell counts. Immunohistochemistry analysis demonstrated interaction and replication of YHV mainly with the granule-containing hemocytes and little to none in hyaline cell. These granule-containing hemocytes are proposed to be YHV targets providing the first line of defense to viral infection. Protein expression profiling of granule-containing hemocytes revealed several immune-responsive proteins including antimicrobial protein crustins (crustinPm1 and crustinPm4), alpha-2-macroglobulin, and kazal-type serine proteinase inhibitor. During an early phase of YHV infection at 6 hpi crustinPm1 illustrated a significant increase of mRNA and protein expression level in plasma. The results suggest that an antimicrobial crustinPm1 may participate in shrimp defense mechanism against YHV, especially on the granule-containing hemocytes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using Biotin-NSH-LC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect biotinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. CrustinPm1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustinPm1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. CrustinPm1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreaks in shrimp aquaculture, we studied the effect of β-glucan administration to activate the prophenoloxidase (proPO) enzymatic cascade prior to WSSV challenge. Injection of a single dose of β-glucan (5 μg/g) prior to WSSV challenge resulted in activation of the proPO system and reduced shrimp mortality (25-50%) when compared to controls (100%). By contrast, no significant reduction was observed using yellow head virus (YHV) in a similar protocol. We subsequently hypothesized that administration of a second dose of β-glucan after WSSV challenge might reduce shrimp mortality further. Surprisingly, the opposite occurred, and mortality of the WSSV-infected shrimp increased to 100% after the second β-glucan dose. Both immunofluorescence and RT-PCR assays revealed low WSSV levels in hemocytes of shrimp collected after the second dose of β-glucan administration, suggesting that the cause of increased mortality was unlikely to be increased WSSV replication. We found from measured phenoloxidase acitivity (PO) and H2O2 production that the higher mortality may have resulted from a combination of WSSV infection plus over-production of reactive oxygen species (ROS) stimulated by two doses of β-glucan. Thus, caution may be prudent in continuous or prolonged activation of the shrimp immune system by β-glucan administration lest it exacerbate shrimp mortality in the event of WSSV infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp., (Copyright © 2013 Elsevier Ltd. All rights reserved.)