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A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1994. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod Hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, the negative ISH result with the intranuclear inclusions has not been further elucidated. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics have revealed a novel ssDNA genome sequence of 6,723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence demonstrated the ssDNA presented in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence most closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6,723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV).

The genome sequence of Wenzhou shrimp virus 8 (WzSV8) (GenBank recordKX883984.1) was described in 2015 from wide screening for RNA viruses in aquatic animals. A closely related sequence (GenBank recordOK662577.1) from the whiteleg shrimpPenaeus vannameiwas deposited in 2021 under the name Penaeus vannamei picornavirus (PvPV). In 2022 another closely related sequence (GenBank accession:OP265432) was submitted under the name Penaeus vannamei solinvivirus (PvSV). In 2021, prior to the publication of PvPV and PvSV, we used an RT-PCR method devised from the sequence of KX883984.1 (described herein) to screen for WzSV8 in specimens of cultivated shrimp. Samples that gave positive RT-PCR results were subsequently tested byin situhybridization (ISH) analysis to identify virus target tissues. Several tissues gave positive ISH results within morphologically normal nuclei. Thus, these tissues were of no use for diagnosis of WzSV8 by normal histological analysis. However, unique basophilic, cytoplasmic inclusions within vacuoles in the hepatopancreatic E-cells were also found in the same WzSV8-positive shrimp specimens, sometimes accompanied by a smaller eosinophilic inclusion. We call these Lightner double Inclusions (LDI) that can be considered pathognomonic for diagnosis of WzSV8 infection when detected using the light microscope. Although no current proof of WzSV8 is the cause of disease, it is important to investigate new viruses and related tissue anomalies, even from normal cultivated shrimp, to determine whether they may have any relationship to significant negative effects on the production of cultivated shrimp.

White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10

BackgroundShrimp 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 draft genome of the giant tiger shrimp (Penaeus monodon)(GenBank record JABERT000000000) for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV).ResultsThe shrimp draft genome contained 3 piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in linkage group 35 (LG35). Both LG35 clusters contained multiple DNA fragments 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 LG35 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 linkage group 7 (LG7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against infectious IHHNV.ConclusionsOur 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 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.

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.

Summary Early mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pirvp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 105 colony‐forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA–DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012T genome revealed no homologues of the Pirvp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp., A new species, Shewanella khirikhana has been isolated from shrimp during the early mortality syndrome (EMS) in 2012 in Thailand. It causes distinctive histopathological lesions at the hepatopancreas, which is the same target with EMS/AHPND‐ inducing bacteria (VP‐AHPND) resulting in severe mortality of shrimp in the infected pond.

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.

White feces syndrome (WFS) in shrimp cultivation ponds is characterized by the occurrence of shrimp with abnormal, white intestines (midguts) combined with large floating mats of white, shrimp fecal strings. The etiology for WFS is complex, similar to diarrhea in humans. EHP-WFS is a type of WFS characterized by massive quantities of spores from the microsporidian parasite Enterocytozoon hepatopenaei (EHP) together with mixed, unidentified bacteria in the shrimp hepatopancreas, midgut and fecal strings. However, WFS does not always develop in shrimp with severe EHP infections in controlled laboratory challenges. Further, in EHP-WFS outbreak ponds, some shrimp show white midguts (WG) while others in the same pond show grossly normal midguts (NG). We hypothesized that comparison of the microbial flora between WG and NG from the same EHP-WFS pond would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this hypothesis, we selected a pond exhibiting a severe EHP-WFS outbreak in cultivated Penaeus vannamei and used a combination of microscopic and microbial profiling analyses to compare WG and NG samples. By histology, EHP plasmodia and spores were confirmed in the hepatopancreas (HP) and midgut of WG and NG shrimp, but pathological severity and spore quantity was higher in the WG shrimp. In addition, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium quantity in the HP of WG shrimp was significantly higher (P = 1.08e-5) than in NG shrimp (4,506 vs. 3 copies /100 ng DNA, respectively). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as a potential eukaryote-prokaryote pathobiome that causes EHP-WFS in P. vannamei.HighlightsWhite feces syndrome (WFS) shrimp often harbor the microsporidian Enterocytozoon hepatopenaei (EHP)The hepatopancreas (HP) and midgut of EHP-WFS shrimp had more EHP copies and spores than EHP-non.-WFS shrimpVibrio spp., Propionigenium sp. and EHP dominated in HP microbiomes of EHP-WFS shrimpPropionigenium copy numbers were uniquely high in the HP of EHP-WFS shrimpEHP-WFS shrimp also showed intestinal microbiomes of reduced diversity but more heterogeneity

Infectious myonecrosis virus (IMNV) was first discovered in the Americas in 2004 as a new lethal pathogen of cultivated whiteleg shrimp Penaeus vannamei, but infections were not lethal for the giant tiger shrimp Penaeus monodon. In 2007, it was reported in diseased P. vannamei cultivated in Indonesia but, until recently, not from other countries in Asia. Decapod iridescent virus (DIV1) was first reported from China in 2016 and is lethal for the crayfish Cherax quadricarinatus and Procambarus clarkii, for the penaeid shrimp P. vannamei and P. chinensis and for the palaemonid shrimp Macrobrachium rosenbergii and Exopalaemon carinicauda. It has not yet been reported from other Asian countries. Here we describe the occurrence of positive test results for IMNV and DIV1 using polymerase chain reaction (PCR) technology during screening of grossly normal, broodstock-size, wild P. monodon captured from the Indian Ocean and held in a biosecurity facility for screening. Amplicons for each virus were obtained from two widely separated targets in the relevant viral genomes listed at GenBank, and sequencing revealed 99-100% identity to the targets for each virus. Based on these results, the captured specimens were destroyed. The results raised the possibility that grossly normal, captured P. monodon might serve as potential vehicles for introduction of IMNV and/or DIV1 to shrimp hatcheries and farms. Thus, we recommend that appropriate precautions be taken to avoid this possibility.

Parvoviruses that infect the hepatopancreas (HP) of the penaeid shrimp Penaeus chinensis, P. monodon, and P. merquiensis were previously called hepatopancreatic parvoviruses (HPV). They are now classified in the family Parvoviridae, sub-family Hamaparvovirinae as members of the same genus called Hepanhamaparvovirus and referred to as decapod hepanhamaparvovirus, designated here as DHPV. However, a virus that causes similar lesions in the HP of the giant river prawn Macrobrachium rosenbergii resembles hepanhamaparvoviruses by microscopy and histochemistry. Unfortunately, no genome information is yet available and PCR detection methods that work for DHPV in P. monodon do not work with M. rosenbergii. For hatchery samples of M. rosenbergii in Thailand with DHPV-like lesions, we hypothesized it might be possible to design primer pairs from 8 full DHPV genome sequences at GenBank for use in PCR detection of DHPV in M. rosenbergii. Using this strategy, we successfully designed a new set of primers and a PCR protocol called the DHPV-U method that gave an amplicon with DNA extracts from larvae of M. rosenberigii samples that showed DHPV-like lesions, while extracts from normal larvae gave none. DNA extracts from P. monodon infected with DHPV also gave amplicons. At the same time, the normal PCR method for DHPV in P. monodon gave no amplicon with the M. rosenbergii DNA extracts. The DHPV-U amplicons from P. monodon and M. rosenbergii shared 99% sequence identity, and in situ hybridization (ISH) assays using the DIG-labeled amplicon gave positive histochemical results in the HP tissue of both P. monodon and M. rosenbergii. The DHPV-U method is now being used in Thailand for detection of DHPV in both P. monodon and M. rosenbergii. Overall, the results support the proposal that the HP virus in M. rosenbergii is also a hepanhamaparvovirus. Based on 100% sequence identity of the target region in the currently published DHPV sequences at GenBank, the DHPV-U method may also work for detection of other DHPV isolates.

Aquaculture is the fastest‐growing animal production sector, and shrimp production already exceeds that of the capture fishery. Viruses and bacteria account for the majority of disease losses for shrimp farmers. Viral pandemics in the mid 1990s and, more recently, a bacterial pandemic from 2009 to 2015 have led to the conclusion that future, sustainable shrimp aquaculture will depend on the development of more efficient, biosecure production facilities that cultivate specific pathogen‐free (SPF) shrimp, genetically improved for growth and disease tolerance or resistance. Major requirements for development, maintenance, and use of SPF stocks in aquaculture are effective pathogen surveillance and disease prevention methods. When protective measures fail and diseases occur in production ponds, there are currently only a few approved and practical therapeutic methods available for use with bacterial pathogens and none so far for viral pathogens. To improve existing methods of prevention and therapy and to develop new ones, research is being carried out on the nature of shrimp–pathogen interactions. Promising results have been obtained at the laboratory level for possible applications involving the use of immunostimulants for “immune priming” or “trained immunity” of RNA interference and of endogenous viral elements. Some of these promising new directions are discussed.

The viral accommodation hypothesis for crustaceans and insects was first proposed in 1998/2001, stimulated by observations that shrimp and insects or insect cell lines can coexist with both DNA or RNA viruses without showing any signs of disease (i.e., they tolerate, single to multiple, persistent infections, sometimes for a lifetime). A review of tests of the hypothesis up to 2007 was previously published in DCI. This was followed by a major revision in 2009 when the elusive memory element required by the hypothesis was proposed to reside in non-retroviral fragments of extant viruses, now called endogenous viral elements (EVE) that are autonomously inserted into the host genome as cDNA copied from viral mRNA. Here, progress in research on viral accommodation in crustaceans and insects over the decade following 2009 is reviewed. It culminates with a discussion of exiting research results from insects in 2019 that prove the existence of specific, adaptive and heritable immunity, at least in mosquitoes. It remains to be determined whether the same mechanisms also govern EVE acquisition and its protective RNA production in shrimp. The wide-ranging consequences of the revealed mechanisms for viral disease control in economic crustaceans and insects is discussed.

Spiroplasma eriocheiris was first named as the cause of tremor disease in the Chinese mitten crab Eriocheir sinensis in China in 2003. It was subsequently reported from China to infect also the crayfish Procambarus clarkii, the giant river prawn Macrobrachium rosenbergii, the oriental prawn Macrobrachium nipponensis and the whiteleg shrimp Penaeus vannamei. Unfortunately, none of these publications give clear pictures of the histopathology caused by the pathogen, even though this is one of the first diagnostic procedures often performed for newly reported diseases. Here we describe distinctive histological lesions caused in the hepatopancreatic tissue of M. rosenbergii infected with S. eriocheiris. In the hepatopancreas (HP), these consist of basophilic or mixed basophilic/eosinophilic lesions in cells confined to the interstitial spaces of the HP and not involving the hepatopancreatic tubule epithelial cells. Examination of the lesions by transmission electron microscopy revealed the presence of colonies of intracellular, un-walled bacterial cells of variable shape (but many crescent shaped) and approximately 150 nm in diameter. PCR using group-specific primers for the SSU rRNA followed by cloning and sequencing revealed 99.9% sequence identity to the matching sequence of S. eriocheiris. PCR tests for presence of S. eriocheiris in hepatopancreatic tissue samples gave positive test results, and in situ hybridization assays using a probe derived from the PCR product gave positive signals in the interstitial spaces of the HP, corresponding to the regions of the distinctive histological lesions. Less severe lesions containing cells with eosinophilic/basophilic inclusions were also found and confirmed by in situ hybridization in heart muscle, skeletal muscle, gills, connective tissue and the ventral nerve cord. Recognition of the distinctive histological lesions in interstitial spaces of the hepatopancreatic tissue of M. rosenbergii by histological analysis can be used as a strong presumptive indication of infection by S. eriocheiris and may help in the control of this rapidly-spreading, emerging pathogen.

Early mortality syndrome (EMS) refers to acute mortality within approximately 35 days of shrimp culture. It is often equated with acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio isolates that produce Pir-like toxins. To better understand EMS, 200 Thai shrimp ponds were randomly selected prior to stocking and subsequently sampled from 19/08/2013 to 23/04/2014 to determine the pond prevalence of various pathologies, and particularly those targeting the shrimp hepatopancreas (HP). Ponds exhibiting acute mortality within 35 days were labeled EMS ponds while others were labeled non-EMS ponds. Shrimp samples from each pond (10) were subjected to histological analysis focused on hepatopancreatic (HP) lesions. Similar samples (10) were tested by PCR for the microsporidian Enterocytozoon hepatopenaei (EHP), white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio that cause AHPND. Preservation failed for 4 ponds. Results from the 196 ponds remaining were divided, into 4 groups based largely on HP histology: (1) ponds positive for AHPND by histology and/or PCR at 20.9% prevalence (41/196); (2) ponds showing HP bacterial lesions at 14.8% prevalence (29/196); (3) ponds showing collapsed HP tubule epithelia at 25.0% prevalence (49/196); and (4) ponds showing no HP pathology (normal HP) at 39.3% prevalence (77/196) ponds. The overall prevalence of EMS ponds (i.e., mortality ≤35 days) was 16.3% (32/196), but only 18/32 (56.3%) of these fell within AHPND Group 1. The other 14 EMS ponds (43.7%) were divided among the remaining three groups: Group 2 with HP bacterial lesions (3/32 = 9.4%), Group 3 with collapsed HP tubule epithelia (1/32 = 3.1%) and Group 4 with normal HP histology (a surprising 10/32 = 31.2%). Across all groups, prevalence of ponds exhibiting aggregated transformed microvilli (ATM) was high at 79.8%, as was prevalence of EHP at 60.7%. The overall prevalence of WSSV was low at 4.1% (8/196), while YHV was not found. Overall, the results indicated that equating EMS with AHPND without confirmatory analysis could lead to overestimation of its impact on shrimp aquaculture and to failure in recognizing other important causes of early mortality.

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intracellular spore-forming parasite, which has recently become a serious threat of cultivated penaeid shrimp in Asia. These studies were aimed to generate comprehensive study on EHP infection as well as establishing innovative strategies to reduce its viability and potential infectivity in shrimp farms. The EHP spore purification method by using Percoll gradient separation with ultracentrifugation step at 15 °C has been developed. The viability of the purified spores has been evaluated by its polar tube extrusion with Phloxin B staining. The active EHP spores were exposed to different temperature (−20 °C, 4 °C and 33 °C) and chemical treatments including calcium hypochlorite, formalin, potassium permanganate (KMnO4) and ethanol to identify the conditions that can be used to inhibit the extrusion of the polar tube. Complete inhibition of activity was demonstrated either by freezing the spores at −20 °C for at least 2 h or by treating them with chemicals. The chemicals that yielded 100% inhibition were 15 ppm KMnO4 for 15 min, 40 ppm of 65% active chlorine for 15 min or 10 ppm of 65% active chlorine for 24 h and 20% ethanol for 15 min. However 200 ppm formalin resulted in a maximum reduction of 95.33%. Taken together, our protocol demonstrates for the first time that living EHP spores can be isolated and purified, providing a potential platform for future testing and development of EHP's control strategies.

Detailed histopathological analysis (HA) and in situ DNA hybridization assays (ISH) were carried out with adjacent tissue sections from the whiteleg shrimp Penaeus vannamei that had been infected with decapod iridescent virus 1 (DIV1) in the laboratory. Results with two ISH methods confirmed those recently published in China indicating that unique, basophilic, cytoplasmic lesions pathognomonic for DIV1 disease were produced in the hematopoietic tissue (HPT). In addition, both methods revealed that the lymphoid organ (LO) was also a prime target for DIV1, although the lesions produced in the LO were not sufficiently unique to be considered pathognomonic for DIV1 infection. However, the HPT lesions of DIV1 often require use of a 100× objective lens to confirm their presence. In contrast, the LO can be quickly located and lesions can be recognized using a 10× objective lens and confirmed with a 40× objective lens. Once confirmed, a diagnostician can switch to the HPT (usually relatively nearby) to search for pathognomonic lesions with 40× and 100× objective lenses. This will speed up histological screening for DIV1 infections. However, the LO is unique to penaeid shrimp, so this approach would not be applicable to the many other species of crustaceans that are susceptible to DIV1 infection.

Infectious myonecrosis virus (IMNV) was first discovered in the Americas in 2004 as a new lethal pathogen of cultivated whiteleg shrimp Penaeus vannamei, but infections were not lethal for the giant tiger shrimp Penaeus monodon. In 2007, it was reported in diseased P. vannamei cultivated in Indonesia but, until recently, not from other countries in Asia. Decapod iridescent virus (DIV1) was first reported from China in 2016 and is lethal for the crayfish Cherax quadricarinatus and Procambarus clarkii, for the penaeid shrimp P. vannamei and for the palaemonid shrimp Macrobrachium rosenbergii and Exopalaemon carinicauda. It has not yet been reported from other Asian countries. Here we describe the occurrence of positive test results for IMNV and DIV1 using polymerase chain reaction (PCR) technology during screening of grossly normal, broodstock-size, wild P. monodon captured and held in a biosecurity facility for screening. Amplicons for each virus were obtained from two widely separated targets in the relevant viral genomes listed at GenBank, and sequencing revealed 99–100% identity to the targets for each virus. Due to the positive results, the captured specimens were immediately destroyed within the quarantine facility. The results raised the possibility that grossly normal, captured P. monodon might serve as potential vehicles for introduction of IMNV and/or DIV1 to shrimp hatcheries and farms. Thus, we recommend that appropriate precautions be taken and that surveillance programs for farmed and wild populations be undertaken to avoid this possibility.

Many bacteria, including Vibrio pathogens of shrimp, need to colonize and/or form biofilms in hosts or the environment to cause disease. Thus, one possible control strategy for shrimp Vibriosis is biofilm inhibition. With this objective, an extract from the Japanese fermented soybean product, Natto was tested with the luminescent shrimp pathogen Vibrio harveyi (VH) for its ability to inhibit or degrade biofilm and to interfere with cell growth in broth. Natto is a traditional fermentation product of Bacillus subtilis var Natto (BSN1). Using 96 well microtiter plates coated with 0.4% chitosan, we found that biofilm formation by VH was inhibited, while growth in parallel broth cultures was not. When an extract from Natto prepared using BSN1 was mixed with feed for the whiteleg shrimp Penaeus vannamei before immersion challenge with V. harveyi at 106 cfu/ml, survival was significantly higher (p≤0.05) than for control shrimp given feed without these additives. Further work done to test whether d-amino acids were involved in biofilm formation as previously reported for B. subtilis, Staphylococus aureus and Pseudomonas aeruginosa gave negative results. In conclusion, we discovered that Natto extract can inhibit Vibrio biofilm formation and that it or BSN1 alone added to shrimp feed can significantly reduce shrimp mortality in immersion challenges with pathogenic VH. This shows some promise for possible application against Vibriosis in shrimp since Natto is generally regarded as safe (GRAS) for human consumption.

Using post-larvae derived from specific pathogen free (SPF) stocks in penaeid shrimp farming has led to a dramatic increase in production. At the same time, new pathogens of farmed shrimp are continually being discovered. Sometimes these pathogens are carried by shrimp and other crustaceans as persistent infections without gross signs of disease. Thus it is that a 5-generation stock of Penaeus monodon SPF for several pathogens was found, post-stock-development, to be persistently-infected with newly-discovered Laem Singh virus (LSNV). In this situation, the stock developers were faced with destroying their existing stock (developed over a long period at considerable cost) and starting the whole stock development process anew in order to add LSNV to its SPF list. As an alternative, it was hypothesized that injection of complementary dsRNA into viral-infected broodstock prior to mating might inhibit replication of the target virus sufficiently to reduce or eliminate its transmission to their offspring. Subsequent selection of uninfected offspring would allow for post-clearing of LSNV from the existing stock and for conversion of the stock to LSNV-free status. Testing this hypothesis using the LSNV-infected stock described above, we found that transmission was substantially reduced in several treated broodstock compared to much higher transmission in buffer-injected broodstock. Based on these results, the model is proposed for post-clearing of SPF stocks using dsRNA treatment. The model may also be applicable to post-clearing of exceptional, individual performers from grow-out ponds for return to a nucleus breeding center.

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.

Parvoviruses that infect the penaeid shrimp previously called hepatopancreatic parvoviruses (HPV) are now called Hepanhamaparvovirus and referred to as decapod hepanhamaparvovirus, designated here as DHPV. However, a virus that causes similar lesions to the hepatopancreas of Macrobrachium rosenbergii has previously been known since 1990 and resemble what is now called hepanhamaparvovirus by microscopy and histochemistry. Unfortunately, no genome information is yet available and PCR detection methods that work for DHPV in P. monodon do not work with M. rosenbergii. For hatchery samples of M. rosenbergii in Thailand with DHPV-like lesions, we hypothesized it might be possible to design primer pairs from 8 full DHPV genome sequences at GenBank for use in PCR detection of DHPV in M. rosenbergii. Using this strategy, we successfully designed a new set of primers and a PCR protocol called the DHPV-U method that produced an amplicon of the expected size with DNA extracts from larvae of M. rosenberigii samples that showed DHPV-like lesions, while extracts from normal larvae gave none. DNA extracts from P. monodon infected with DHPV also gave amplicons of the expected size. At the same time, the conventional PCR method for DHPV in P. monodon gave no amplicon with the M. rosenbergii DNA extracts. The DHPV-U amplicons from P. monodon and M. rosenbergii shared 99% sequence identity, and in situ hybridization (ISH) assays using the DIG-labeled amplicon gave positive histochemical results in the HP tissue of both P. monodon and M. rosenbergii. The DHPV-U method is now being used in Thailand for detection of DHPV in both P. monodon and M. rosenbergii. Overall, the results support the proposal that the HP virus in M. rosenbergii is also a hepanhamaparvovirus. Based on 100% sequence identity of the target region in the currently published DHPV sequences at GenBank, the DHPV-U method may also work for detection of other DHPV isolates.

Laem Singh virus (LSNV) was discovered in 2006 and proposed as a necessary but insufficient cause of retarded growth in the giant tiger shrimp Penaeus monodon. Its closest relatives were plant viruses including an unassigned Sobemovirus and viruses in the family Luteoviridae. During succeeding years, attempts to obtain the full LSNV genome sequence by genome walking failed. However, recent publication of the full sequence of Wenzhou shrimp virus 9 (WZSV 9) at GenBank revealed that LSNV sequences in our database shared 99% sequence identity with it. Thus, we hypothesized that LSNV and WZSV 9 were different isolates of the same virus species. Here we confirm that hypothesis by cloning and sequencing of the full genome of LSNV from P. monodon and by showing that it consists of two fragments each with 99% identity to the matching fragments of WZSV.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevance This work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers.

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 EVE366, suggesting that it was heritable, since shrimp sperm (non-motile) do not contain mitochondria. However, to prove this hypothesis that EVE366 was heritable and located in chromosomal DNA, it was necessary to carry out mating tests to show that EVE366 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 EVE366 while the male was negative, and in Cross 2, both the female and male were PCR-positive for EVE366. Individual analysis of the offspring of Cross 1 revealed a distribution of 1:1 for EVE366, indicating that the EVE366-positive female parent was heterozygous for EVE366. In the second cross, the distribution of EVE366 in the offspring was 3:1, indicating that both PCR-positive parents were heterozygous for EVE366. These results supported the hypothesis that EVE366 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.

Several shrimp diseases are new or newly emerged in Asia, including acute hepatopancreatic necrosis disease (AHPND), hepatopancreatic microsporidiosis (HPM), hepatopancreatic haplosporidiosis (HPH), aggregated transformed microvilli (ATM) and covert mortality disease (CMD). In addition to these, white spot disease (WSD), yellow head disease (YHD) and infectious myonecrosis (IMN) continue as the most serious viral threats to shrimp farmers in the region. Other diseases such as monodon slow growth syndrome (MSGS), white tail disease (WTD) and abdominal segment deformity disease (ASDD) are of less concern. In contrast, Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) have become innocuous due to the widespread use of highly tolerant specific pathogen free (SPF) stocks of Penaeus ( Litopenaeus ) vannamei that dominate production. Similarly, diseases caused by monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) appear not to affect P. vannamei . Spread of diseases has been promoted by the use of live or fresh broodstock feeds such as polychaetes and clams. Also, shortages in the supply of imported SPF broodstock led some entrepreneurs to employ post larvae (PL) of imported SPF stocks to produce 2nd generation broodstock in open shrimp ponds where they became contaminated and were then used to produce PL for stocking ponds. These practices left the whole shrimp industry vulnerable to rapid spread of the new and newly emerging diseases and resulted in the current crisis in Asian shrimp culture. The situation has been exacerbated since 2009 by an almost exclusive focus on AHPND, which is only partially responsible for what has been widely called early mortality syndrome (EMS). The purpose of this review is to summarize progress of research on AHPND bacteria and also to encourage a wider focus on additional pathogens that are causing farm losses. The significance of these diseases and their implications for the future of shrimp aquaculture are discussed. Statement of relevance This review summarizes recent information about new and newly emerging diseases of cultured shrimp in Asia and discusses the biosecurity lapses that led to the current shrimp production crisis. All industry stakeholders must be aware of this situation and of the need for regional and global collaborative efforts to stem this crisis and prevent future development of another.

The viral accommodation hypothesis proposes that endogenous viral elements (EVE) from both RNA and DNA viruses are being continually integrated into the shrimp genome by natural host processes and that they can result in tolerance to viral infection by fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we hypothesized that previously reported microarray results for the presence of white spot syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427 in a domesticated giant tiger shrimp (Penaeus monodon) breeding stock might have represented expression from EVE, since the stock had shown uninterrupted freedom from white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens from a current stock generation were confirmed for freedom from WSSV infection using two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128 specimens (26%) positive for at least one of the ORF at very high sequence identity (95-99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated RNA templates. This revealed variable RNA expression in individual shrimp including no RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2) or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens producing putative imRNA. The variable types and numbers of the EVE and the variable RNA expression (including potential imRNA) support predictions of the viral accommodation hypothesis that EVE are randomly produced and expressed. Positive nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp sperm (germ cells), indicated that they were heritable.

In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (accession numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2 h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes.

White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55–214) of Penaeus monodon caspase ( Pm Casp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length Pm Casp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with Pm Casp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon ( Pm caspase) did not block apoptosis induced by Pm Casp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon .

The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mortality (often called early mortality syndrome or EMS) in cultivated shrimp were responsible for a 33% drop in shrimp production during the first quarter of 2013. Similar early mortality in Vietnam was ascribed to specific isolates of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND) but the status of EMS/AHPND in Thailand was unclear. Here we describe the isolation and characterization of bacteria isolated from the hepatopancreas (HP) of shrimp collected from an early mortality outbreak farm in Thailand. Four independent bacterial isolates were identified as V. parahaemolyticus by BLAST analysis and by gene-specific marker detection of a lecithin dependent hemolysin (LDH) considered to be specific for the species. Immersion challenges with 3 of these and a reference isolate, obtained from China in 2010, using a previously published laboratory infection model caused very high mortality accompanied by characteristic AHPND histopathology in the shrimp HP. Tests with one of these isolates (5HP) revealed that rate of mortality was dose dependent. Using the same challenge protocol, the 4th isolate (2HP) also caused high mortality, but it was not accompanied by AHPND histopathology. Instead, it caused a different histopathology of the HP including collapsed epithelia and unique vacuolization of embryonic cells (E-cells). These results revealed the possibility of diversity in isolates of V. parahaemolyticus that may cause early mortality in shrimp cultivation ponds. Genomic and episomic DNA of these isolates and isolates of V. parahaemolyticus that cause no disease need to be compared to better understand the molecular basis of bacterial virulence in AHPND.

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein–protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

We have shown previously that ultrafiltrates (5 kDa cutoff) of cell-free medium from mosquito cell cultures persistently infected with DENV serotype 2 (DENV-2) contained a novel antiviral agent (called viprolaxikine) that could protect pre-treated, naive mosquito cells from DENV infection. Here, we show that viprolaxikine also reduced DENV-2 titers by almost 4 logs (>99.9%) when compared to Vero cells mock-treated with ultrafiltrates from cultures of uninfected mosquito cells. Protease treatment removed the anti-DENV-2 activity. Pre-incubation for 48-h was required to obtain the maximum, dose-dependent protection against DENV-2, indicating that the antiviral activity was based on the interaction between Vero cells and viprolaxikine rather than direct action of viprolaxikine on DENV-2. Activity was highest against DENV-2, but there was also significant activity against the 3 other DENV serotypes. LC–MS–MS analysis revealed that the active viprolaxikine fraction contained anionic, antiviral peptides, each comprised of 7 amino acids (DDHELQD, DETELQD and DEVMLQD or DEVLMQD) and with a common sequence motif of D-D/E-X-X-X-Q-D. These sequences do not occur in the dengue virus genome, suggesting that the peptides are produced by the host insect cells when persistently infected with DENV-2. These peptides represent a new class of anionic, insect-derived, antiviral peptides with activity against a flavivirus in both mammalian and insect cells.

Whitening of muscle tissue in farmed whiteleg shrimp Penaeus (Litopenaeus) vannamei can result from stress-induced muscle cramps and from viral infections caused by infectious myonecrosis virus (IMNV) or Penaeus vannamei nodavirus (PvNV). A similar viral-induced whitening of muscles can be caused in the river prawn Macrobrachium rosenbergii and in larvae of the penaeid shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus by a mixed infection with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here we describe mixed infections of IMNV and MrNV in juvenile cultivated P. vannamei from Indonesia, detected by nested RT-PCR and immunohistochemical analysis. Muscle lesions in the dually-infected shrimp gave positive immunohistochemical reactions for both IMNV and MrNV, while connective tissue in the same samples gave positive reactions for MrNV only, indicating some differences in tissue specificity between the two viruses. Although it is not known whether the dual infections are more lethal to shrimp than single IMNV infections, this is possible since earlier work has shown that MrNV alone can increase mortality of P. vannamei under stress.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.

High mortality of juvenile Penaeus (Litopenaeus) vannamei less than one month old in Indonesia has been observed in shrimp farms and hatcheries since 2007. The gross signs of disease included shrinkage of the hepatopancreas, flaccid bodies, cuticular melanization and retarded growth. Overall loss in the severely-infected farms was 60 to 90% due to progressive mortality. The loss since 2007 is estimated to be more than 5 million US dollars. PCR testing of shrimp samples for major viral pathogens gave negative results, but histopathological analysis of hepatopancreatic (HP) tissue showed evidence of a haplosporidian parasite indicated by cytoplasmic multinucleate plasmodia and trophonts in tubule epithelial cells. Plasmodia caused disruption of HP tubule epithelial cells when they discharged trophonts into the tubular lumen. To confirm the presence of haplosporidia, PCR was performed to amplify a small subunit rRNA gene (SSU rDNA) fragment and a cloned 1.5 kb amplicon showed 96% sequence identity to that of a haplosporidian previously described in P. vannamei from Belize, Central America suggesting that the Indonesian haplosporidian was either conspecific or very closely related. In situ hybridization assays using a probe based on this amplicon gave positive results specifically with the putative haplosporidian lesions. Prevalence of infections in broodstock and ponds from 2004 to 2010 suggested that the haplosporidian disease outbreaks had resulted from stocking of infected post-larvae.

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight.

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(®), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.