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Since 2010, sexual precocity, a typical sign of the iron prawn syndrome (IPS), resulting in the reduced size of farmed giant freshwater prawns Macrobrachium rosenbergii, has caused substantial production losses. However, the cause of IPS was not clear. We ran tests for eight major shrimp pathogens, but none were detected from IPS-affected prawns. We performed the histopathological examination of tissues and identified an eosinophilic inclusion in the perinuclear cytoplasm of cells in various tissues associated with nervous and endocrinal functions in the compound eyes. A subsequent bioassay with viral extracts of IPS-affected samples reproduced the gross signs of IPS. Metatranscriptomic sequencing identified a novel virus of Flaviviridae in all IPS-affected M. rosenbergii prawns, which was not found in samples without IPS. This virus contains a positive-sense, single-stranded RNA genome of 12,630 nucleotides (nt). Phylogenetic analysis of the conserved RdRp and NS3 domains showed that it may belong to a new genus between Jingmenvirus and Flavivirus. Under transmission electron microscopy (TEM), putative virus particles showed as spherical with a diameter of 40 to 60 nm. In situ hybridization found hybridization signals consistent with the histopathology in the compound eyes from IPS-affected M. rosenbergii. We provisionally name this virus infectious precocity virus (IPV) and propose the binominal Latin name Crustaflavivirus infeprecoquis gen. nov., sp. nov. We developed a nested reverse transcription-PCR diagnostic assay and confirmed that all IPS-affected prawns tested IPV positive but normal prawns tested negative. Collectively, our study revealed a novel virus of Flaviviridae associated with sexual precocity in M. rosenbergii. IMPORTANCE The iron prawn syndrome (IPS), also described as sexual precocity, results in the reduced size of farmed prawns at harvest and significant economic losses. IPS has been frequently reported in populations of farmed Macrobrachium rosenbergii since 2010, but the cause was heretofore unknown. Here, we reported a novel virus identified from prawns with IPS using infection experiments, metatranscriptomic sequencing, and transmission electron microscopy and provisionally named it infectious precocity virus (IPV). Phylogenetic analysis showed that IPV represents a new genus, proposed as Crustaflavivirus gen. nov., in the family Flaviviridae. This study provides novel insight that a viral infection may cause pathological change and sexual maturation and subsequently affect crustacean growth. Therefore, we call for quarantine inspection of IPV in transboundary trade of live M. rosenbergii and enhanced surveillance of IPV in aquaculture in the region and globally.

Acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease of marine shrimp that has caused severe economic losses to aquaculture producers in affected areas. AHPND was originally found to be caused by virulent strains of Vibrio parahaemolyticus (VpAHPND). These pathogenic strains carry pirABvp genes, which produce a binary toxin and are located on pVA1-type plasmid. Subsequently, it was found that AHPND can also be caused by pathogenic strains of other Vibrio species including: V. harveyi-like, V. campbellii, V. punensis, and V. owensii, all of which harbor a pVA1-type plasmid. Here we hypothesize that the pVA1-type plasmid can be transferred horizontally among Vibrio species. We carried out laboratory studies confirming the horizontal transfer of pVA1-type plasmid from AHPND-causing V. campbellii strain 20130629003S01 (VcAHPND-3S01) to a non-AHPND V. owensii strain 20160513VC2W (Vo-VC2W). ANI analysis revealed that strain Vo-VC2W is closely related to the V. owensii strain XSBZ03 isolated from corals in the South China Sea. However, the pirABvp genes on the transferred plasmid were unstable and lost from the pVA1-type plasmid of Vo-VC2W after 2 sub-culture passages. The sequences of the pVA1-type plasmid in AHPND-Vc3S01 and ΔpirABvpVo-VC2W were highly homologous (99.9% identical), indicating that this plasmid acquired by the V. owensii was from the AHPND-V. campbellii strain. In addition, complete genome sequence of the ΔpirABvpVo-VC2W showed that it contains 2 chromosomes and 3 plasmids; its detailed genomic characteristics will provide relevant information for better understanding of a V. owensii isolate that harbors transferred pVA1-type plasmid.

Interspecies transmission of viruses, where a pathogen crosses species barriers and jumps from its original host into a novel species, has been receiving increasing attention. Viral covert mortality disease, caused by covert mortality nodavirus (CMNV), is an emerging disease that has recently had a substantial impact on shrimp aquaculture in Southeast Asia and Latin America. While investigating the host range of CMNV, we found that this virus is also capable of infecting populations of the farmed Japanese flounder Paralichthys olivaceus, a vertebrate host. The infected fish were being raised in aquaculture facilities that were also producing marine shrimp. Through RT-nPCR, targeting the RNA-dependent RNA polymerase (RdRp) gene of CMNV, we found that 29 % of the fish sampled were positive. The amplicons were sequenced and aligned to the RdRp gene of shrimp CMNV and were found to have 98 % identity. Histopathological examination indicated that CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain, as well as extensive necrosis of cardiac muscle. In situ hybridization showed positive reactions in tissues of the eye, brain, heart, liver, spleen and kidney of infected fish. Transmission electron microscopy showed the presence of CMNV-like particles in all of the above-mentioned tissues, except for brain. The novel finding of a shrimp alphanodavirus that can also infect farmed P. olivaceus indicates that this virus is capable of naturally crossing the species barrier and infecting another vertebrate. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Covert mortality nodavirus (CMNV), a novel aquatic pathogen, causes viral covert mortality disease (VCMD) in shrimps and also known to infect farmed marine fish. To date, there has no report regarding the ability of this virus to infect freshwater fish. In this study, we screened and discovered CMNV-positive freshwater zebrafish individuals by reverse transcription-nested PCR (RT-nPCR). The sequence of CMNV amplicons from zebrafish was found to share 99% identity with RNA-dependent RNA polymerase (RdRp) gene of the original CMNV isolate. Histopathological examination of the CMNV-positive zebrafish samples revealed extensive vacuolation and karyopyknosis lesions in the retina of the eye and the midbrain mesencephalon. CMNV-like virus particles were visualized in these tissues under transmission electron microscope. Different degrees of pathological damages were also found in muscle, gills, thymus and ovarian tissues. Strong positive signals of CMNV probe were observed in these infected tissues by in situ hybridization. Overall, all results indicated that zebrafish, an acknowledged model organism, could be infected naturally by CMNV. Thus, it is needed to pay close attention to the possible interference of CMNV whether in assessment of toxic substances, or in studying the developmental characterization and the nerval function, when zebrafish was used as model animal.

The ecological risks of white spot syndrome virus (WSSV), an important aquatic pathogen, has been causing increasing concern recently. A continuous survey on the prevalence of WSSV in the wild crustaceans of the Bohai Sea was conducted in present study. The result of loop-mediated isothermal amplification detection showed that WSSV positivity rates of sampling sites were determined to be 76.73%, 55.0% and 43.75% in 2016, 2017 and 2018, respectively. And the WSSV positivity rates of samples were 17.43%, 12.24% and 7.875% in 2016, 2017 and 2018, respectively. Meanwhile, the investigation revealed that 11 wild species from the sea were identified to be WSSV positive. The WSSV infection in wild crustacean species was confirmed by transmission electron microscopy analysis. The results of this study suggest that WSSV had been colonized in wild species offshore and the impact caused by WSSV to the wild marine ecosystem cannot be ignored.

Tubulins are major components of microtubules, structures that are involved in many cellular processes. The gene β-tubulin, which encodes a β-tubulin protein, is conserved, and yet with sufficient variability to be useful for phylogenetic studies and for use in molecular diagnostic protocols. We sequenced and analyzed a fragment (870-bp) of β-tubulin gene of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp and associated with growth retardation. The sequence was then used to conduct a phylogenetic study to determine the position of EHP within the Microsporidia and to develop primers for an EHP nested PCR method. Blast analysis revealed a 75% nucleotide identity of EHP β-tubulin to that of E. bieneusi. Similar to other studies based on the small subunit rRNA sequence, the phylogenetic analysis using β-tubulin sequences clustered EHP with E. bieneusi and two other crustacean microsporidia, Agmasoma penaeii and Hepatospora eriocheir. As for the diagnostic use, a nested PCR method was developed that successfully detected EHP in infected Penaeus vannamei from Indonesia, Thailand, Vietnam, and India and in some cases of Artemia biomass. There were no cross-reactions to the shrimp and other crustacean genomes. This nested PCR is 100-fold more sensitive than the one-step PCR and thus suitable for detecting a low level of EHP infection.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has been one of the most problematic diseases in marine shrimp aquaculture throughout Southeast Asia and Latin America. To evaluate the effectiveness of a bacteriophage (phage) treatment for AHPND, a series of bioassays were carried out in a marine shrimp (Penaeus vannamei) model using an AHPND-V. parahaemolyticus strain that is highly pathogenic to shrimp. We monitored the mortality and histopathological changes during phage treatment. Shrimps treated with phage prophylaxis and phage therapy displayed significant protection from AHPND and survived a lethal bacterial challenge.

The impacts of mariculture on coastal ecosystems are increasingly raising attention recently. To study the prevalence of white spot syndrome virus (WSSV) in wild crustaceans, a pathogenic agent of cultured shrimps, continuous and large-scale surveys in the Bohai Sea were firstly conducted from 2016 to 2018. A total of 820 samples were collected from 59 sampling sites. The results of loop-mediated isothermal amplification (LAMP) assay showed that 11 of the 19 wild species were identified to be WSSV positive. Among them, Alpheus distinguendus had the highest WSSV-positive rate at 21.9%. The percentage of WSSV positive sampling sites was 76.7%, 55.0%, 43.7% in 2016, 2017, 2018, respectively. The percentage of WSSV positive samples was 17.4%, 12.2% and 7.8%, respectively. The multiple sequence alignment and phylogenetic tree analysis showed that the WSSV VP26 gene cloned from the wild crustaceans was consistent with those from WSSV strains previously reported in India, China, Brazil, Thailand, as well as Mexico, which was clustered tightly into one branch. The presence of WSSV virions in the epithelial cells of wild southern rough shrimp Trachypenaeus curvirostris was confirmed by TEM assay, which further verifying the occurrence of WSSV infection in wild crustaceans. The results of present study demonstrated that WSSV had colonized in the populations of offshore wild crustaceans in the Bohai Sea, and the potential negative impact of WSSV prevalence on the wild crustacean populations together with marine ecosystem deserved close attention and further investigation.

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intracellular parasite that has become a critical threat to the shrimp farming industry in SE Asia. This parasite replicates in the hepatopancreas and midgut, and infected shrimp exhibit reduced feeding and severely retarded growth. In this study, we describe the first case of EHP-infected Penaeus vannamei cultured in Venezuela. Its histopathology is very similar to that of SE Asia EHP, with infected shrimp showing basophilic inclusions in hepatopancreas. Upon in situ hybridization of an EHP 18S rRNA gene fragment labeled with digoxigenin, the probe reacted intensely to the basophilic inclusions within the cytoplasm of the infected cells. We also compared the nucleotide sequence similarities in 18S rRNA gene (1095-bp), in β-tubulin (583-bp) and spore wall protein (431-bp) genes between the Venezuela EHP and SE Asia isolates; and the results showed 99%, 93% and 91% identities, respectively. This suggests that the Venezuela EHP was not recently introduced from SE Asia. In a shrimp farm that was affected by EHP, Taura syndrome virus (TSV) was also detected in the P. vannamei by RT-qPCR and histological examinations. TSV is a major pathogen in penaeid shrimp aquaculture and has caused substantial economic losses during the last 2 decades, however, TSV has not been reported since 2011. This TSV was determined to be a Venezuela genotype based on the phylogenetic analysis in the capsid protein 2 gene and it showed to have sequence identities of 97% (nucleotide) and 98% (amino acid) to a Venezuela isolate found in 2005. The emergence of EHP and occurrence of TSV in the Venezuela will have a significant impact on the shrimp production if they spread to other farms. Therefore, the shrimp cultured within the country should be monitored closely for the presence of EHP and TSV.

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X14/15, X23/24) (N75-N125-N94) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (595014/15, 1097123/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (595014/15, 1097123/24) (375-6125-794). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.

The hepatopancreatic microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that affects cultured shrimp Penaeus vannamei in several SE Asian countries including China, Vietnam, Thailand, Indonesia, India and Malaysia. EHP infections are often accompanied by opportunistic infections of Vibrio spp. Laboratory challenges and a case control study were used to determine the effects of EHP infection on two Vibrio diseases: acute hepatopancreatic necrosis disease (AHPND) and septic hepatopancreatic necrosis (SHPN). To determine the effect of EHP on AHPND, two independent experimental infections were carried out. EHP-infected shrimp (EHP-AHPND group) and healthy shrimp (AHPND group) were challenged with 2.4 × 105 CFU/mL of AHPND-causing Vibrio parahaemolyticus. The results of the experimental infections showed that EHP-AHPND group exhibited higher mortalities (60 and 44%) than the AHPND group (0 and 18%). The pathological effects of AHPND vs. EHP-AHPND groups were compared during the first 12 h post infection, 57% of the EHP-AHPND group displayed severe hepatopancreas necrosis and sloughing, features characteristic of AHPND infection while only 11% of AHPND group showed these features. This indicated that EHP-infected shrimp have a higher susceptibility to AHPND infection. To determine the effect of EHP on SHPN, we reviewed the histopathology of samples collected where EHP is endemic; and a case control study was carried out to determine the association between SHPN and EHP. We compared individual shrimp displaying histological signs of SHPN with the shrimp from the same ponds without these signs. A strong association was found between SHPN and EHP, indicating that shrimp with EHP have an increased susceptibility to SHPN. These findings suggested that EHP infection is a risk factor for both AHPND and SHPN.

Two Vibrio parahaemolyticus virulence genes, pir A vp and pir B vp , are known to encode a binary photorhabdus insect-related (Pir) toxin that causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. These genes are flanked with repeats of a mobile element (insertion sequence) in a large plasmid. This insertion sequence is closely (92%) related to the known insertion sequence ISVal1. The pir AB vp genes and the flanking ISVal1 forms a 5535-bp composite transposon, designated Tn6264. There are pir AB vp gene deletions in some strains of V . parahaemolyticus . During 2013–2016, we found 2 types of pir AB vp deletion mutants from AHPND-affected farms. The type I mutants included 3 strains with deletions (4.4-kb or 6.0-kb) of entire pir AB vp genes and the downstream ISVal1, and these mutants were named pir AB vp (‐). The type II mutants included 3 strains with smaller deletions (1.5-kb or 1.6-kb) including a pir A vp gene and a partial pir B vp gene, and were named pir A vp (‐). In laboratory bioassays, these were not pathogenic to shrimp confirming that both pir A vp and pir B vp are required for AHPND pathogenicity. During 2016, we also isolated 4 V . campbellii strains carrying pir AB vp genes from diseased shrimp. These V . campbellii stains were found, through laboratory bioassays and histological evaluation, to cause AHPND.

Acute hepatopancreatic necrosis disease (AHPND, also known as early mortality syndrome, EMS) has caused substantial mortality, up to 100%, in populations of penaeid shrimp cultured in SE Asia and in Latin America. The disease is caused by the bacterium Vibrio parahaemolyticus , which secretes binary toxins (PirA vp and PirB vp ) resulting in the deterioration of the hepatopancreas tissue of infected shrimp. Diagnosis, screening, and monitoring of AHPND in shrimp populations involve sacrificing individuals to obtain tissue samples. This sampling method is undesirable when applied to valuable populations of broodstock. Here, we evaluated a non-invasive diagnostic method based on shrimp fecal samples that are analyzed by PCR. Small groups of Pacific white shrimp Penaeus vannamei were exposed to low levels of AHPND-bacteria and their feces were collected prior to any mortality observed (in the bioassays #1 and #2). Two protocols were evaluated. In one, DNA extracted from the fecal samples was directly analyzed by PCR. In the other, the fecal samples were cultured in TSB+ for 6 h to enrich the bacterial populations, then the enriched bacterial broth was used for PCR analyses. Our results showed that the presence of V. parahaemolyticus could be detected both in fecal DNA samples and in the enriched bacterial broth, but the bands from the bacterial broth showed stronger amplification than the DNA; 12 strong positive in the enriched bacterial broth, but only 7 strong positive in the fecal DNA samples. Also, the AHPND bacteria present in the feces is infectious, determined by a bioassay of feeding specific pathogen free indicator shrimp with AHPND-feces (in the bioassay #3), and this proves that the AHPND can be transmitted through a fecal-oral route.

Acute hepatopancreatic necrosis disease (AHPND) has caused sharp declines in aquaculture industries of whiteleg shrimp Penaeus vannamei in Asia and the Americas since 2010. Vibrio parahaemolyticus, V. campbellii, V. owensii and V. punensis have been proved to cause AHPND. However, the mechanisms underlying the burgeoning number of Vibrio species that cause AHPND is not known. All of AHPND-causing Vibrio bacteria (VAHPND) harbor a highly homologous plasmid (designated as pVA1-type) carrying pirABvp toxin genes. In this study, we demonstrate conclusively that the pVA1-type plasmid can be transferred from VAHPND to non-pathogenic bacteria. We constructed a pVPGX1-Cmr plasmid (a pVA1-type plasmid) by adding a chloramphenicol resistance gene as a marker in a donor AHPND-causing V. parahaemolyticus 20130629002S01 (Vp2S01). Horizontal transfer of this plasmid was successfully performed from the AHPND-Vp2S01 to a non-pathogenic strain of V. campbellii at the transfer efficiency of 2.6 x 10-8 transconjugant/recipient, and DNase I treatment did not eliminate the transfer. The recipient V. campbellii acquired the pVA1-type plasmid and was shown to produce pirABvp RNA and proteins. Challenge studies using the transconjugant caused 100% mortality in exposed groups of P. vannamei. The challenged shrimp, infected with the transconjugant bacteria, showed typical gross signs and histological lesions of AHPND. This is the first report demonstrating the conjugative transfer of an AHPND pVA1-type plasmid. It provides timely information for explaining the increased species of AHPND-causing Vibrio bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND.

Acute hepatopancreatic necrosis disease (AHPND, also known as early mortality syndrome, EMS) caused by Vibrio parahaemolyticus has resulted in severe marine shrimp mortality and significant economic losses in related aquaculture throughout Southeast Asia and Central America. As no viable remedy has yet been reported, the main objective of this study was to develop an effective bacteriophage (phage)-based method of controlling AHPND/EMS. To determine the bacteriolytic activity of phage pVp-1 against V. parahaemolyticus strains causing AHPND/EMS, its infectivity was tested on 22 strains isolated from geographically diverse regions (5 of the Asian type and 17 of the Mexican type). This phage was able to infect 90.9% (20 strains among 22 strains) of the AHPND/EMS-related V. parahaemolyticus strains used in this study, and demonstrated substantial bacteriolytic activity against three strains known to be highly pathogenic. To the best of our knowledge, this is the first report of a virulent phage infecting V. parahaemolyticus strains responsible for AHPND/EMS, and indicates the potential utility of pVp-1 in phage therapy.

A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.

A quantitative polymerase chain reaction (qPCR) assay was developed, based on a TaqMan probe, to detect and quantify a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The assay uses a pair of PCR primers, which amplify a 135-bp DNA fragment, and a TaqMan probe selected from the plasmid pir A-like gene. This qPCR assay reacted with AHPND-pathogenic isolates of V. parahaemolyticus collected from Vietnam and Mexico, but not with non-pathogenic strains of Vibrio spp. For quantification, a plasmid (pVpPirA-1) containing the target pir A-like gene was constructed, purified and serially diluted to be used as a standard. With this standard, the qPCR assay was then used to quantify the virulence plasmid in shrimp samples collected from different farms. Up to 5.8 × 10 5 copy per mg tissue were detected in AHPND-affected shrimp collected from Vietnam. Lower quantities, up to 1.5 × 10 4 copies per mg of tissues were detected in affected shrimp collected from a Chinese farm. In the laboratory bioassays, similar plasmid quantities, 1.8 × 10 3 to 4.7 × 10 6 copies of plasmid per mg of tissues were found in the moribund/dead shrimp, 3.5 × 10 2 to 2.2 × 10 6 copies of plasmid per mL were detected in the water samples. This assay is specific with high sensitivity (10 copies of virulence plasmid) and can be used to detect AHPND-pathogenic V. parahaemolyticus in shrimp and water samples.

We report here the complete genome sequence of Vibrio campbellii , isolated from Penaeus vannamei cultured in a Latin American country. The Tn 3 -like transposon and pirAB genes were encoded on the plasmid pLA16-2. These data support the geographical variations in the virulence plasmid found among acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio isolates from Latin America and Asia.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

We developed a qPCR assay based on the β-tubulin gene sequence for the shrimp microsporidian parasite Enterocytozoon hepatopenaei (EHP). This assay reacted with the hepatopancreas (HP) of EHP-infected shrimps, and the highest copy numbers were found in HP and feces samples from Southeast Asian countries (106–108 copies mg−1), while HP samples from Latin America, Artemia, and EHP-contaminated water showed lower amounts (101–103 copies mg−1 or mL−1 of water). No false positive was found with the normal shrimp genome, live feeds, or other parasitic diseases. This tool will facilitate the management of EHP infection in shrimp farms.

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tan- dem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We catego- rized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment.

Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS reappeared as a significant disease. In 2010, an apparently new strain of TSV (designated as CO 10) was collected in Colombia. Its genome was sequenced and compared with six other fully sequenced isolates. This analysis revealed that the TSV CO 10 is closely related to the isolates from Hawaii and Venezuela. Phylogenetic analysis based on capsid protein 2 (CP2) region from 59 TSV isolates shows that the recent Colombian isolates (2006–2010) form a new cluster and differ from the previous Colombia isolates (1994–1998) by 4% in nucleotide sequence. The virulence of this CO 10 isolate was similar to a Belize TSV determined through experimental infection in P. vannamei showing 100% mortalities and similar survival curves. By RT-qPCR for TSV, the viral loads were also close in the infected shrimp from both CO 10 and Belize at the order of 1 × 1010 copies per μl RNA. To develop TSV-resistant lines, the candidate shrimp should be challenged with virus strains that have been isolated most recently from the regions where they will be cultured. This study suggests that the TSV present in Colombian shrimp farms during the last 5 years is a new TSV strain with high virulence.

Samples of microsporidia-infected shrimps exhibiting clinical signs of cotton shrimp disease were collected from Madagascar, Mozambique, and the Kingdom of Saudi Arabia from 2005 to 2014. The tails of the infected shrimps appeared opaque and whitish; subsequent histological examination revealed the presence of cytoplasmic inclusions and mature spores in tissues of the muscle, hepatopancreas, gills, heart, and lymphoid organ. PCR analysis targeting the small subunit rDNA (SSU rDNA) from infected samples resulted in the amplification of a 1.2 kbp SSU rDNA sequence fragment 94% identical to the corresponding region in the genome of the microsporidian Perezia nelsoni, which infects populations of Penaeus setiferus in the USA. Its SSU rDNA sequence was 100% identical among isolates from Madagascar and Saudi Arabia, indicating that shrimps from the Red Sea and Indian Ocean were infected with the same microsporidium, the novel Perezia sp. A 443 bp fragment of the SSU rDNA sequence was cloned, labeled with digoxigenin and subjected to an in situ hybridization assay with tissue sections of Perezia sp.-infected Penaeus monodon from Madagascar and Mozambique, and P. indicus from Saudi Arabia. The probe hybridized to the mature spores in the hepatopancreas and muscle from which the spores had been obtained for DNA isolation. This assay was specific, showing no reaction to another microsporidium, Enterocytozoon hepatopenaei (EHP), infecting the hepatopancreas of shrimp P. stylirostris cultured in SE Asian countries. We also developed an SSU rDNA-based PCR assay, specific for the novel Perezia sp. This PCR did not react to EHP, nor to genomic DNA of shrimp and other invertebrates.

White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.

Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the eastern USA via frozen commodity products and it reached the main shrimp farming countries of the Americas located on the Pacific side of the continents by the same mechanism in 1999. As is the case in Asia, WSD is the dominant disease problem of farmed shrimp in the Americas. The most recent disease to emerge in the Americas was infectious myonecrosis caused by IMN virus. As had happened before, within 3years of its discovery, the disease had been transferred to SE Asia with live P. vannamei, and because of its impact on the industry and potential for further spread in was listed by the OIE in 2005. Despite the huge negative impact of disease on the shrimp farming industry in the Americas, the industry has continued to grow and mature into a more sustainable industry. In marked contrast to 15-20years ago when PLs produced from wild adults and wild PLs were used to stock farms in the Americas, the industry now relies on domesticated lines of broodstock that have undergone selection for desirable characteristics including disease resistance.

The Penaeus vannamei nodavirus (PvNV), which causes muscle necrosis in Penaeus vannamei from Belize, was identified in 2005. Infected shrimp show clinical signs of white, opaque lesions in the tail muscle. Under transmission electron microscopy, the infected cells exhibit increases in various organelles, including mitochondria, Golgi stacks, and rough endoplasmic reticulum. Cytoplasmic inclusions containing para-crystalline arrays of virions were visualized. The viral particle is spherical in shape and 19 to 27 nm in diameter. A cDNA library was constructed from total RNA extracted from infected shrimp. Through nucleotide sequencing from the cDNA clones and northern blot hybridization, the PvNV genome was shown to consist of 2 segments: RNA1 (3111 bp) and RNA2 (1183 bp). RNA1 contains 2 overlapped open reading frames (ORF A and B), which may encode a RNA-dependent RNA polymerase (RdRp) and a B2 protein, respectively. RNA2 contains a single ORF that may encode the viral capsid protein. Sequence analyses showed the presence of 4 RdRp characteristic motifs and 2 conserved domains (RNA-binding B2 protein and viral coat protein) in the PvNV genome. Phylogenetic analysis based on the translated amino acid sequence of the RdRp reveals that PvNV is a member of the genus Alphanodavirus and closely related to Macrobrachium rosenbergii nodavirus (MrNV). In a study investigating potential PvNV vectors, we monitored the presence of PvNV by RT-PCR in seabird feces and various aquatic organisms collected around a shrimp farm in Belize. PvNV was detected in mosquitofish, seabird feces, barnacles, and zooplankton, suggesting that PvNV can be spread via these carriers.

A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in Penaeus vannamei . A pair of primers which amplify a 67 bp DNA fragment and a TaqMan probe were selected from the 16 S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 10 8 copies. An NHP-B challenge test using two different populations, Colombia (COL) and Specific Pathogen Free KONA line (KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs 1 × 10 5 copies).

In early 2005, the Aquaculture Pathology Laboratory of the University of Arizona received samples of diseased shrimp, Penaeus vannamei, from a Taura syndrome (TS) outbreak in the Lake Maracaibo region of Venezuela. Histopathology and in situ hybridization (ISH) were performed and the results confirmed the presence of Taura syndrome virus (TSV). The viral isolate was sequenced and presented a 93% similarity with the TSV reference strain from Hawaii (TSV-HI94). Immunohistochemistry (IHC), dot blot immunoassay and bioassays were also performed. While processed samples reacted only faintly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassay, the Venezuelan isolate of TSV (TSV-VE05) presented mortality comparable to TSV-HI94 in P. vannamei SPF Kona stock. These data indicate that a new variant of the virus was responsible for the outbreak of TS in Venezuela.

A segment of Madagascar hepatopancreatic parvovirus (HPV) genomic sequence (5742 nucleotides) was determined through PCR and direct sequencing. This nucleotide sequence was compared to isolates from Australia, Thailand, Korea, and Tanzania, and the mean distance was determined to be 17%. The Madagascar HPV is closest to the Tanzania isolate (12%), followed by isolates from Korea (15%), Australia (17%) and Thailand (20%). Analysis of the genomic structure revealed that this HPV sequence is comprised of one partial Left open reading frame (ORF) (349 amino acids, aa) and complete Mid (578 aa) and Right (820 aa) ORFs. The amino acid sequences of the 3 ORFs were compared among isolates. The Right ORF was found to have the highest variation with a mean distance of 24%. This was followed by the Left and Mid ORF with distances of 13 and 7%, respectively. A phylogenetic analysis based on the amino acid sequence of the Right ORF divides 7 HPV isolates into 3 well-separated groups: Korea, Thailand, and Australia. The Madagascar HPV clustered with the Korea and Tanzania isolates. In Madagascar, HPV has been detected by histological examination since the 1990s. PCR analysis of a recent (2007) sampling showed a 100% prevalence. HPV was also detected in Mozambique with a 100% prevalence. High (95%) prevalence of HPV was found in wild Penaeus merguinesis collected from New Caledonia. These results indicate that HPV displays a high degree of genetic diversity and is distributed worldwide among populations of penaeid shrimp.

A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes mus- cle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from labora- tory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.

Infectious myonecrosis, caused by Infectious myonecrosis virus (IMNV), is an important emerging disease of shrimp that has affected the production of cultured Litopenaeus vannamei in Northeast Brazil. In this study we report the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe to detect this virus in shrimp. The real-time RT-PCR showed a strong linear correlation (r2 = 0.986) between threshold cycles (CT) and RNA quantities. The assay gave negative results for other viruses, including Yellow head virus (YHV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), and White spot syndrome virus (WSSV) and the necrotizing hepatopancreatic bacterium (NHPB). This real-time RT-PCR assay can detect as few as 10 IMNV copy numbers/μl RNA, while the nested RT-PCR can detect no fewer than 1000 IMNV copy numbers/μl RNA. Specific-pathogen-free L. vannamei were used in the infectivity assay. There were one control group (Group 1) and one viral challenged group (Group 2), from which shrimp were sampled for RT-PCR and histological analysis. The RNA from dead shrimp was extracted and tested for IMNV by nested and real-time RT-PCR. The shrimp in Group 1 showed 100% survival, while those in Group 2 showed a 0% survival. The first mortality in the Group 2 was observed at Day 8 post-inoculation (p.i.); and the mortalities dramatically increased after Day 40 p.i. Histological sections from Group 2 shrimp taken at intervals throughout the study exhibited acute to chronic phase lesions of IMNV infection, and consecutive tissue sections reacted to the IMNV-specific cDNA probes by in situ hybridization. The real-time RT-PCR detected the presence of IMNV in all 30 of the challenged specimens in Group 2. In contrast, the nested RT-PCR detected the presence of IMNV in 23 of the 30 specimens. The real-time RT-PCR revealed that the 7 specimens not detected by nested RT-PCR contained relatively low IMNV copy numbers compared to the other 23 specimens. These results demonstrate that the real-time RT-PCR developed in this study is a sensitive diagnostic method for IMNV.

Acute hepatopancreatic necrosis disease (AHPND) has caused severe mortalities in farmed penaeid shrimp throughout SE Asia and Mexico. The causative agent of AHPND is the marine bacterium Vibrio parahaemolyticus, which secretes PirA- and PirB-like binary toxin that caused deterioration in the hepatopancreas of infected shrimp. The genes responsible for the production of this toxin are located in a large plasmid residing within the bacterial cells. We analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 unspecified Central American countries, but not in SE Asian isolates from China, Vietnam, and Thailand. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type based on the presence of the Tn3-like transposon. The SSR is found within the coding region of a hypothetical protein and has either 4, 5, or 6 repeat units. SSRs with 4 repeat units were found in isolates from Vietnam, China, and Thailand. SSRs with 5 repeat units were found in some Vietnamese isolates, and SSRs with 6 repeat units were only found in the Mexican isolates.

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13-028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syn- drome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated pro- teins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which sug- gests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In labora- tory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10 5 CFU ml �1 and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.

New sequencing studies of the nonsegmented dsRNA genome of penaeid shrimp infectious myonecrosis virus (IMNV), a tentatively assigned member of the family Totiviridae, identified previously unread sequences at both genome termini in three previously analyzed IMNV strains, one from Brazil (the prototype strain of IMNV) and two from Indonesia. The new sequence determinations add >600 nt to the 5′ end of the genomic plus strand of each strain, increasing the length of the 5′ nontranslated region to at least 469–472 nt and the length of the upstream open reading frame (ORF1) translation product by at least 48 aa. These new findings are similar to recent ones for two other IMNV strains (GenBank KF836757.1 and KJ556923.1) and thereby corroborate important amendments to the full-length IMNV genome sequence.

Penaeus monodon-type baculovirus (MBV) infects and causes disease in hatchery-reared larvae and early stages of juvenile P. monodon. In this study, an improved 1-step PCR method was developed for the detection of MBV. The oligonucleotide primers, assigned as 261F and 261R, were designed from a genomic clone of MBV. Using extracted DNA from hepatopancreas or feces of ten geographic isolates of MBV infected shrimp, the PCR reaction yields a 261 bp fragment specific to the MBV sequence. Primers 261F/R did not cross-react with DNA from other shrimp viruses or with uninfected shrimp tissue. The detection limit of this primer pair is 100 copies of positive control plasmid DNA. This improved molecular method provides a diagnostic tool for identification of MBV in infected shrimp tissue collected from different geographic regions.

A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72 bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples.

White spot syndrome virus (WSSV) is highly virulent to penaeid shrimp and has been responsible for serious economic losses on shrimp farms throughout the world. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a small DNA virus that also infects penaeid shrimp; and, although once virulent to culture stocks of Penaeus stylirostris , it has not been associated with mass mortalities in recent years. Through three laboratory challenge studies, we discovered an interference interaction between these two viruses in P. stylirostris. In the first bioassay, juvenile P. stylirostris were infected with IHHNV by feeding them with IHHNV-infected tissue. These shrimp, along with a group of non-infected control shrimp, were then fed WSSV-infected tissue. Two days after the WSSV challenge, mortalities began to occur. All of the control shrimp, which were not exposed to IHHNV, died within 5 days. In contrast, replicated challenge groups of the IHHNV pre-infected shrimp had 31% and 44% survival. Quantitation by real-time PCR determined that the surviving shrimp had high levels (10 9 copies per μg DNA) of IHHNV and very low levels (50–400 copies per μg DNA) of WSSV DNA. Moribund shrimp had high levels (10 6 –10 7 copies per μg DNA) of WSSV and low levels (10 4 –10 6 copies per μg DNA) of IHHNV DNA. A second challenge study with P. stylirostris that were heavily infected with IHHNV showed 21 of 23 (91%) shrimp survived 19 days after exposure to WSSV. Ten of these surviving shrimp were re-exposed to WSSV-infected tissue, and six were still alive after 3 weeks. The third challenge study showed that 28% of the IHHNV pre-infected P. stylirostris survived 22 days after exposure to WSSV. In situ hybridization analysis in all the challenge studies confirmed that the surviving shrimp were strongly infected with IHHNV and had, at most, only low levels of WSSV infection. Laboratory WSSV challenges showed that there were no survivals in either IHHNV pre-infected Penaeus vannamei or in IHHNV-resistant P. stylirostris (SuperShrimp™). In conclusion, juvenile P. stylirostris that are highly infected with IHHNV show resistance to WSSV infection.

Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P. vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii. In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.

A reovirus (tentatively designated as Callinectes sapidus reovirus, CsRV) was found in the blue crabs C. sapidus collected in Chesapeake Bay in 2005. Histological examination of hepatopancreas and gill from infected crabs revealed eosinophilic to basophilic, cytoplasmic, inclusions in hemocytes and in cells of connective tissue. A cDNA library was constructed from total RNA extracted from hemolymph of infected crabs. One clone (designated as CsRV-28) with a 532-bp insert was 75% identical in nucleotide sequence (and 95% similar in translated amino acid sequence) to the quanylytransferase gene of the Scylla serrata reovirus (SsRV). The insert of CsRV-28 was labeled with digoxigenin-11-dUTP and hybridized to sections of hepatopancreas and gill of infected C. sapidus, this probe reacted to hemocytes and cells in the connective tissue. No reaction was seen in any of the tissues prepared from uninfected crabs. Thus, this in situ hybridization procedure can be used to diagnose CsRV.

Three insecticidal toxin complex (tc)-like genes were identified in Vibrio parahaemolyticus 13-028/A3, which can cause acute hepatopancreatic necrosis disease in penaeid shrimp. The three genes are a tcdA-like gene (7710 bp), predicted to code for a 284-kDa protein; a tcdB-like gene (4272 bp), predicted to code for a 158-kDa protein; and a tccC3-like gene (2916 bp), predicted to encode a 107-kDa protein. All three predicted proteins contain conserved domains that are characteristic of their respective Tc proteins. By RT-PCR, all three tc-like genes were found to be expressed in this bacterium. Through genome walking and the use of PCR to join contigs surrounding these three genes, a genomic island (87 712 bp, named tc-GIvp) was found on chromosome II localized next to the tRNA Gly. The GC content of this island, which is not found in other Vibrio species, is 40%. The tc-GIvp is characterized to have 60 ORFs encoding regulatory or virulence factors. These include a type 6 secretion protein VgrG, EAL domain-containing proteins, fimbriae subunits and assembly proteins, invasin-like proteins, peptidoglycan-binding proteins, and Tc proteins. The tc-GIvp also contains 21 transposase genes, suggesting that it was acquired through horizontal transfer from other organisms.

A susceptibility study of postlarvae (PL) and juvenile Super Shrimp®, a selected line of Penaeus stylirostris , was conducted to compare their resistance to infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection to that of a specific pathogen free (SPF) population of P. vannamei . Super Shrimp® PLs were fed with IHHNV-infected shrimp tissue for 2 days and then maintained on a pelletized ration for an additional 28 days. PLs were sampled at days 0, 1, 2, 3, 4, 6, 10, 15, 20, 25 and 30. There was no apparent mortality during the experimental period. Tissue DNA extracted from the PLs was analyzed for the presence of IHHNV by PCR. Low levels of IHHNV were detected only in DNA extracts from samples at days 1, 2, and 3. No IHHNV DNA was detected from days 4 to 30. The days that the PLs were weakly IHHNV-PCR positive were during the period that they were being fed with IHHNV-tissue, and thus, the IHHNV DNA signal was suspected to be from the infected tissue used as a feed. Through both histology and in situ hybridization, we confirmed that tissues of Super Shrimp® PLs were not infected with IHHNV. PCR results of another IHHNV challenge study with juveniles of Super Shrimp® were similar to those with PLs. These results indicate that IHHNV did not replicate in the PL and juvenile Super Shrimp®. In contrast, P. vannamei juveniles, which were used as a positive control, showed a more intense IHHNV infection, as determined by PCR detection, beginning at 6 days postchallenge and increasing throughout the remainder of the study. In addition, the IHHNV-infected P. vannamei at 30 days postchallenge showed histological changes characteristic of IHHNV infection and had a positive reaction for IHHNV with in situ hybridization. Our studies show that Super Shrimp® are resistant to IHHNV infection. This is the first unequivocal demonstration of resistance to viral infection in shrimp.

A competitive polymerase chain reaction (PCR) method for quantification of white spot syndrome virus (WSSV) genome was developed. A pair of WSSV primers, designated WSSV341F/R, was selected to amplify a 341-bp DNA fragment from the WSSV genome. For a competitive internal standard, we constructed and cloned a 289-bp DNA fragment, the result of a 52-bp deletion from the 341-bp amplicon. In a competitive PCR reaction, we co-amplified the target WSSV DNA with known concentrations of the internal standard using WSSV341F/R primers. The amplicons from WSSV DNA and from internal standard DNA differed in size and could be distinguished after gel electrophoresis. The concentration of WSSV genomes was determined from its relation to the concentration of the internal standard. The log–log plot of the ratio of the amplicons (internal standard: WSSV) on the internal standard concentration was linear. Using this competitive PCR procedure, we quantified WSSV DNA in the samples of hemolymph and tissues of the cephalothorax of individual WSSV-infected shrimp. The number of WSSV genomes in both hemolymph and tissues corresponded to the severity of infection determined by histological evaluation. In addition, the changes in number of WSSV genomes in the hemolymph during the course of the infection were determined.

Since 1992, white spot syndrome virus (WSSV) and yellow head virus (YHV) have caused mortalities in cultured shrimp throughout Asia. By 1995, WSSV was detected in Texas and South Carolina, and the virus has also been recently reported in Central and South America (Nicaragua, Honduras, Guatemala, Panama, Colombia, Peru, and Ecuador). The importation of live infected shrimp is the principal mechanism by which exotic viruses may be introduced to new geographic regions. However, another probable mechanism is via the importation of infected commodity shrimp from regions where the pathogens are enzootic. Ten different lots of imported frozen tails of Penaeus monodon were screened for WSSV and YHV by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis. In 8 of 10 samples tested, WSSV was detected, and YHV was found in 3 out of the 10 samples. Six of the 10 sample sets of frozen shrimp gave strong positive tests for WSSV or YHV by PCR, and these were selected for bioassay with spe...

Taura syndrome virus (TSV)-specific primers, designated as 7171F/7511R, were designed to improve the sensitivity of RT-PCR detection. This pair of primers was shown to detect TSV isolates representative of four phylogenetic lineages: Belize, Americas, SE Asia, and Venezuela. Its detection limit was determined to be 20 copies of the TSV genome, 100 times more sensitive than the TSV primers 9992/9195 currently being used by many laboratories and recommended for surveillance and diagnostic applications by the Office International des Epizooties. Primers 7171F/7511R were found to be specific to TSV and did not to react to either Infectious hypodermal and hematopoietic necrosis virus (IHHNV), White spot syndrome virus (WSSV), Yellow head virus (YHV), or Infectious myonecrosis virus (IMNV). This new RT-PCR method was shown to detect TSV in chronically infected Penaeus vannamei that had survived up to 236 days after exposure to TSV.

White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly patho genic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Ara- bia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV iso- lates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analy- sis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a spe- cific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing com- plete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock.

Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome virus (TSV) and then challenged with yellow head virus (YHV) acquired partial protection from yellow head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.