Shrimp injection with dsRNA targeting the microsporidian EHP polar tube protein reduces internal and external parasite amplification.

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

Molecular and cellular characterization of four putative nucleotide transporters from the shrimp microsporidian Enterocytozoon hepatopenaei (EHP).

Microsporidia are obligate intracellular parasites that lost several enzymes required in energy production. The expansion of transporter families in these organisms enables them to hijack ATP from hosts. In this study, nucleotide transporters of the microsporidian Enterocytozoon hepatopenaei (EHP), which causes slow growth in economically valuable Penaeus shrimp, were characterized. Analysis of the EHP genome suggested the presence of four putative nucleotide transporter genes, namely EhNTT1, EhNTT2, EhNTT3, and EhNTT4. Sequence alignment revealed four charged amino acids that are conserved in previously characterized nucleotide transporters. Phylogenetic analysis suggested that EhNTT1, 3, and 4 were derived from one horizontal gene transfer event, which was independent from that of EhNTT2. Localization of EhNTT1 and EhNTT2 using immunofluorescence analysis revealed positive signals within the envelope of developing plasmodia and on mature spores. Knockdown of EhNTT2 by double administration of sequence specific double-stranded RNA resulted in a significant reduction in EHP copy numbers, suggesting that EhNTT2 is crucial for EHP replication in shrimp. Taken together, the insight into the roles of NTTs in microsporidian proliferation can provide the biological basis for the development of alternative control strategies for microsporidian infection in shrimp.

Molecular characterization of turtle-like protein in whiteleg shrimp (Litopenaeus vannamei) and its role in Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp hepatopancreas, causing growth retardation and disease susceptibility. Knowledge of the host-pathogen molecular mechanisms is essential to understanding the microsporidian pathogenesis. Turtle-like protein (TLP) is part of the immunoglobulin superfamily of proteins, which is widely distributed in the animal kingdom. TLP has multiple functions, such as cell surface receptors and cell adhesion molecules. The spore wall proteins (SWPs) of microsporidia are involved in the infection mechanisms. Some SWPs are responsible for spore adherence, which is part of the activation and host cell invasion processes. Previous studies showed that TLP from silkworms (Bombyx mori) interacted with SWP26, contributing to the infectivity of Nosema bombycis to its host. In this study, we identified and characterized for the first time, the Litopenaeus vannamei TLP gene (LvTLP), which encodes an 827-aa protein (92.4 kDa) composed of five immunoglobulin domains, two fibronectin type III domains, and a transmembrane region. The LvTLP transcript was expressed in all tested tissues and upregulated in the hepatopancreas at 1 and 7 days post-cohabitation (dpc) and at 9 dpc in hemocytes. To identify the LvTLP binding counterpart, recombinant (r)LvTLP and recombinant (r)EhSWP1 were produced in Escherichia coli. Coimmunoprecipitation and enzyme-linked immunosorbent assays demonstrated that rLvTLP interacted with rEhSWP with high affinity (KD = 1.20 × 10-7 M). In EHP-infected hepatopancreases, LvTLP was clustered and co-localized with some of the developing EHP plasmodia. Furthermore, LvTLP gene silencing reduced the EHP copy numbers compared with those of the control group, suggesting the critical role of LvTLP in EHP infection. These results provide insight into the molecular mechanisms of the host-pathogen interactions during EHP infection.

False mussels (Mytilopsis leucophaeata) can be mechanical carriers of the shrimp microsporidian Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite causing hepatopancreatic microsporidiosis (HPM) in cultivated shrimp in Asian countries. One strategy to control EHP is to identify and eliminate biological reservoir(s) in shrimp ponds. Several marine and brackish-water organisms, including false mussels (Mytilopsis) have been reported to test positive for EHP using the PCR method. Thus, we tested Thai false mussel Mytilopsis leucophaeata collected from the 6 ponds with EHP-infected shrimp for the presence of EHP using SWP-PCR. Results revealed the sampled mussels from all 6 ponds were PCR positive. Subsequent bioassays were carried out to study EHP transmission between mussels and shrimp. Firstly, the naïve mussels were cohabitated with EHP-infected shrimp and all mussels were SWP-PCR positive at day 20 post cohabitation. One batch of such PCR-positive mussels was transferred for cohabitation with naïve shrimp and 37.5% EHP-positive shrimp were observed within 10 days. Tissue analysis of the SWP-PCR-positive mussels using light microscopy, in situ hybridization technique and electron microscopy did not confirm EHP infection. In summary, there was no evidence demonstrating that Mytilopsis leucophaeata was itself infected with EHP. However, the false mussels were apparently capable of carrying infectious spores for some period after ingestion and serving as a mechanical or passive carrier. The results support previous reports warning of the danger of feeding living or fresh bivalves to broodstock shrimp in hatcheries or shrimp in rearing ponds without prior heating or freezing.

Co-expression of double-stranded RNA and viral capsid protein in the novel engineered Escherichia coli DualX-B15(DE3) strain.

BACKGROUND: Viruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell. RESULTS: A novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA. CONCLUSION: Co-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA.

The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.

The hematopoietic organ of Macrobrachium rosenbergii: Structure, organization and immune status.

The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 µm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph.

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia.

Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first sequenced genome from the Enterocytozoonidae - a major family of human and animal-infecting microsporidians - appeared to have lost genes for glycolysis. Here, we sequence and analyse genomes from additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei, a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri, a pathogen that lives exclusively inside epithelial cell nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri's adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host-pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.

Movement of the ß-hairpin in the third zinc-binding module of UvrA is required for DNA damage recognition.

Nucleotide excision repair (NER) is distinguished from other DNA repair pathways by its ability to process various DNA lesions. In bacterial NER, UvrA is the key protein that detects damage and initiates the downstream NER cascade. Although it is known that UvrA preferentially binds to damaged DNA, the mechanism for damage recognition is unclear. A ß-hairpin in the third Zn-binding module (Zn3hp) of UvrA has been suggested to undergo a conformational change upon DNA binding, and proposed to be important for damage sensing. Here, we investigate the contribution of the dynamics in the Zn3hp structural element to various activities of UvrA during the early steps of NER. By restricting the movement of the Zn3hp using disulfide crosslinking, we showed that the movement of the Zn3hp is required for damage-specific binding, UvrB loading and ATPase activities of UvrA. We individually inactivated each of the nucleotide binding sites in UvrA to investigate its role in the movement of the Zn3hp. Our results suggest that the conformational change of the Zn3hp is controlled by ATP hydrolysis at the distal nucleotide binding site. We propose a bi-phasic damage inspection model of UvrA in which movement of the Zn3hp plays a key role in damage recognition.

Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP).

BACKGROUND: Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. It is probably endemic in Australasia and was first characterized and named from the giant or black tiger shrimp Penaeus monodon from Thailand in 2009. Later, it was also found to infect exotic Penaeus vannamei imported for cultivation in Asia. HPM is not normally associated with shrimp mortality, but information from shrimp farmers indicates that it is associated with significant growth retardation that is not clearly noticeable until 2-3 months of cultivation. In order to study modes of HPM transmission and to test possible control measures, a laboratory challenge model was needed that would mimic the mode of infection in shrimp ponds. RESULTS: We describe successful transmission in a cohabitation model with natural E. hepatopenaei (EHP)-infected shrimp in closed, perforated plastic containers placed in aquaria together with free-swimming, uninfected shrimp. After a period of 14 days all the free-swimming shrimp tested positive by PCR (approximately 60% with heavy infections evident by 1-step PCR positive test results) and gave positive histological and in situ hybridization results for E. hepatopenaei (EHP) in the hepatopancreas. CONCLUSIONS: A laboratory cohabitation model for studying E. hepatopenaei (EHP) has been developed and used to confirm that E. hepatopenaei (EHP) can be directly transmitted horizontally among shrimp via water. The model will facilitate studies on methods to prevent the E. hepatopenaei (EHP) transmission.

Potential of RNAi applications to control viral diseases of farmed shrimp.

Viral pathogens pose a primary threat to global shrimp aquaculture. Despite the urgent industry need for them, practical anti-viral control methods are unavailable due, in part, to lack of an adaptive immune response in crustaceans that renders conventional vaccination methods ineffective. One currently studied method of high interest for protecting shrimp against viral infection relies on the post-transcriptional gene silencing mechanism called RNA interference (RNAi) that is induced by gene-specific constructs of double stranded RNA (dsRNA). Although this approach was first described for successful protection of shrimp against white spot disease (WSD) by injecting dsRNA specific to genes of white spot syndrome virus (WSSV) into shrimp in the laboratory in 2005 no practical method for use of dsRNA in shrimp farms has been developed to date. The apparent bottleneck for farm-scale applications of RNAi-mediated viral control in shrimp aquaculture is the lack of simple and cost-effective delivery methods. This review summarizes recent studies on use and delivery of dsRNA to shrimp via injection and oral routes in hatcheries and on farms and it discusses the research directions that might lead to development of practical methods for applications with farmed shrimp. Oral delivery methods tested so far include use of dsRNA-expressing bacteria as a component of dry feed pellets or use of living brine shrimp (Artemia) pre-fed with dsRNA before they are fed to shrimp. Also tested have been dsRNA enclosed in nanocontainers including chitosan, liposomes and viral-like particles (VLP) before direct injection or use as components of feed pellets for hatchery or pond-reared shrimp.

A Nested PCR Assay to Avoid False Positive Detection of the Microsporidian Enterocytozoon hepatopenaei (EHP) in Environmental Samples in Shrimp Farms.

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.

The hexameric helicase DnaB adopts a nonplanar conformation during translocation.

DNA polymerases can only synthesize nascent DNA from single-stranded DNA (ssDNA) templates. In bacteria, the unwinding of parental duplex DNA is carried out by the replicative DNA helicase (DnaB) that couples NTP hydrolysis to 5' to 3' translocation. The crystal structure of the DnaB hexamer in complex with GDP-AlF(4) and ssDNA reported here reveals that DnaB adopts a closed spiral staircase quaternary structure around an A-form ssDNA with each C-terminal domain coordinating two nucleotides of ssDNA. The structure not only provides structural insights into the translocation mechanism of superfamily IV helicases but also suggests that members of this superfamily employ a translocation mechanism that is distinct from other helicase superfamilies. We propose a hand-over-hand mechanism in which sequential hydrolysis of NTP causes a sequential 5' to 3' movement of the subunits along the helical axis of the staircase, resulting in the unwinding of two nucleotides per subunit.