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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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•Current insights into EHP life cycles and transmission methods are discussed.•Methodologies of EHP diagnosis are comprehensively reviewed.•Infection with EHP and white feces syndrome ...appear to be conditionally related.•Practical control strategies and urgent research needs are summarized.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat for shrimp culture. We hypothesized that commercial feeds processed at 75 °C or higher would present no risk for EHP ...transmission, even if they test positive for EHP by PCR. To test this hypothesis, the purified EHP spores (1 × 106 spores) were incubated at 25 °C (non-heated) or 75 °C (heated) for 45–60 s prior to determining viability by polar tube extrusion assay. Heat at 75 °C prevented extrusion when compared to spores incubated at 25 °C, confirming inactivation even though PCR detection of EHP was still positive. Further tests were carried out using oral injection of heated and non-heated spores. At day 14 post injection, SYBR Green-qPCR based on the EHP spore wall protein (SWP) sequence revealed 1063–36,449 copies of EHP per ng DNA in the hepatopancreatic tissues of 3 from 4 shrimp given non-heated spores while shrimp given spores heated at 75 °C for 45 s or 60 s showed no detectable copies. In another experiment, shrimp were fed EHP-PCR-positive, commercial feed samples (CS1 and CS2) containing the equivalent of 2.2 × 106 and 4.8 × 105 EHP copies/day for 21 days. The positive control consisted of EHP-PCR-negative feed supplemented with active EHP spores (2.0 × 105 spores/day) fed for 14 days followed by feeding with un-supplemented, EHP-PCR-negative feed from day 15 to 21. The negative control consisted of EHP-PCR-negative, commercial feed. After 21 days, all shrimp in the positive control group showed EHP infection by SYBR Green-qPCR and in situ hybridization. In contrast, shrimp fed with the negative control feed and the two PCR-positive commercial feed samples (CS1 and CS2) showed no signs of EHP infection. The results supported our hypothesis that CS1 and CS2 feeds contained inactive EHP DNA. Considering the heat exposure during manufacture (equal to or exceeding 75 °C for 60 s), such feeds should be regarded as posing no risk for EHP transmission.
•EHP spores are highly sensitive to heat inactivation at 75 °C for one minute.•After heating at 75 °C for 1 min, the EHP spore extrusion rate and infectivity were abolished.•commercial feeds processed at ≥75 °C present no risk for EHP transmission, even if they test positive for EHP by PCR.
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Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a highly prevalent and hard to eradicate parasite in major shrimp aquaculture hubs. Laboratory models to study ...the spread of EHP have revealed horizontal transmission via spores released from infected shrimp into rearing water. However, infectivity duration for the released spores is unknown. Thus, we carried out experiments using aquaria in which shrimp heavily-infected with EHP were held for 4 weeks after which the presence of spores in the water was confirmed by SybrGreen qPCR, and spore activity was determined by polar tube extrusion assay. After the shrimp were removed, the EHP-water was used directly (100%), diluted 50% or diluted 75% before adding naïve PL-12 and rearing for 16 days. Infection was determined by PCR and in situ hybridization (ISH) and compared to the results obtained using a standard EHP-cohabitation model. Infections developed at all dilutions by day 12 (100, 66 and 33% positive samples respectively). During the second experiment, the EHP-water was subjected to chemical (20 ppm calcium hypochlorite) and to resting with aeration for 5 days and 10 days before addition of naïve PL-12 to determine its infectivity. Within 16 days, the PL-12 in untreated EHP-water and in 5-days rested water developed EHP infections (50% and 10%, respectively), while the PL-12 cultured in EHP-water treated with 20 ppm calcium hypochlorite and in 10-days rested water remained uninfected. Altogether results showed that EHP contaminated water is a potential transmission risk but that chemical disinfection or resting for a suitable period in the absence of carriers can substantially reduce its infectivity.
•Spores released from EHP-infected shrimp can reach millions per liter in culture water.•Contaminated culture water remains infective for 5 days but is not infective after 10 days of resting.•In contrast, treatment with 20 ppm chlorine rapidly eliminates infectivity.
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•EHP infections could be transmitted between mussels, Mytilopsis leucophaeata and shrimp detected by the SWP-PCR method.•No indications of any EHP developmental stages in the ...digestive glands and other tissue of SWP-PCR positive mussels demonstrated by histology or ISH.•The false mussel can serve as a passive carrier to transmit infective spores to naïve shrimp in the pond.
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.
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Unlike that of vertebrates, the penaeid shrimp stomach is of ectodermic origin and is thus covered by a cuticle that is sloughed upon molting. It is composed of two chambers, here called the anterior ...and posterior stomach chambers, ASC and PSC, respectively. The PSC contains a filtration structure variously called a pyloric filter, filter press, gastric filter or gastric sieve (GS), and the last of these will be used here. The GS resembles an elongated, inverted-V, dome-like, chitinous structure with a midline ridge that is integral to the ventral base of the PSC. The dome surface is covered with a carpet-like layer of minute, comb-like setae bearing laterally branching setulae. This carpet serves as a selective filter that excludes large partially digested food particles but allows smaller particles and soluble materials to enter hepatopancreatic ducts that conduct them into the shrimp hepatopancreas (HP), where further digestion and absorption of nutrients takes place. Although the GS function is well known, its exclusion limit for particulate material has not been clearly defined. Using histological and ultra-structure analysis, we show that the GS sieve pore diameter is approximately 0.2-0.7 µm in size, indicating a size exclusion limit of substantially less than 1 µm. Using fluorescent microbeads, we show that particles of 1 µm diameter could not pass through the GS but that particles of 0.1 µm diameter did pass through to accumulate in longitudinal grooves and move on to the HP, where some were internalized by tubule epithelial cells. We found no significant difference in these sizes between the species
and
or between juveniles and adults in
This information will be of value for the design of particulate feed ingredients such as nutrients, therapeutic drugs and toxin-absorbing materials that may selectively target the stomach, intestine or HP of cultivated shrimp.
Early mortality syndrome (EMS) refers to acute mortality within approximately 35 days of shrimp culture. It is often equated with acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio ...isolates that produce Pir-like toxins. To better understand EMS, 200 Thai shrimp ponds were randomly selected prior to stocking and subsequently sampled from 19/08/2013 to 23/04/2014 to determine the pond prevalence of various pathologies, and particularly those targeting the shrimp hepatopancreas (HP). Ponds exhibiting acute mortality within 35 days were labeled EMS ponds while others were labeled non-EMS ponds. Shrimp samples from each pond (10) were subjected to histological analysis focused on hepatopancreatic (HP) lesions. Similar samples (10) were tested by PCR for the microsporidian Enterocytozoon hepatopenaei (EHP), white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio that cause AHPND. Preservation failed for 4 ponds. Results from the 196 ponds remaining were divided, into 4 groups based largely on HP histology: (1) ponds positive for AHPND by histology and/or PCR at 20.9% prevalence (41/196); (2) ponds showing HP bacterial lesions at 14.8% prevalence (29/196); (3) ponds showing collapsed HP tubule epithelia at 25.0% prevalence (49/196); and (4) ponds showing no HP pathology (normal HP) at 39.3% prevalence (77/196) ponds. The overall prevalence of EMS ponds (i.e., mortality ≤35 days) was 16.3% (32/196), but only 18/32 (56.3%) of these fell within AHPND Group 1. The other 14 EMS ponds (43.7%) were divided among the remaining three groups: Group 2 with HP bacterial lesions (3/32 = 9.4%), Group 3 with collapsed HP tubule epithelia (1/32 = 3.1%) and Group 4 with normal HP histology (a surprising 10/32 = 31.2%). Across all groups, prevalence of ponds exhibiting aggregated transformed microvilli (ATM) was high at 79.8%, as was prevalence of EHP at 60.7%. The overall prevalence of WSSV was low at 4.1% (8/196), while YHV was not found. Overall, the results indicated that equating EMS with AHPND without confirmatory analysis could lead to overestimation of its impact on shrimp aquaculture and to failure in recognizing other important causes of early mortality.
•Determination of shrimp pathogens and pathologies were carried out in the 200 pre-selected Thai shrimp ponds.•Among 196 ponds examined, 32 ponds were reported to be EMS ponds.•Only 18 in 32 EMS ponds were found to be positive for AHPND by histology and/or PCR.•Shrimp EMS is not equal to AHPND.
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The microsporidian Enterocytozoon hepatopenaei (EHP) is an intracellular spore-forming parasite, which has recently become a serious threat of cultivated penaeid shrimp in Asia. These studies were ...aimed to generate comprehensive study on EHP infection as well as establishing innovative strategies to reduce its viability and potential infectivity in shrimp farms. The EHP spore purification method by using Percoll gradient separation with ultracentrifugation step at 15 °C has been developed. The viability of the purified spores has been evaluated by its polar tube extrusion with Phloxin B staining. The active EHP spores were exposed to different temperature (−20 °C, 4 °C and 33 °C) and chemical treatments including calcium hypochlorite, formalin, potassium permanganate (KMnO4) and ethanol to identify the conditions that can be used to inhibit the extrusion of the polar tube. Complete inhibition of activity was demonstrated either by freezing the spores at −20 °C for at least 2 h or by treating them with chemicals. The chemicals that yielded 100% inhibition were 15 ppm KMnO4 for 15 min, 40 ppm of 65% active chlorine for 15 min or 10 ppm of 65% active chlorine for 24 h and 20% ethanol for 15 min. However 200 ppm formalin resulted in a maximum reduction of 95.33%. Taken together, our protocol demonstrates for the first time that living EHP spores can be isolated and purified, providing a potential platform for future testing and development of EHP's control strategies.
•Active EHP spores were successfully purified using a Percoll gradient.•A polar tube extrusion assay was developed to determine the EHP spore activity.•Spore activity can be inhibited by freezing at −20 °C for 2 h onwards.•Spore activity can be inhibited by using low doses of chlorine, KMnO4 and ethanol.
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•The shrimp microsporidian EHP alone does not cause white feces syndrome (WFS)•16S rRNA-NGS sequencing was used to screen for potential component bacterial causes.•Higher burdens of ...EHP and Propionigenium and Vibrio species occurred in WFS shrimp than non-WFS shrimp.•Specific Propionigenium occurred in significant numbers in WFS hepatopancreas.•Specific Vibrio isolates likely occurred in significant numbers in WFS hepatopancreas.
White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10-5) than in NG shrimp (12 copies/100 ng DNA). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as potential component causes of EHP-WFS in P. vannamei.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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