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Enterocytozoon hepatopenaei (EHP) is a parasite in shrimp farming. EHP mainly parasitizes the hepatopancreas of shrimp, causing slow growth, which severely restricts the economic income of shrimp farmers. To explore the pathogenic mechanism of EHP, the host subcellular construction, molecular biological characteristics, and mitochondrial condition of Litopenaeus vannamei were identified using transmission electron microscopy (TEM), real-time qPCR, an enzyme assay, and flow cytometry. The results showed that EHP spores, approximately 1 μm in size, were located on the cytoplasm of the hepatopancreas. The number of mitochondria increased significantly, and mitochondria morphology showed a condensed state in the high-concentration EHP-infected shrimp by TEM observation. In addition, there were some changes in mitochondrial potential, but apoptosis was not significantly different in the infected shrimp. The qPCR results showed that the gene expression levels of hexokinase and pyruvate kinase related to energy metabolism were both upregulated in the diseased L. vannamei. Enzymatic activity showed hexokinase and lactate dehydrogenase were significantly increased in the shrimp infected with EHP, indicating EHP infection can increase the glycolysis process and decrease the oxidative phosphorylation process of L. vannamei. Previous transcriptomic data analysis results also support this conclusion.

The objective of this work was to investigate, for the first time, the antioxidant effect of a mixture of natural antimicrobials in an Enterocytozoon hepatopenaei (EHP) shrimp-gut model of infection and the biological mechanisms involved in their way of action. The study approach included investigations, firstly, in vitro, on shrimp-gut primary (SGP) epithelial cells and in vivo by using EHP-challenged shrimp. Our results show that exposure of EHP spores to 0.1%, 0.5%, 1%, and 2% AuraAqua (Aq) significantly reduced spore activity at all concentrations but was more pronounced after exposure to 0.5% Aq. The Aq was able to reduce EHP infection of SGP cells regardless of cells being pretreated or cocultured during infection with Aq. The survivability of SGP cells infected with EHP spores was significantly increased in both scenarios; however, a more noticeable effect was observed when the infected cells were pre-exposed to Aq. Our data show that infection of SGP cells by EHP activates the host NADPH oxidases and the release of H2O2 produced. When Aq was used during infection, a significant reduction in H2O2 was observed concomitant with a significant increase in the levels of CAT and SOD enzymes. Moreover, in the presence of 0.5% Aq, the overproduction of CAT and SOD was correlated with the inactivation of the NF-κB pathway, which, otherwise, as we show, is activated upon EHP infection of SGP cells. In a challenge test, Aq was able to significantly reduce mortality in EHP-infected shrimp and increase the levels of CAT and SOD in the gut tissue. Conclusively, these results show, for the first time, that a mixture of natural antimicrobials (Aq) can reduce the EHP-spore activity, improve the survival rates of primary gut-shrimp epithelial cells and reduce the oxidative damage caused by EHP infection. Moreover, we show that Aq was able to stop the H2O2 activation of the NF-κB pathway of Crustins, Penaeidins, and the lysozyme, and the CAT and SOD activity both in vitro and in a shrimp challenge test.

Enterocytozoon hepatopenaei (EHP), a microsporidian responsible for hepatopancreatic microsporidiosis, is a major pathogen in commercial shrimp production. Among the affected species, Palaemon carinicauda (formerly Exopalaemon carinicauda) is commercially important in China and represents a potential research model for studying crustaceans. However, little information is available on its response to EHP infection. Hence, this study analyzed the transcriptome and metabolome of P. carinicauda’s hepatopancreas using high-throughput sequencing and liquid chromatograph-mass spectrometry (LC-MS) to determine its response during the early stage of infection. The transcriptomic analyses identified 730 differentially expressed genes, of which those associated with EHP infection were enriched in metabolic pathways as well as detoxification and antioxidant pathways. In addition, 144 differential metabolites were identified using a combination of positive and negative ion modes in LC-MS. The Kyoto encyclopedia of genes and genomes pathway analyses further indicated that the degradation of aromatic compounds, the AMP-activated protein kinase signaling pathway and C5-branched dibasic acid metabolism were significantly enriched after EHP infection. These results could provide useful insights into the effects of EHP on shrimps during the early stages of infection and help to understand the mechanisms underlying the stunted growth of shrimps after infection.

In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.

White feces syndrome (WFS), a gastrointestinal disorder of cultivated penaeid shrimp, causes severe economic and production losses worldwide. Shrimp with WFS usually show a reduced feed consumption and growth rate, hepatopancreatic discoloration, and loose shells. Recently, WFS has been said to be related to an infection with Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, and other opportunistic bacteria, such as the Vibrio species. Vibrio spp. may play an important role in WFS occurrence; nevertheless, Vibrio sp., as a single pathogen, cannot reproduce this syndrome under laboratory conditions. To date, no novel treatment has been found for WFS; therefore, preventive measures, such as strict biosecurity systems to eliminate or inactivate EHP spores and limit the total Vibrio number, have been suggested. This review summarizes the information regarding the association between WFS and water parameters, gut microbiomes, EHP and Vibrio, and disease control strategies.

Infectious diseases are considered the greatest threat to the modern high-density shrimp aquaculture industry. Specificity, rapidity, and sensitivity of molecular diagnostic methods for the detection of asymptomatic infected shrimp allows preventive measures to be taken before disease outbreaks. Routine molecular detection of pathogens in infected shrimp can be made easier with the use of a direct polymerase chain reaction (PCR). In this study, four direct PCR reagent brands were tested, and results showed that the detection signal of direct PCR in hepatopancreatic tissue was more severely affected. In addition, portable capillary electrophoresis was applied to improve sensitivity and specificity, resulting in a pathogen detection limit of 25 copies/PCR-reaction. Juvenile shrimp from five different aquaculture ponds were tested for white spot syndrome virus infection, and the results were consistent with the Organization for Animal Health’s certified standard method. Furthermore, this methodology could be used to examine single post larvae shrimp. The overall detection time was reduced by more than 58.2%. Therefore, the combination of direct PCR and capillary electrophoresis for on-site examination is valuable and has potential as a suitable tool for diagnostic, epidemiological, and pathological studies of shrimp aquaculture.

Enterocytozoon hepatopenaei, a spore-forming and obligate intracellular microsporidium, mainly infects shrimp and results in growth retardation and body length variation, causing huge economic losses to the Asian shrimp aquaculture industry. However, the lack of a full understanding of the surface proteins of spores associated with host infection has hindered the development of technologies for the detection of EHP. In this study, the surface proteins of EHP spores were extracted using the improved SDS method, and 130 proteins were identified via LC-MS/MS analysis. Bioinformatic analysis revealed that these proteins were enriched in biological processes (67), cellular components (62), and molecular functions (71) based on GO terms. KEGG pathway analysis showed that 20 pathways, including the proteasome (eight proteins) and the fatty acid metabolism (15 proteins), were enriched. Among 15 high-abundance surface proteins (HASPs), EhSWP3 was identified as a novel spore wall protein (SWP), and was localized on the endospore of the EHP spores with an indirect immunofluorescence and immunoelectron microscopy assay. Polyclonal antibodies against EhSWP3 showed strong species specificity and high sensitivity to the hepatopancreas of EHP-infected shrimp. As a specific high-abundance protein, EhSWP3 is therefore a promising target for the development of immunoassay tools for EHP detection, and may play a crucial role in the invasion of EHP into the host.

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

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 (10 6 -10 8 copies mg -1 ), while HP samples from Latin America, Artemia , and EHP-contaminated water showed lower amounts (10 1 -10 3 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.