Enterocytozoon Hepatopenaei Infection Research Articles

A comparative analysis of different challenge routes against Enterocytozoon hepatopenaei infection in Penaeus vannamei

A comparative analysis of different challenge routes against Enterocytozoon hepatopenaei infection in Penaeus vannamei

Immune signaling of Litopenaeus vannamei c-type lysozyme and its role during microsporidian Enterocytozoon hepatopenaei (EHP) infection.

The microsporidian Enterocytozoon hepatopenaei (EHP) is a fungi-related, spore-forming parasite. EHP infection causes growth retardation and size variation in shrimp, resulting in severe economic losses. Studies on shrimp immune response have shown that several antimicrobial peptides (AMPs) were upregulated upon EHP infection. Among those highly upregulated AMPs is c-type lysozyme (LvLyz-c). However, the immune signaling pathway responsible for LvLyz-c production in shrimp as well as its function against the EHP infection are still poorly understood. Here, we characterized major shrimp immune signaling pathways and found that Toll and JAK/STAT pathways were up-regulated upon EHP infection. Knocking down of a Domeless (DOME) receptor in the JAK/STAT pathways resulted in a significant reduction of the LvLyz-c and the elevation of EHP copy number. We further elucidated the function of LvLyz-c by heterologously expressing a recombinant LvLyz-c (rLvLyz-c) in an Escherichia coli. rLvLyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus. Interestingly, we found an antifungal activity of rLvLyz-c against Candida albican, which led us to further investigate the effects of rLvLyz-c on EHP spores. Incubation of the EHP spores with rLvLyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that rLvLyz-c possibly digest a chitin coat on the EHP spores. Transmission electron microscopy analysis revealed that an endospore layer, which is composed mainly of chitin, was digested by rLvLyz-c. Lastly, we observed that EHP spores that were treated with rLvLyz-c showed a significant reduction of the spore germination rate. We hypothesize that thinning of the endospore of EHP would result in altered permeability, hence affecting spore germination. This work provides insights into shrimp immune signaling pathways responsible for LvLyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.

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.

Study of microsporidians in white faecal syndrome affected farmed shrimp (Penaeus vannamei) Boone, 1931 of West Bengal, India

Enterocytozoon hepatopenaei (EHP), an intracellular microsporidian parasite, has been reported in cultivated Penaeus vannamei infected with white faecal syndrome (WFS) in south-east Asian shrimp producing countries. The appearance of white faecal strings floating on the surface of the ponds indicates a severe microsporidian infection. In the present study suspected shrimps and floating faecal strings were collected from culture ponds of study area located in Purba Medinipur district of West Bengal. This study aims to unveil the presence of microsporidians in hepatopancreas and associated histopathological changes in it using light and scanning electron microscopy, and microsporidian identification by nested PCR. Microscopic study revealed the presence of oval-shaped microsporidian spores in the hepatopancreas and nested PCR study confirmed that these spores belong to E. hepatopenaei. The spores isolated from the floating faecal strings exhibited similar morphology with those found in hepatopancreas. Histopathology of the hepatopancreas of WFS-affected shrimp exhibited alterations in hepatopancreatic tissues along with haemocytic infiltration into the inter-tubular spaces and evidence of necrosis, vacuolation and degeneration. The findings from the nested PCR analysis indicated a total of 18 positive results (69.23%) out of 26 samples studied. WFS infection can be considered a gastrointestinal disorder caused by severe EHP infection, which is significant in the grow-out ponds in the regions under study.

Enterocytozoon hepatopenaei Infection in Shrimp: Diagnosis, Interventions, and Food Safety Guidelines.

The emergence of disease in shrimp has governed much concern in food safety and security among consumers with the recent reports on hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP). The microsporidians present in shrimp remain a silent pathogen that prevents optimal shrimp growth. However, the biggest threat is in its food safety concerns, which is the primary focus in ensuring food biosecurity and biosafety. Hence, the objective of this review is to summarise the current knowledge of EHP and its infection in shrimp with food safety concerns. This paper provides an analysis of the diagnostic methods for detecting EHP infections in shrimp aquaculture. Interventions with current molecular biology and biotechnology would be the second approach to addressing EHP diseases. Finally, a systematic guideline for shrimp food safety using diagnostic and intervention is proposed. Thus, this review was aimed to shed light on effective methods for the diagnosis and prevention of EHP infection in shrimp. We also include information on molecular and genomics tools as well as innate immune biomolecules as future targets in the intervention strategies on the microsporidsosis life cycle in shrimp and its environment. Overall, this will result in reduced disease outbreaks in shrimp aquaculture, ensuring the shrimp food safety in the future.

The shrimp C-type lectins modulate intestinal microbiota homeostasis in microsporidia infection

Enterocytozoon hepatopenaei (EHP) is abundant in the shrimp hepatopancreas and intestines and can be transmitted through the digestive system. But the relationship between EHP and intestines or gut microbiota is still unclear. In this study, the microbiome and proteome of shrimp Penaeus vannamei intestine were used to analyze the changes of the gut microbiome and the immune protein after EHP infection. Our results showed that EHP infection brought about a marked increase in the proportion of pathogenic bacteria, such as Shigella, Aeromonas, Faecalibacterium, and Streptococcus, suggesting that EHP infection would lead to the increasing of pathogenic bacteria. Proteome results indicated showed that down-regulated proteins were mainly concentrated in immune-related domains such as C-type lectins (CTLs), hemoglobin, and glutathione transferase, suggesting that EHP might lead to a weakening of the innate immunity of shrimp. In shrimp, CTLs can involve in innate immunity by regulating the transcription of various antimicrobial peptides, and directly participate in the inhibition of pathogens through binding and agglutination. On this basis, after successfully cloned and recombination expressed, three down-regulated CTLs (perlucin, mannose receptor 1 and c-type lectin domain family 17 with typical CTL domains) were proved to have binding and agglutinin ability against four pathogenic bacteria (Enterococcus faecalis, Shigella castellani, Salmonella enteritidis, Aeromonas hydrophila). These results suggested that EHP infection led to decrease the expression of three CTLs and increase secondary infection caused by pathogenic bacteria. And CTLs played important roles in maintaining the balance of shrimp gut microbiome in microsporidia infection. In a word, these results are helpful for us to better understand the interaction between EHP and intestinal microbiota of P. vannamei and will gain new insights into prevention and control of hepatopancreatic microsporidiosis.

Enterocytozoon hepatopenaei (EHP) Infection Alters the Metabolic Processes and Induces Oxidative Stress in Penaeus vannamei

Enterocytozoon hepatopenaei (EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the differences in histology, metabolism, oxidative stress and growth between healthy and EHP-infected Penaeus vannamei were analyzed using an EHP challenge experiment. Histology showed that EHP caused lesions in the hepatic tubules of P. vannamei, such as hepatic tubular atrophy and epithelial cell shedding, with mature spores. Meanwhile, white feces may appear when the infection is severe. Furthermore, the content of total protein, glycogen, ATP and glucose in the EHP challenge group was significantly reduced. The qPCR results showed that EHP infection changed the expression of key genes in glucose metabolism, among which hexokinase (HK), phosphofructokinase (PFK), pyruvatekinase (PK), citrate synthase (CS) and isocitric dehydrogenase (IDH) were significantly down-regulated, while phosphoenolpyruvate carboxykinase (PEPCK), fructose bisphosphatase (FBP) and glucose-6-phosphatase (G6P) were significantly up-regulated. Obviously, the expression of growth-related genes was disordered. Simultaneously, the antioxidant genes manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferases (GST) and nuclear factor E2-related factor2 (Nrf2) were up-regulated to varying degrees in the EHP challenge group, and EHP infection induced significant increases in the oxidative damage products lipid peroxide (LPO) and malondialdehyde (MDA). Ultimately, the shrimp weight of the challenge group was 6.85 ± 0.86 g, which was significantly lower than that of the control group (8.95 ± 0.75 g). Taken together, we speculate that EHP changes the substance metabolism and growth process by causing oxidative damage to the hepatopancreas, which may lead to the growth retardation of P. vannamei.

Dynamic Interplay of Metabolic and Transcriptional Responses in Shrimp during Early and Late Infection Stages of Enterocytozoon hepatopenaei (EHP)

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects Litopenaeus vannamei, causing severe hepatopancreatic microsporidiosis (HPM) and resulting in significant economic losses. This study utilizes a combined analysis of transcriptomics and metabolomics to unveil the dynamic molecular interactions between EHP and its host, the Pacific white shrimp, during the early and late stages of infection. The results indicate distinct immunological, detoxification, and antioxidant responses in the early and late infection phases. During early EHP infection in shrimp, immune activation coincides with suppression of genes like Ftz-F1 and SEPs, potentially aiding parasitic evasion. In contrast, late infection shows a refined immune response with phagocytosis-enhancing down-regulation of Ftz-F1 and a resurgence in SEP expression. This phase is characterized by an up-regulated detoxification and antioxidant response, likely a defense against the accumulated effects of EHP, facilitating a stable host–pathogen relationship. In the later stages of infection, most immune responses return to baseline levels, while some immune genes remain active. The glutathione antioxidant system is suppressed early on but becomes activated in the later stages. This phenomenon could facilitate the early invasion of EHP while assisting the host in mitigating oxidative damage caused by late-stage infection. Notably, there are distinctive events in polyamine metabolism. Sustained up-regulation of spermidine synthase and concurrent reduction in spermine levels suggest a potential role of polyamines in EHP development. Throughout the infection process, significant differences in genes such as ATP synthase and hexokinase highlight the continuous influence on energy metabolism pathways. Additionally, growth-related pathways involving amino acids such as tryptophan, histidine, and taurine are disrupted early on, potentially contributing to the growth inhibition observed during the initial stages of infection. In summary, these findings elucidate the dynamic interplay between the host, Litopenaeus vannamei, and the parasite, EHP, during infection. Specific phase differences in immune responses, energy metabolism, and antioxidant processes underscore the intricate relationship between the host and the parasite. The disruption of polyamine metabolism offers a novel perspective in understanding the proliferation mechanisms of EHP. These discoveries significantly advance our comprehension of the pathogenic mechanisms of EHP and its interactions with the host.

Identification of Enterocytozoon hepatopenaei in Mud Crabs (Scylla serrata) Co-infected with White Spot Syndrome Virus in Taiwan

Background: The high mortality rate and stunted growth of marine shrimp in the farm have raised concerns about the outbreak of co-infection involving Enterocytozoon hepatopenaei (EHP) and white spot syndrome virus (WSSV), which could lead to significant economic loss. In a mixed-culture farm consisting of Scylla serrata and Peneaus vannamei located in Yunlin Country, Taiwan, were reported mortality cases on culture animals. Methods: Based on clinical signs, histology and molecular analyses, the disease was diagnosed in this study. In the histopathology examination, infected organs, such as the carapace and gills, were stained with hematoxylin and eosin (H and E), then observed under microscope. For the molecular analysis, SalI DNA fragment (WSSV) and spore wall protein (SWP) gene (EHP) were targeted to detected the pathogen in animal host. Result: The cumulative mortality data for this co-infection indicates that S. serrata (1.5% mortality) exhibits higher susceptibility compared to P. vannamei ( greater than 99% mortality). Phylogenetic tree analysis reveals that the spore wall protein (SWP) gene of EHP and the SalI DNA fragment of WSSV strain extracted from S. serrata are genetically similar to those extracted from Peneaus spp., indicating the potential for cross-species infection. Histopathological examination of lesion tissue observed the presence of WSSV basophilic intranuclear inclusion bodies in the carapace tissue and gill fragments of S. serrata. The source of the disease outbreak is highly suspected to be the feed used, which consisted of raw shrimp and fish. This study provides the first documented case of natural EHP infection with co-infection of WSSV in S. serrata.

A comparative transcriptome analysis of how shrimp endure and adapt to long-term symbiosis with Enterocytozoon hepatopenaei infection

Enterocytozoon hepatopenaei (EHP) is a prevalent microsporidian pathogen responsible for hepatopancreatic microsporidiosis (HPM) in Litopenaeus vannamei. This infection not only leads to slowed growth in shrimp abut aslo inflicts substantial economic losses in the global aquaculture industry. However, the molecular mechanisms by which EHP influences the host during various infection stages remain unclear. This study employed comparative transcriptomics to examine the effects of EHP infection on Litopenaeus vannamei between early and late stage of infection groups. Utilizing transcriptomic approaches, we identified differentially expressed genes (DEGs) with notable biological significance through the COG, GO, KEGG, GSEA, and Mufzz time-series methodologies. The results reveal that EHP infection considerably influences host gene expression, with marked differences between early and late infection across distinct timeframes. Key processes such as detoxification, cell apoptosis, and lipid metabolism are pivotal during host-parasite interactions. Hexokinase and phosphatidic acid phosphatase emerge as key factors enabling invasion and sustained effects. Cytochrome P450 and glucose-6-phosphate dehydrogenase could facilitate infection progression. EHP significantly impacts growth, especially through ecdysteroids and 17β-estradiol dehydrogenase. By delineating stage-specific effects, we gain insights into interaction between EHP and Litopenaeus vannamei, showing how intracellular pathogens reprogram host defenses into mechanisms enabling long-term persistence. This study provides a deeper understanding of host-pathogen dynamics, emphasizing the interplay between detoxification, metabolism, immunity, apoptosis and growth regulation over the course of long-term symbiosis.

Metagenomic Studies Reveal the Evidence of Akkermansia muciniphila and Other Probiotic Bacteria in the Gut of Healthy and Enterocytozoon hepatopenaei (EHP)-Infected Farmed Penaeus vannamei.

Penaeus vannamei (whiteleg shrimp) is the most widely cultured shrimp globally. Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, infects P. vannamei and causes severe growth retardation, subsequent production, and economic losses in the shrimp culture. The influence of EHP infection in the shrimp gut microbiota is poorly studied, and this would be an interesting area to investigate since the gut microbiome of shrimp influences a number of key host processes such as digestion and immunity. In this study, a metagenomic approach was followed to compare the overall species richness of the gut microbiota of EHP-infected and healthy P. vannamei. Bacterial genomic DNA from the healthy and EHP-infected gut sample were profiled for the bacterial 16S rRNA gene, targeting the V3-V4 conserved region. Operational taxonomic units (OTUs), an approximation of definitive taxonomic identity, were identified based on the sequence similarity within the sample reads and clustered together using a cut-off of 97% identity using UCLUST. The OTUs were then used for the computation of alpha diversity and beta diversity for each sample. EHP-infected gut sample showed lower bacterial abundance throughout the family, class, order, genus, and species levels when compared to healthy gut sample. This study shows that the shrimp gut microbiota is sensitive and exhibits a high level of plasticity during a microsporidian infection like EHP. Furthermore, Akkermansia muciniphila, a novel probiotic bacterium, has been reported in the shrimp gut for the first time.

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 spore wall protein 1 of (rEhSWP1) 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.

Characterization of a crustin-like peptide involved in shrimp immune response to bacteria and Enterocytozoon hepatopenaei (EHP) infection in Palaemon carinicauda.

Crustins represent one type of antimicrobial peptides (AMPs) that are key components of the innate immune process of crustaceans. This study successfully identified a novel crustin-like peptide, EcCrustin2, in ridgetail white prawn, Palaemon carinicauda (formerly Exopalaemon carinicauda). EcCrustin2 was found to be 1082 bp in length, with a 378 bp open reading frame (ORF) encoding 125 amino acids. The deduced amino acid sequence of EcCrustin2 exhibited characteristics of crustins in crustacean, including a Cys-rich region at the N-terminus as well as a whey acidic protein domain at the C-terminus. Phylogenetic analysis revealed that the EcCrustin2 was first clustered with Type I crustins, then with other crustins. Expression of EcCrustin2 was mainly detected in immune tissues, including hemocytes, gill and stomach. The expression level of EcCrustin2 was also significantly up-regulated after being exposed to lipopolysaccharide (LPS), lipoteichoic acid (LTA), Vibrio parahaemolyticus and Staphylococcus aureus. EHP infection could also induce EcCrustin2 expression in P. carinicauda. Knockdown of EcCrustin2 with siRNA increased the mortality of V. parahaemolyticus challenged shrimp. Finally, the recombinant EcCrustin2 protein was obtained and demonstrated a wide spectrum of antibacterial activity in vitro. These results indicated that EcCrustin2 takes part in the immune response against bacteria and EHP infection.

In vivo and in silico investigations on the efficacy of albendazole against Enterocytozoon hepatopenaei (EHP) infecting Penaeus vannamei

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intercellular spore former that causes hepatopancreatic microsporidiosis (HPM), a commercially important disease in Penaeus vannamei (white leg shrimp) farming. In this study, albendazole an inhibitor of β-tubulin polymerization in microsporidians was evaluated for its efficacy in reducing EHP infection in P. vannamei. EHP-infected shrimps were exposed to 1, 5, 10, 25, 50 and 75 mg L−1 of albendazole and evaluated for the reduction in EHP DNA copy numbers at 3, 6, 9, 12, 15, 18, 21, 24 days post-exposure (dpe) by absolute quantitative real-time PCR (RT-qPCR) assay. The expressions of antioxidant enzyme genes viz., cytosolic manganese superoxide dismutase (cMnSOD), catalase (CAT) and glutathione peroxidase (GPx); haemolymph aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assessed at 24 dpe. A maximum of about 98% reduction in EHP DNA copies was observed in the shrimps treated with 75 mg L−1 per day for 24 days. A significant upregulation in relative expressions of GPx genes and a downregulation cMnSOD and CAT genes were observed in shrimps exposed to 25, 50 and 75 mg L‐1 at 24 dpe. A significant reduction in haemolymph AST and ALT levels was observed upon treatment with 25, 50 and 75 mg L−1 at 24 dpe and there are no significant changes in ALP. A virtual molecular docking between albendazole and EHP encoded β-tubulin revealed a best binding interaction with the presence of 11 hydrogens bonds, multiple non-covalent bonds and a very much lower solvation-free energy gain of −15.6 kcal mol−1 in the active sites of protein interactions. Thus, this study identified albendazole as a potential drug to control EHP infection in shrimp, which might help to combat the production losses caused by EHP.

Host and transmission route of Enterocytozoon hepatopenaei (EHP) between dragonfly and shrimp

Enterocytozoon hepatopenaei (EHP) is a shrimp pathogen that causes huge economic losses. In the present study, the hosts of EHP were investigated using polymerase chain reaction (PCR) in an aquaculture farm located in Maoming, China. EHP was detected in Litopenaeus vannamei, Penaeus monodon, Varuna litterata, Mytilopsis leucophaeata, and three dragonfly species (Anax parthenope, Pantala flavescens, and Ischnura senegalensis). In the histopathological examination using hematoxylin–eosin (HE) staining, EHP spores were found in nymphs and adult dragonflies naturally infected with EHP that were collected from the shrimp farm. Fluorescence in situ hybridization (FISH) results showed a positive signal for EHP infection in the fat body of dragonfly nymphs. Immature and mature microsporidian spores and late sporogonial plasmodium were observed in the cytoplasm of dragonfly nymphs using transmission electron microscopy (TEM). The transmission of EHP from shrimp to dragonfly nymphs was confirmed via cohabitation challenge experiments in which EHP-free dragonfly nymphs were cohabited with EHP-infected shrimp, and the transmission of EHP from dragonfly nymphs to shrimp was demonstrated via the cohabitation of EHP-infected dragonfly nymphs with EHP-free shrimp and oral administration challenge experiments. This study confirms that dragonflies can act as natural EHP hosts, and a novel EHP horizontal transmission route exists between dragonflies and shrimp.

Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.

Analysis of differentially expressed proteins after EHP-infection and characterization of caspase 3 protein in the whiteleg shrimp (Litopenaeus vannamei)

Whiteleg shrimp (Litopenaeus vannamei) is the most important species of shrimp farmed worldwide in terms of its economic value. Enterocytozoon hepatopenaei (EHP) infects the hepatopancreas, resulting in the hepatopancreatic microsporidiosis (HPM) of the host, which causes slow growth of the shrimp and poses a threat to the farming industry. In this study, differentially expressed proteins (DEPs) between EHP-infected and uninfected shrimp were investigated through proteomics sequencing. A total of 9908 peptides and 2092 proteins were identified. A total of 69 DEPs were identified in the hepatopancreas (HP), of which, 28 were upregulated and 41 were downregulated. Our results showed that the differences among the level of multiple proteins involved in the apoptosis were significant after the EHP infection, which indicated that the apoptosis pathway was activated in whiteleg shrimp. In addition, expression leve of caspase 3 gene were identified related to the EHP infection. Furthermore, predictions of spatial structure, analysis of phylogeny and chromosome-level linearity of the caspase 3 protein were performed as well. In conclusion, a relatively complete proteomic data set of hepatopancreas tissues in whiteleg shrimp were established in this study. Findings about genes involved in the apoptosis here will provide a further understanding of the molecular mechanism of EHP infection in the internal immunity of whiteleg shrimp.

Identification techniques to prevent the current emerging disease hepatopancreatic microsporidiosis in white shrimp Penaeus vannamei: an overview

Aquaculture combines techniques for breeding and harvesting aquatic organisms used in shrimp production. It is important as a source of income and for generating foreign exchange in the regions where it is practiced. However, the timely detection of diseases continues to be a great challenge for aquaculture and fisheries production. In recent years, Enterocytozoon hepatopenaei (EHP) has emerged as a major pathogen of the Pacific white shrimp Penaeus vannamei in many Asian countries (Vietnam, China, Indonesia, Malaysia, Thailand, India, and Korea). In Latin America, only in Venezuela, and to date, there is no report of its presence in Mexico. It is transmitted directly from shrimp to shrimp by oral or fecal means, cannibalism, or exposure to contaminated water. Hepatopancreatic microsporidiosis (HPM) is mainly associated with stunted growth and severe infections that can cause a poor production cycle, mortality, and problems in larva-producing laboratories. This review aims to overview the main microsporidian parasites and diseases found in white shrimp, including the clinical signs, control and prevention measures for EHP infection, and the detection of HPM using different techniques. In order to offer timely detection tools, different techniques are available for the detection and study of microsporidia. Such as optical microscopy, transmission electron microscopy, and histology; however, for diagnostic purposes, molecular methods are preferred due to their sensitivity, specificity, and short-time analysis. Our review suggests that constant monitoring in shrimp hatcheries and farms is essential to avoid the entry or transference of infected organisms, affecting shrimp production and the ideal development of healthy shrimp.

Comparative transcriptome analysis of non-germinated and germinated spores of Enterocytozoon hepatopenaei (EHP) in vitro

Enterocytozoon hepatopenaei (EHP), an obligate intracellular parasite classified as microsporidia, is an emerging pathogen with a significant impact on the global shrimp aquaculture industry. The understanding of how microsporidia germinate has been a key factor in exploring its infection process. However, the germination process of EHP was rarely reported. To gain insight into the germination process, we conducted a high-throughput sequencing analysis of purified EHP spores that had undergone in vitro germination treatment. This analysis revealed 137 differentially expressed genes, with 84 up-regulated and 53 down-regulated genes. While the functions of some of the genes remain unknown, this study provides important data on the transcriptomic changes before and after EHP germination, which can aid in further studies on the EHP infection mechanism.

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP)

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.