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.

Background: Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. Results: A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. Conclusions: Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

Microsporidia are a large group of mysterious obligate intracellular eukaryotic parasites. The microsporidian spore can survive in the absence of nutrients for years under harsh conditions and germinate within seconds under the stimulation of environmental changes like pH and ions. During germination, microsporidia experience an increase in intrasporal osmotic pressure, which leads to an influx of water into the spore, followed by swelling of the polaroplasts and posterior vacuole, which eventually fires the polar filament (PF). Infectious sporoplasm was transported through the extruded polar tube (PT) and delivered into the host cell. Despite much that has been learned about the germination of microsporidia, there are still several major questions that remain unanswered, including: (i) There is still a lack of knowledge about the signaling pathways involved in spore germination. (ii) The germination of spores is not well understood in terms of its specific energetics. (iii) Limited understanding of how spores germinate and how the nucleus and membranes are rearranged during germination. (iv) Only a few proteins in the invasion organelles have been identified; many more are likely undiscovered. This review summarizes the major resolved and unresolved issues concerning the process of microsporidian spore germination.

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 10³ copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 10⁴ copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Microsporidia are a class of obligate intracellular parasitic unicellular eukaryotes that infect a variety of hosts, even including humans. Although different species of microsporidia differ in host range and specificity, they all share a similar infection organelle, the polar tube, which is also defined as the polar filament in mature spores. In response to the appropriate environmental stimulation, the spore germinates with the polar filament everted, forming a hollow polar tube, and then the infectious cargo is transported into host cells via the polar tube. Hence, the polar tube plays a key role in microsporidian infection. Here, we review the origin, structure, composition, function, and application of the microsporidian polar tube, focusing on the origin of the polar filament, the structural differences between the polar filament and polar tube, and the characteristics of polar tube proteins. Comparing the three-dimensional structure of PTP6 homologous proteins provides new insight for the screening of additional novel polar tube proteins with low sequence similarity in microsporidia. In addition, the interaction of the polar tube with the spore wall and the host are summarized to better understand the infection mechanism of microsporidia. Due to the specificity of polar tube proteins, they are also used as the target in the diagnosis and prevention of microsporidiosis. With the present findings, we propose a future study on the polar tube of microsporidia. Graphical Abstract

Microsporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism.