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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.

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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.

Insect-associated microorganisms play important roles in the health and development of insects. This study aimed to investigate the similarities and differences in bacterial community structure and composition between the larval gut of Osmia excavata, nest soil, and brood provision from the nest tube. We sequenced larvae gut and their environments’ microorganisms of O. excavata from four locations based on full-length 16S rRNA gene amplicons. The results showed 156, 280, and 366 bacterial OTUs from gut, brood provision, and nest soil, respectively, and three groups shared 131 bacterial OTUs. In the gut, the top two dominant bacteria were Sodalis praecaptivus (68.99%), Lactobacillus micheneri (17.95%). In the brood provision, the top two dominant bacteria were S. praecaptivus (26.66%), Acinetobacter nectaris (13.05%), and in the nest soil, the two most abundant bacteria were Gaiella occulta (4.33%), Vicinamibacter silvestris (3.88%). There were significant differences in diversity between the brood provision groups and the nest soil groups, respectively. Three of the four locations did not differ for gut microbial diversity. Bacteria similar to other solitary bees also existed in the gut of the larvae. Results indicated when the habitat environments were similar, the bacterial community diversity of the gut of O. excavata was similar, despite significant differences among brood provisions and soils, respectively.