Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

Abstract

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

“…), microsporidium (Ding et al . ) and spiroplasma (another common pathogen in E. sinensis ; Ding et al . ).…”

“…This holding system had its own filtration unit to maintain adequate water quality. Hemolymph of the crabs was sampled aseptically and examined initially by real-time PCR assays for the detection of WSSV (Ding et al 2016a), microsporidium (Ding et al 2016b) and spiroplasma (another common pathogen in E. sinensis; Ding et al 2014). Crabs negative for these pathogens were chosen for the experiments.…”

Lipid metabolism disorders contribute to the pathogenesis of Hepatospora eriocheir in the crab Eriocheir sinensis

“…In the crab, its hepatopancreas colour changes from yellow‐golden to nearly white, which represents an obvious disease sign and an apparent tissue‐specific feature of infection. The preferred accumulation of H. eriocheir in hepatopancreas was also confirmed by systematic analysis and quantifications (Ding et al., 2017). However, the functional consequences of such tissue tropism remain poorly studied, although tissue tropism of parasitic diseases has been associated with parasite proliferation, transmission enhancement and treatment failure (Pereira et al., 2019).…”

“…Besides, they were processed with 4% paraformaldehyde fixation (pH 7.3) for histological analysis, and 4% glutaraldehyde fixation (dissolved within the 0.3 M sodium phosphate buffer, pH 7.4) for transmission electron microscopy (TEM). Further, another six crabs that showed no signs were checked through detecting the common pathogenic agents, such as spiroplasma (Wang et al., 2011), WSSV (Bateman et al., 2012) and H. eriocheir (Ding et al., 2017). Crabs that showed negative results for such pathogenic agents were screened into the control group.…”

“…In situ hybridization was carried out following the protocol of Ding et al., 2017. Overnight hybridization with the DIG‐labelled probe was done in a humid chamber at 42°C.…”