Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 10¹ copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens.

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.

The sustainability of shrimp aquaculture can be achieved through the development of greenhouse and aquaponic rearing modes, which are classified as heterotrophic and autotrophic bacterial aquaculture systems. However, there have been few investigations into the discrepancies between the intestinal and water microbiota of these two rearing methods. In this study, we collected shrimp samples from greenhouse-rearing (WG) and aquaponic-rearing (YG) ponds, and water samples (WE, YE), and investigated the intestinal and water microbiota between the two rearing modes. The results, through alpha and beta diversity analyses, reveal that there was basically no significant difference between shrimp intestine WG and YG (p > 0.05) or between rearing water WE and YE (p > 0.05). At the phylum and genus levels, the common bacteria between WE and WG differed significantly from those of YE and YG. The analysis of the top six phyla shows that Proteobacteria and Patescibacteria were significantly more abundant in the WG group than those in the YG group (p < 0.05). Conversely, Actinobacteriota, Firmicutes, and Verrucomicrobiota were significantly more abundant in the YG group than those in the WG group (p < 0.05). Venn analysis between WE and WG shows that Amaricoccus, Micrococcales, Flavobacteriaceae, and Paracoccus were the dominant bacteria genera, while Acinetobacter, Demequina, and Rheinheimera were the dominant bacteria genera between YE and YG. Pathways such as the biosynthesis of secondary metabolites, microbial metabolism in different environments, and carbon metabolism were significantly more upregulated in WG than those in YG (p < 0.05). In addition, pathways such as sulfate, chloroplast, phototrophy, and the nitrogen metabolism were significantly different between the WE and YE samples. These findings suggest that the greenhouse mode, a typical heterotrophic bacterial model, contains bacterial flora consisting of Amaricoccus, Micrococcales, Flavobacteriaceae, and other bacteria, which is indicative of the biological sludge process. Conversely, the aquaponic mode, an autotrophic bacterial model, was characterized by Acinetobacter, Demequina, Rheinheimera, and other bacteria, signifying the autotrophic biological process. This research provides an extensive understanding of heterotrophic and autotrophic bacterial aquaculture systems.