Highlights
- The transcriptomic response of shrimp to EHP was investigated through infection experiments.
- Differentially expressed genes (DEGs) related to immunity and metabolism were analyzed.
- EHP infection can activate the shrimp immune system.
- EHP enhances lipid metabolism but inhibits carbohydrate and amino acid metabolism.
Abstract
- Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite that primarily infects the hepatopancreas of shrimp, causing severe growth retardation. In this study, Penaeus vannamei shrimp were experimentally infected with EHP, and differentially expressed genes and biological pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed using transcriptome sequencing.
- The results showed a total of 240 significantly differentially expressed genes, including 99 upregulated genes and 141 downregulated genes. Immunity-related genes such as Astakine, lysozyme, NACHT, LRR and PYD domain-containing protein 3 (NLRP3), and macrophage mannose receptor 1 (MMR) were all upregulated. Concurrently, the expression levels of genes related to lipid metabolism such as lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA) and adiponectin receptor protein (AdipoR) also increased.
- Conversely, several genes related to carbohydrate and protein metabolism were downregulated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1).
- These results indicate that EHP infection in shrimp can strongly activate the immune system, but simultaneously disrupt energy metabolism and metabolic processes. The study identified a large number of genes and biological pathways associated with EHP infection, providing important data for elucidating the molecular mechanisms of growth retardation in shrimp.
Introduction
- As an important source of high-quality protein, shrimp is the most commercially significant aquatic species in international seafood products. Penaeus vannamei is the most widely farmed and economically efficient species globally, accounting for approximately 6% of total aquaculture production. However, microorganisms in the aquatic environment have caused enormous economic losses to the shrimp farming industry, typically including White Spot Syndrome Virus (WSSV), Enterocytozoon hepatopenaei (EHP), Vibrio parahaemolyticus, and many other agents. Therefore, studying disease mechanisms through molecular biotechnology is crucial for the sustainable development of the shrimp farming industry.
- Microsporidia are a group of parasites widely distributed in nature, with a diverse host range, from protozoa to mammals. Previously, there was much debate about whether microsporidia belonged to protozoa or fungi, until recently they were reclassified as fungi rather than protozoa. EHP is a branch of microsporidia, first discovered in 2009 in farmed black tiger shrimp in Thailand and subsequently spread rapidly. There have now been numerous reports of EHP in Southeast Asia, China, and other regions. EHP-infected shrimp exhibit slow growth rates, with significantly smaller body length and weight compared to shrimp cultured at the same time, but without causing mass mortality. This leads to significant feed waste and considerable economic losses for the shrimp farming industry.
- Histopathological studies show that EHP primarily parasitizes the epithelial cells of the shrimp hepatopancreatic tubules – this is the main site of EHP residence – while only small amounts are found in the stomach and intestine. Under an electron microscope, EHP spores are approximately 0.7 × 1.1 μm in size, consisting of a single nucleus, 5–6 polar filament coils, a posterior vacuole, and a thick, dense cell wall. Furthermore, horizontal transmission of EHP has been documented through co-culturing healthy shrimp with infected shrimp, as well as feeding healthy shrimp with EHP-infected shrimp. Due to the small size and long incubation period of EHP, current detection methods primarily rely on PCR and LAMP tests combined with fluorescent dyes (SYTO-16). In addition, in situ hybridization and histological sections are also commonly used to detect EHP infection in shrimp. Recently, a new EHP diagnostic method has been proposed based on the analysis of EHP polar tubule characteristics.
- Importantly, when pathogenic microorganisms such as bacteria and viruses invade, the body's immune system is activated to perform its host defense function. Microsporidia, as intracellular parasites, can reproduce and infect hosts through their characteristic polar tubules. Shrimp are invertebrates, primarily relying on their innate immune system to respond to the invasion of pathogenic microorganisms. The shrimp innate immune system mainly includes the microbial recognition system and the prophenoloxidase (proPO) activation system. EHP invasion can trigger a series of immune responses, thereby initiating the host's defense system.
- The hepatopancreas, acting as the shrimp's "metabolic factory," plays an irreplaceable role in immunity and metabolism. When shrimp are infected with EHP, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) significantly increase, while immune factors such as lysozyme and C-type lectin are activated to combat the pathogen. Furthermore, during its evolutionary adaptation to the host, EHP has gradually lost mitochondria as well as enzymes and genes related to glycolysis, oxidative phosphorylation, and carbon metabolism, and must rely on hijacking host ATP to sustain its own energy requirements.
- In recent years, next-generation sequencing platforms have become increasingly sophisticated and demonstrated clear technical advantages. Analyzing gene transcription levels has made fundamental molecular biology research more accessible and reliable. However, there are still relatively few studies on the transcriptomic analysis of P. vannamei under experimental EHP infection conditions, and the host's immune and metabolic response mechanisms to EHP are not yet fully understood. Therefore, utilizing next-generation transcriptome sequencing technology to construct the P. vannamei transcriptome under EHP infection conditions will facilitate a deeper analysis of the molecular mechanisms of physiological responses in crustaceans when infected with pathogens.
- In this study, we artificially infected healthy P. vannamei shrimp with EHP and confirmed the EHP infection status using PCR on DNA extracted from the shrimp hepatopancreas. Subsequently, the hepatopancreas of EHP-infected and healthy shrimp were separately sequenced to identify differentially expressed genes related to physiological responses, and some key gene expression patterns were also validated. The objective of this study was to clarify the changes in the immune and metabolic processes of P. vannamei during EHP infection, thereby contributing to elucidating the molecular mechanisms of growth retardation and providing an experimental basis for further research in related fields.
Experimental Animals
P. vannamei shrimp used for the experiment were collected from an aquaculture pond in Dongying City, Shandong Province, with a body length of 3 ± 1 cm and a weight of 2.8 ± 0.1 g. Before the experiment, the shrimp were tested by PCR for WSSV, Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), and EHP to ensure they were pathogen-free and suitable for the experiment. This was to ensure the normal growth and development of this batch of P. vannamei.
Detection of EHP Infection in Shrimp
Through comparison between the experimental group and the control group, it was easily observed that the body length and weight of shrimp in the experimental group were significantly smaller than those in the control group. No other distinct differences were recorded, and no mortality occurred. DNA extracted from the hepatopancreas of shrimp in both groups was amplified using conventional PCR.
Discussion
Since EHP was first discovered, the global shrimp farming industry has suffered enormous economic losses. Because EHP is difficult to detect in early infection stages and easily transmitted horizontally or vertically, it can ultimately lead to widespread infection throughout the stock. Furthermore, EHP disrupts energy metabolism and hinders the normal growth and development of shrimp, resulting in significantly smaller body length and weight in infected shrimp compared to healthy ones. EHP has attracted significant research interest.
Author Contributions Statement
- Zheng Cao: Performed transcriptome data analysis, conducted experiments, and wrote the manuscript.
- Yang Gao: Assisted in EHP infection experiments, detection experiments, and transcriptome data analysis.
- Jiahui Xu: Assisted in EHP infection and detection experiments.
- Ning Yang: Assisted in conducting the experiments.
Declaration of Competing Interest
The authors declare that there are no financial conflicts of interest or personal relationships that have influenced or could influence the reporting in this article.
Acknowledgments
We thank Yingying Zhang for her contributions to the EHP infection experiments. We also express our gratitude to Zihao Hou and Jiyue Yu for their assistance in sample preparation. This research was funded by the Natural Science Foundation of Shandong Province (ZR2020QC213), the Yantai Engineering Laboratory for the Development and Application of Characteristic Marine Organisms (No. 2023), and the Ludong University Scientific Research Start-up Fund (20220022).
