| Litopenaeus vannamei occupies a significant position in China's aquatic product market. However, continuous intensification has led to increasingly serious eutrophication of water bodies, resulting in frequent occurrences of cyanobacteria blooms. These blooms release microcystins, which can pollute water and cause significant deaths among cultured organisms, posing a serious threat to agriculture and public health. Microcystin-LR (MC-LR) is the most prevalent and highly toxic variant. It has a significant toxic effect on aquatic organisms and can impact terrestrial organisms and human health in various ways This has attracted widespread attention in the academic community. MC-LR is hepatotropic and highly accumulated in the hepatopancreas after entering the body. Numerous reports have described the toxicological effects of MC-LR from various perspectives, but its potential biological processes are still highly complex and may involve alterations in multiple signaling pathways in L.vannamei.
In many experiments investigating the acute exposure of shrimp, most of them use the method of blood sinus injection or intramuscular injection. However, these methods have the potential to cause tissue damage and changes in immune indicators. Reverse?gavage greatly reduces tissue damage and can directly affect the hepatopancreas. It can also better simulate the process of causing toxic reactions under natural conditions. First , the MC-LR solution was mixed with red edible dye, and then the mixture was slowly dripped into the anal cavity of the experimental group using an automatic pipette. The inoculation solution was observed to enter the midgut from the hindgut and ceased when the red color appeared in the hepatopancreas. After 24 hours of MC-LR treatment, the cephalothoraxes was dissected to obtain the hepatopancreas tissue. Subsequently, transcriptome sequencing technology was used to identify the differentially expressed genes, related signaling pathways, and metabolic pathways in the hepatopancreas of L.vannamei under MC-LR treatment. The results of the transcriptome sequencing were validated using quantitative real-time PCR technology.
The study revealed that MC-LR induced significant differential expression of 1194 genes compared to the control group. Of these, 1164 were up-regulated and 830 were down-regulated. The differentially expressed genes of L.vannamei were categorized based on biological processes, cellular components, and molecular functions using the Gene Ontology (GO) database, resulting in 33 functional entries. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database metabolic pathway analysis revealed that the transcriptome of L.vannamei had 240 differentially expressed genes annotated across 6 categories: metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, and human disease. The main metabolic pathways include carbohydrate metabolism, lipid metabolism, protein translation, signal transduction, cell growth and apoptosis, transport and catabolism, immune system, and endocrine system. GO functional enrichment analysis revealed that the functions of significantly differentially expressed genes were primarily enriched in catalytic activity, heterocyclic compound binding, carbohydrate derivatives, small molecule binding, protein folding, and RNA metabolism. Among the top 20 pathways of KEGG enrichment, the ribosome biosynthesis pathway was significantly enriched. Additionally, pathways related to lipid and atherosclerosis, protein processing in the endoplasmic reticulum, and purine metabolism were also enriched. The number of differentially expressed genes is higher. These pathways may be involved in the metabolism, processing of environmental information, and cellular processes of L.vannamei under microcystin stress. Ten genes with the most significant differential expression were further screened. It is worth noting that the expression of zinc finger protein 761-like was down-regulated. The statistical analysis of differential gene expression indicated that several genes belonging to the zinc finger protein family were significantly down-regulated. This suggests their potential involvement in the microcystin response of L.vannamei.
In conclusion, this study provides a method ological reference for shrimp exposure experiments and basic data for revealing the molecular regulation mechanism of L.vannamei in response to microcystins. However, the reverse?gavage method requires further improvement, and a methodological comparison of different approaches is also necessary to better understand the advantages and disadvantages of reverse?gavage methods. At the same time, it is necessary to further verify these differentially expressed genes to determine their close relationship with the response of L.vannamei to microcystin stress. This will enable more in-depth exploration and improvement of the toxicological mechanism of L.vannamei. |
