EFFECTS OF BACTERIA IN DIGESTIVE SYSTEM ON PATHOGENESIS AND IMMUNITY OF BLACK TIGER SHRIMP Penaeus monodon

Abstract

The outbreak of shrimp diseases causes massive mortality, and a huge loss of income from the shrimp exporting industry. Since most of the symptoms appear in the gastrointestinal tract, understanding the interaction between pathogenic bacteria and shrimp immune system in the digestive system is crucial to possible find means to prevent the infection of the gut. Scanning electron microscopy (SEM) was used to investigate the interaction between bacteria, both pathogenic and non-pathogenic, and the inner surface of the gastrointestinal tract of P. monodon. The differential expression of immune-related genes during an infection was examined using suppression subtractive hybridization (SSH) as well as RNA sequencing (RNA-seq). Results showed that an infection by Vibro harveyi 1526 was initiated by a proliferation of the bacteria in the biofilm-like formations in the stomach, the upper and middle midgut, but neither in the posterior midgut nor the hindgut. SEM also revealed the induced production of peritrichous pili-like structures by the Vibrio parahaemolyticus attaching to the stomach lining, whilst only a single polar fibre was seen forming an apparent physical bridge between V. harveyi and the host’s epithelium. In contrast, no adherences or linkages were seen when trials were conducted with non-pathogenic Vibrio sp. or with Micrococcus luteus, and no obvious resultant changes to the host’s gut surface was observed. A total of 612 randomly recombinant clones from SSH was matched to 69 known transcripts. The expressions of known genes, i.e. salivary alkaline phosphatase, pacifastin heavy chain precursor, ubiquitin-conjugating enzyme H5b, ferritin, astakine variant 1 and dicer 2 homologues were significantly up-regulated in stomach at 6 hours post infection. A set of 1,514 DEGs (1,122 unique genes up-regulated and 392 unique genes down-regulated) in the stomach was reported during V. parahaemolyticus (3HP) infection. Among these significantly differentially expressed genes, 141 unique genes could be classified as immune-related genes, and their functions were categorized into 10 functions including antimicrobial peptides, signal transduction pathways, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptosic tumor-related proteins, pathogen recognition immune regulators, blood clotting system, adhesive proteins and heat shock proteins. A novel isoform of anti-lipopolysaccharides was significantly increased in the stomach at 6 and 12 hours post infection. After characterization, this novel isoform contains 369 bp of the open reading frame, and was named PmALF7. Additionally, a C-type lectin receptor from an older P. monodon library was identified in hepatopancreas, and it is likely an important gene in the immune response of the hepatopancreas. The C-type lectin receptor contains 522 bp of the open reading frame, and its expression was significantly decreased in the hepatopancreas at 6 hours post infection. The current dissertation suggests that pathogens of P. monodon must be able to colonize in the digestive tract, particularly the stomach, where chitin is present, and then they use an array of virulent factors and enzymes to establish an infection in their host and P. monodon responds to pathogenic bacteria through physical barriers and activation of immune processes. These studies about interaction between bacteria and gastrointestinal tract of P. monodon increase the knowledge of how the shrimp stomach responds to bacteria and might provide new strategies for controlling and managing shrimp diseases.