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Identification, Characterization, and Gene Expression Analysis of Thioester-Containing Proteins (TEPs), and the Host Response to Environmentally Evolved Bacteria in the Squid Euprymna scolopes

Thuraisamy, T;

The squid Euprymna scolopes forms a mutualistic symbiotic association with the bioluminescent marine bacteria Vibrio fischeri. Various host and microbial factors regulate symbiotic associations and host immune components and are believed to play a crucial role in the establishment and maintenance of these symbiotic associations. E. scolopes is an invertebrate animal and does not contain a classical antibody dependent adaptive immune system. Thus, it relies only on its innate immune system, which has two major types of components: humoral and cellular. Among these components, pattern recognition receptors (PRRs) are a large group of first-line sensors that allow the host to recognize and distinguish foreign agents from self-molecules and are involved in both humoral and cellular immune responses. Thioester containing proteins (TEPs), are one of the most phylogenetically ancient innate immune PRRs molecules and found in both vertebrate and vii invertebrate organisms. However, many of these molecules have not been well characterized and immune functions of these molecules in the animal-beneficial bacteria associations have not been well studied. The first objective of this study was to investigate the presence and diversity of TEP molecules in E. scolopes and their possible role in symbiosis. It was hypothesized that E. scolopes contains diverse TEP molecules and that these molecules are modulated in the presence of symbiotic bacteria to allow for efficient colonization and maintenance in the dynamics of symbiosis. In silico methods were used to identify and initially characterize the TEP molecules in E. scolopes. Transcriptome analysis resulted in the identification of eight different TEP molecules (A2M-1, A2M-2, C3-1, C3-2, TEP-1, TEP-2, TEP-3, and MCR-1) in E. scolopes that were categorized into four subfamilies. In this study, E. scolopes A2M and MCR molecules were identified for the first time. To verify the expression of TEP molecules, end point polymerase chain reaction (PCR) was used to amplify TEP transcripts in uncolonized and colonized juveniles, and in one adult tissue. Six out of the eight TEPs (A2M-1, A2M-2, C3-1, C3-2, TEP-1, TEP-2, and MCR-1) were successfully amplified in all three types of samples tested: whole body of uncolonized and colonized juveniles, and adult gill tissue. TEP-2 was not able to be amplified in any of the three samples, while TEP-3 was amplified only in 24 h-colonized juveniles and in adult gills. Real time-quantitative PCR (RT-qPCR) results showed that three genes (C3-1, C3-2, and MCR-1) were downregulated, with statistical significance, in 48 h-colonized juveniles, supporting the hypothesis that exposure to V. fischeri bacteria results in the modulation of the squid host’s immune genes to allow for the symbiont colonization and persistence. The next phase of this research study focused on investigating the effects of environmentally evolved symbiotic V. fischeri on the development, survival, and health of the squid. The highly viii specific squid-Vibrio symbiotic association provides an experimental model to investigate the role of symbionts on the development of animal host tissues. In nature, E. scolopes acquires its symbiont V. fischeri horizontally from the surrounding seawater within few hours of hatching. Then, the bacteria will colonize the crypt spaces, a specialized region in the squid’s light organ (LO). Following colonization by V. fischeri, the juvenile LO undergoes multiple developmental changes, including the regression and apoptosis of the ciliated epithelial appendages (CEA). It was hypothesized that environmentally evolved V. fischeri strains that respond to environmental stressors such as temperature changes will adapt and have a better ability to colonize the squid’s LO compared to the ancestral strains. To test this hypothesis, two objectives were formulated: (1) to examine the timeline progression of morphological changes in the juvenile squid LO following colonization with temperature-evolved V. fischeri strains, and compare to that of their ancestral strains; and (2) to assess squid’s health and survival upon exposure to temperature evolved strains and compare to that of their ancestral strains. Analysis of regression of the appendages surface epithelium indicated that the temperature evolved strains of V. fischeri ES114 and EM17 showed faster morphological changes in the LO, suggesting that these temperature evolved strains could be more efficient in initiating the developmental process in the squid than their corresponding ancestral strains. Survival and health of juvenile squid were not affected by the colonization by temperature evolved strains. Furthermore, these findings suggest that the squid-Vibrio symbiosis can be adapted and sustained despite abiotic changes in their environment and provide a framework for future studies on the symbiosis between animals and environmentally adapted bacteria.