Date of Award

12-1-2017

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Rader, Bethany

Abstract

The binary beneficial symbiotic relationship between the Hawaiian bobtail squid Euprymna scolopes and the Gram negative bioluminescent bacteria, Vibrio fischeri is governed by recognition of microbial molecular patterns by pattern recognition receptors (PRRs) of the host’s innate immune system. PRRs such as toll like receptors (TLRs), have been shown to promote long term beneficial host-symbiont interactions. To date six TLRs have been identified in E. scolopes (EsTLR1-6), and we hypothesized that EsTLRs play a role in the symbiont colonization and maintenance of the squid-vibrio symbiosis. To elucidate a role for EsTLRs in colonization of the squid we performed reverse transcription PCR (RT-PCR) using primers specific to TLRs 2,3,4,5 and 6 in the light organ of symbiotic or non-symbiotic juvenile animals at 0, 24, 48 and 72 hours post-hatching. Estlrs were expressed starting at 24 hours in both symbiotic and non-symbiotic animals, with expression ceasing at 72 hours in non-symbiotic light organs for Estlr 3 and 5. Immunohistochemistry utilizing antibodies designed to EsTLR specific peptides showed EsTLR2 alone was seen at in non-symbiotic light organs at 48 and 72 hours post-colonization. To elucidate a role for EsTLRs in the maintenance of the symbiosis we cured adult squid of the symbiont with antibiotics and compared them to symbiotic non-antibiotic treated adults. In general EsTLRs increased in expression in the central cores (light organs) of cured animals as compared to the central cores of symbiotic animals. EsTLR6 is more highly expressed in the symbiotic central core, while Estlrs2, 3 and 5 appear to be more active in the cured central core. To provide more evidence that EsTLRs play a role in symbiont maintenance, we looked at EsTLR expression at different time points during the day/night cycle, 7am (after venting), 1pm, 7 pm, and 1 am, which are predicted to have different bacterial population densities. Although in general EsTLR expression differed between animals, Estlrs4 and 5 were consistently expressed at 7am, when the bacterial population density is low. These data suggest that Estlrs2, 3 and 5 may play a role in recognition of the symbiont, as they are expressed during initial colonization of juveniles as well as more highly expressed after an adult has been cured of the symbiont. Estlrs4 and 5 may play a role in maintenance of the symbiont or at least the venting behavior because they are expressed at 7 am. Continuing investigations will explore Estlr mRNA and protein expression and localization in response to colonization as well as exposure to various environmental symbiotic and non-symbiotic strains.

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