CRABS & ‘CROBES: THE TRIPARTITE RELATIONSHIP OF A HOST, PARASITE, AND THEIR RESPECTIVE MICROBIOMES

Growing evidence suggests that the associated microbiome of organisms (the holobiont) has been shaping the evolutionary pathways of macroorganisms for thousands of years, and that these tiny symbionts can influence species interactions. Yet, while studies have investigated host-parasites and microbiomes separately, how the two systems interact and influence each other has only begun to be explored. This relationship among hosts, parasites, and microbial communities changes the dynamic of host-parasite evolution from a more traditional co-evolution, to a tripartite evolutionary relationship. My research aimed to resolve questions about the community composition and diversity of microbial symbionts associated with host and parasite separately and when combined (parasitized hosts). My research also developed a methodology that can be used in future studies to manipulate microbiomes, in the pursuit of understanding how the loss or manipulation of the microbiome affects parasitism. Developing the methodology for future microbiome manipulation included determining a method for bacterial inhibition and testing the effects of inhibition on community diversity. For the first part of the research, infected and uninfected crab hosts were sampled from a coastal North Carolina oyster reef three times over a four-month period. Tissue samples were collected from four biological groups: uninfected crab viscera, infected crab viscera (i.e. host + parasite), the entire adult parasite externa, and parasite larvae. Microbial DNA was extracted from tissue samples and sequenced using the V6-V8 region of the 16S rRNA microbial gene to determine the community composition and diversity of the microbiome for each biological sample and across time. Microbial community analysis revealed that parasite externae and larvae had very similar microbiomes but were significantly different from the microbiomes of the crabs. Microbiomes of infected versus uninfected crabs were also significantly different. Both adult parasite externae and parasite larvae were found to have bacteria including Pseudoalteromonas species, which provide natural antimicrobial defenses, while uninfected crabs were mainly comprised of Rhodobacteraceae, commonly associated with photo- and chemoautotrophy. To develop the methodology for the second part of the research, modified Kirby-Bauer disk diffusion technique was used to determine the effects of biological sample, antibiotic type, antibiotic concentration, and time to inhibition of bacteria. Three broad spectrum antibiotics (ampicillin, chloramphenicol, and gentamicin) and their combinations (total of seven antibiotics and combinations) were tested across four concentrations against all four biological groups. Chloramphenicol, at 2mg/mL, was found to be the most effective antibiotic at inhibiting microbial growth across all four biological samples. To determine the effect of chloramphenicol on microbial community composition and diversity, this treatment (or a no treatment control) was applied to live infected and uninfected crabs for 24 hours. After which, microbial DNA was extracted from all four biological samples and will be sent for sequencing, thus the results of bacterial community diversity are pending. Understanding the microbial community composition of a host and parasite, and developing a methodology for manipulating those microbiomes, is an important step to beginning to understand the microbiome's role in the host-parasite relationship and determining how the tripartite relationship impacts coevolutionary processes