Date of Completion

8-21-2017

Embargo Period

8-18-2018

Keywords

Symbiosis, Microbiome, 16S, Euprymna, Eggs, Development, Fungus

Major Advisor

Spencer V Nyholm

Associate Advisor

Joerg Graf

Associate Advisor

Jonathan Klassen

Associate Advisor

Daniel Gage

Associate Advisor

Nichole Broderick

Field of Study

Microbiology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Many aquatic organisms deposit their eggs into an environment where successful embryogenesis depends on minimizing biofouling. The female Hawaiian bobtail squid, Euprymna scolopes, harbors a diverse bacterial community within the accessory nidamental gland (ANG), a symbiotic reproductive organ. This community is hypothesized to be environmentally transmitted, and to be deposited from the ANG into the egg jelly coat (JC). Illumina sequencing of the 16S rRNA V4 gene region demonstrated that the ANG bacterial community (n=29) and that of the JC (n=35) were composed primarily of members of the Rhodobacteraceae and Verrucomicrobia, which together comprised on average 86% of the sequences recovered per sample (68% and 18% respectively). JC bacterial communities clustered with the ANG community of the female that produced those eggs, suggesting that bacteria from the ANG are deposited directly into the JC. OTUs representing 94.5% of the average ANG abundance were found in the natural squid environment, consistent with the hypothesis of environmental transmission between generations. The ANG bacterial community gradually changed from a Verrucomicrobia-dominated to an Alphaproteobacteria-dominated community over the course of host sexual development. The surface of the immature ANG was covered in microvilli and contained numerous ciliated invaginations, demonstrating that the immature ANG is poised for colonization by environmental bacteria. Eggs treated with antibiotics over the course of embryogenesis developed a biofilm, primarily composed of the fungus Fusarium keratoplasticum, which led to the death of the embryos (3% viability; n=17 clutches). Fungal challenge experiments on dissected eggs demonstrated that the JC containing the bacterial community is essential for egg defense from fungal bud cells (n=3 trials, 8-10 eggs/treatment). Extracts from ANG/JC bacteria were also able to inhibit F. keratoplasticum in vitro. Taken together, these data suggest that the ANG/JC bacteria protect developing embryos from biofouling. Ongoing work is focused on identifying specific strains and compounds responsible for antifungal activity. This association offers a unique experimental model for understanding mechanisms by which marine invertebrates protect their eggs in the environment. This research has set the background for utilizing the E. scolopes ANG system as a model for studying how consortial symbioses are established and maintained.

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