1 Are they what they eat? A stable GIT microbiome characterized in P. resecata. by Tarah Gustafson A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology (Honors Scholar) Presented November 12, 2020 Commencement June 2021 2 3 AN ABSTRACT OF THE THESIS OF Tarah Gustafson for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology presented on November 12, 2020. Title: Are they what they eat? A stable GIT microbiome characterized in P. resecata. Abstract approved:_____________________________________________________ Ryan Mueller Intertidal herbivores, such as isopods, help regulate and contribute to nutrient cycling and organic carbon flow through the trophic levels in estuaries and coastal ecosystems. Though much is known about the microbiomes of macrophyte leaves that serve as the primary food source for isopods, and (to a lesser extent) the microbiomes of herbivores themselves, little has been studied about the community assembly dynamics of herbivore microbial communities. In this study, the intertidal herbivore P. resecata (order: Isopoda) was fed three different common macrophyte diets (Z. marina, U. lactuca, and L. saccharina) and the 16S rRNA genes of the microbial communities from the leaves of each diet, isopod gastrointestinal (GIT) content, and fecal pellets were amplified and sequenced with Illumina MiSeq sequencing. These data were cleaned using a DADA2 pipeline in R studio and the community complexity, structure, and prevalent taxa were examined for changes between each compartment sampled and based on diet treatment. Fecal pellets and macrophyte leaf samples showed structural differences based on macrophyte type. An authentic, present, and stable GIT content microbiome was found; no changes were found between the GIT microbiome of P. resecata fed different macrophytes, providing support to the growing body of work indicating a GIT-associated microbiome is present in marine isopods. Key Words: microbiome, intertidal isopod, beta diversity Corresponding e-mail address: [email protected] 4 ©Copyright by Tarah Gustafson November 12, 2020 5 Are they what they eat? A stable GIT microbiome characterized in P. resecata. by Tarah Gustafson A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biochemistry and Molecular Biology (Honors Scholar) Presented November 12, 2020 Commencement June 2021 6 Honors Baccalaureate of Science in Biochemistry and Molecular Biology project of Tarah Gustafson presented on November 12, 2020. APPROVED: _____________________________________________________________________ Ryan Mueller, Mentor, representing Microbiology _____________________________________________________________________ Byron Crump, Committee Member, representing Earth, Ocean, and Atmospheric Sciences _____________________________________________________________________ Lydia Baker, Committee Member, representing Microbiology _____________________________________________________________________ Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. _____________________________________________________________________ Tarah Gustafson, Author 7 CONTRIBUTION OF AUTHORS Dr. Ryan Mueller assisted with data analysis and writing. Dr. Gema Hernán, Michael Moses, Dr. Fiona Tomas Nash, Dr. Ryan Mueller performed and designed experiments. Sample collection, processing, and DNA extractions were completed by Dr. Gema Hernán. Additional collaboration was given by Alexis Morris, Christina Mauney, Jen Hayduk, Grant Schwinge, Dr. Winni Wang and MK English. 8 Introduction Understanding the factors that control the digestion and feeding of herbivores can have important implications on overall ecosystem function. Herbivores that directly consume macrophytes within intertidal zones are vital to the regulation of nutrient and organic matter flow within their ecosystems as well as macroecological resource cycling within marine ecosystems (1). The top-down regulation of macrophytes and their epiphytes via herbivory impacts nutrient balancing in marine systems (1-3). Additionally, the detrital matter in herbivore feces settles into the sediment, disperses into the water column, and drifts into nearby ecosystems, which can impact the spread of nutrient resources from the primary producers into the surroundings (4-6). Consumption of herbivores by predators is a key step that acts to transfer the organic carbon fixed by macrophytes and epiphytes to higher trophic levels in these aquatic food webs (3,7). Macrophytes (such as seagrasses and seaweeds), which are commonly found along ocean coastlines, fix a disproportionately high amount of carbon relative to the area they cover and thus act as an important carbon sink in marine systems (8-9). Unlike seagrasses, which are angiosperms and have only a few species, seaweeds consist of brown, red, and green algae that belong to diverse taxonomic groups (10-11). Along the coast of the Northeastern Pacific Ocean, one seagrass, Zostera marina, dominates many areas of coast and estuaries (12). In addition, there are many different types of seaweeds that grow in the littoral zone of these ecosystems and compete with seagrasses for light, nutrients, and space. Microbes are ubiquitous across all components of these intertidal ecosystems, including the water column, the sediment, macrophytes, epiphytes, animals, and detrital matter (13-15) and can have important influences on ecosystem processes, such as nutrient cycling, and the health of the organisms with which they are associated (5,16-19). Despite the structural differences that result in distinct taxa and functions associated with each microenvironment, there are many common microbes shared across these niches, indicating dispersal of microbes between them (20). Microbes that are found intimately associated with different host organisms (i.e., their microbiomes) can form specific relationships with the host in which one or both parties benefit (19). Specialized functional adaptations of hosts and microbes are often implicated as the drivers of symbiotic interactions between 9 hosts and specific microbiota of the microbiome (21). These interactions and the highly selective pressures found in host-associated niches often result in microbiomes that are specific, deterministic, and relatively stable over time (20). Examples of this co-adaptation between microbes and their hosts can be seen in many animal hosts including in aphids, whose microbiomes provide nutrient and digestive assistance and where similarities between microbiomes of different aphid populations relates more to phylogeny than to ecology (22). Despite knowledge of these general principles of community assembly of host-associated microbiomes, specifics such as the characterization of many of these microbial communities or specific beneficial aspects to host/microbe relationships is ongoing (16, 17, 20). Additionally, little is known regarding the effect of interactions between the microbiomes of different hosts when the hosts themselves interact. Isopods, in general, have a variety of feeding strategies and can be omnivores and detritivores, as well as herbivores (1, 23). Pentidotia resecata is a generalist isopod herbivore found in many intertidal zones of the Northeastern Pacific Ocean (3, 24). Due to the difference between the low nitrogen food source and their relatively high tissue composition of nitrogen, herbivores often rely on microbial symbionts to increase their nitrogen intake (25). Generalist herbivores, despite consuming a wide variety of vegetative food sources, frequently have a fairly homogenous and simplistic microbiome, presumably due to specific functional interactions and symbioses (1). Much of the current literature about isopod-microbe interactions is on the microbiome of the terrestrial isopods, due to their unique evolutionary history as the only terrestrial crustacean (26). This work has shown that their microbiome has dramatic impacts on many aspects of their behavior, physiology, and nutrition (19, 21, 26, 27). For example, several bacteria, such as Wolbachia, Rickettsia, Spiroplasma, and Cardinium affect sex ratios of the terrestrial isopods (19). Additionally, several taxa found in the gastrointestinal tract (GIT) of isopods, such as Candidatus Hepatoplasma crinochetorum, Candidatus Hepatincola porcellionum, and Rhabdochlamydia porcellionis, can help with nutrient absorption and degradation of complex molecules, such as lignins, phenolics, and cellulose (16, 17, 25–27). In terrestrial isopods, the origin of an organism’s microbiome is thought to be through ingestion (horizontal transfer) and not through vertical inheritance from the progenitor’s reproduction (28, 29). Much less is known of the microbiomes of aquatic isopods. In fact, there is debate on whether there is an authentic and specific GIT microbiome in marine isopods (16–18, 21, 27). 10 This paper explores this question and considers the possibility of horizontal transfer of the microbiome from macrophyte diets to the P. resecata GIT and investigates differences in microbiome structure leaf surfaces, GIT contents, and fecal pellets as a