The Generation of a Zebrafish Microbial Culture Collection to Facilitate Mechanistic Understanding of Host-Microbe Interactions

A rapidly expanding body of evidence suggests that relationships exist between the microbiome and vertebrate health and disease. The zebrafish is a frequently utilized model organism to support this claim. Specifically, its gut microbiome is an advantageous resource for the study of microbiome health and diversity. While it is an important model for understanding host-microbe interactions, its utility is mitigated by the limited number of cultured isolates made publicly available for zebrafish model systems. Establishment of a culture collection would enhance our ability to conduct empirical assessments of host microbiome interactions in a high-throughput manner. The resulting objective was to generate a foundational zebrafish microbial culture collection and quantify the proportion of zebrafish culturable gut microbial diversity. Gut microbes were cultured from 5D-line zebrafish fecal samples obtained at 5 different time points over a 6-month period. For each isolate we characterized morphological characteristics and oxygen tolerance abilities. To quantify taxonomic diversity of the culture collection we PCR amplified and Sanger Sequenced the 16S rRNA gene of each isolate. In total, 108 zebrafish gut microbes were isolated and preserved, and we were able to assign taxonomy to 78. Together, these represented approximately 50% of the typical zebrafish gut genera

Trophic diversification and parasitic invasion as ecological niche modulators for gut microbiota of whitefish

Introduction: The impact of parasites on gut microbiota of the host is well documented, but the role of the relationship between the parasite and the host in the formation of the microbiota is poorly understood. This study has focused on the influence that trophic behavior and resulting parasitism has on the structure of the microbiome. Methods: Using 16S amplicon sequencing and newly developed methodological approaches, we characterize the gut microbiota of the sympatric pair of whitefish Coregonus lavaretus complex and the associated microbiota of cestodes parasitizing their intestine. The essence of the proposed approaches is, firstly, to use the method of successive washes of the microbiota from the cestode’s surfaces to analyze the degree of bacterial association to the tegument of the parasite. Secondly, to use a method combining the sampling of intestinal content and mucosa with the washout procedure from the mucosa to understand the real structure of the fish gut microbiota. Results and discussion: Our results demonstrate that additional microbial community in the intestine are formed by the parasitic helminths that caused the restructuring of the microbiota in infected fish compared to those uninfected. Using the desorption method in Ringer’s solution, we have demonstrated that Proteocephalus sp. cestodes possess their own microbial community which is put together from “surface” bacteria, and bacteria which are weakly and strongly associated with the tegument, bacteria obtained after treatment of the tegument with detergent, and bacteria obtained after removal of the tegument from the cestodes.info:eu-repo/semantics/publishedVersio

Trophic diversification and parasitic invasion as ecological niche modulators for gut microbiota of whitefish

Introduction: The impact of parasites on gut microbiota of the host is well documented, but the role of the relationship between the parasite and the host in the formation of the microbiota is poorly understood. This study has focused on the influence that trophic behavior and resulting parasitism has on the structure of the microbiome. Methods: Using 16S amplicon sequencing and newly developed methodological approaches, we characterize the gut microbiota of the sympatric pair of whitefish Coregonus lavaretus complex and the associated microbiota of cestodes parasitizing their intestine. The essence of the proposed approaches is, firstly, to use the method of successive washes of the microbiota from the cestode’s surfaces to analyze the degree of bacterial association to the tegument of the parasite. Secondly, to use a method combining the sampling of intestinal content and mucosa with the washout procedure from the mucosa to understand the real structure of the fish gut microbiota. Results and discussion: Our results demonstrate that additional microbial community in the intestine are formed by the parasitic helminths that caused the restructuring of the microbiota in infected fish compared to those uninfected. Using the desorption method in Ringer’s solution, we have demonstrated that Proteocephalus sp. cestodes possess their own microbial community which is put together from “surface” bacteria, and bacteria which are weakly and strongly associated with the tegument, bacteria obtained after treatment of the tegument with detergent, and bacteria obtained after removal of the tegument from the cestodes

The microbiome of the equine roundworm, \u3ci\u3eParascaris\u3c/i\u3e spp.

Parasitic nematodes, including the large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarid, Parascaris spp., was the first ascarid parasite to develop wide-spread anthelmintic drug resistance, with other species slowly following suit. There are no new classes of anthelmintics currently in development, and a solution to the ever-increasing prevalence of resistance is desperately needed. The microbiome has been shown to be an important factor in the fitness and health of many organisms and changes to microbiome composition have been associated with a plethora of diseases. The microbiome is also important to the health of parasitic nematodes, and the endosymbiotic bacterium Wolbachia, whose presence is essential for the viability of filarial nematodes, has been exploited for treatment of filariasis in humans by using both broad-range and, more recently, specific anti-Wolbachial antimicrobial treatments. Despite this success, parasite microbiomes are understudied. The overarching goal of this dissertation was to characterize the microbiome of Parascaris spp. by identifying a common core microbiota, by comparing microbiota diversity metrics for the whole worm at different life stages and in individual organs in male and female parasites, and by assessing the female gonad microbiota in greater detail. Worms, along with jejunal content samples, were collected from foals at necropsy and used for both the whole worm study, which utilized a total of 27 parasites (9 male, 9 female, 9 immature), and in the organ study, which utilized a total of 46 adult parasites (24 male, 22 female). DNA extracted from these samples was used to produce a library using a 16S rRNA metagenomic sequencing protocol, and this library was sequenced using the Illumina MiSeq platform. A bioinformatics pipeline was developed to identify taxa and their relative abundance in the samples, and subsequent data analysis was carried out using R packages including Vegan, DESeq2, corncob, metagenomeSeq, and ANCOM.BC. The 22 female gonad samples were further analyzed using next generation metagenomic sequencing following the same protocol as the other two studies, and then using a kit that targeted to multiple regions and that allowing consensus sequences to be assembled. Additionally, another female worm was also collected, immediately fixed, dissected, and submitted for sectioning and examination by transmission electron microscopy. A common core microbiota consisting of eleven genera was established for Parascaris spp. and consisted of: Acinetobacter, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (ANPR), Clostridium senso stricto 1, Gemella, Janthinobacterium, Lactobacillus, Reyranella, Sarcina, Sphingomonas, Streptococcus, and Veillonella. When comparing organs, Veillonella was differentially abundant when using DESeq2 and ANCOM-BC (p \u3c 0.0001), corncob (p = 0.0008), and metagenomeSeq (p = 0.0118) and Sarcina was differentially abundant across all four analytical methods (p \u3c 0.0001). Alpha and beta diversity for the whole worm microbiota was similar across groups for all three taxonomic levels. Alpha diversity for the organ microbiota was significantly different based upon both sex and location at all three taxonomic levels. Simpson alpha diversity was significantly different between the female intestine (FI) and male gonad (MG) at the phylum (p \u3c 0.0001), family (p = 0.0058) and genus (p = 0.0018) levels, and between both the female gonad (FG) and FI (p \u3c 0.0001) and the FI and male intestine (MI; p = 0.0072) at the phylum level. Shannon alpha diversity was significantly different between the FI and the FG (p \u3c 0.0001), the horse jejunum (HJ; p = 0.0483), the MG (p \u3c 0.0001) and the MI (p = 0.0007) at the phylum level, between the FI and MG (p = 0.0003) at the family level and between the FG and MG (p = 0.0130), the FI and HJ (p = 0.0383) and the FI and MG (p = 0.0001) at the genus level. Beta diversity was significantly different between FI and FG (p = 0.0377) at the phylum level, MG and FG (p = 0.0010), FI (p = 0.0174), and HJ (p = 0430), and FG and MI (p = 0.0061) at the family level, and MG and FG (p = 0.0006), MI and FG (p = 0.0093), and MG and FI (p = 0.0041) at the genus level. Twelve species were identified in the female gonad, and phylogenetic trees were created for the genera Aminobacter, Reyranella, Limosilactobacillus and Ligilactobacillus. Cladograms indicated that consensus sequences from members of these genera were related to species found in soil and water, and to those that had previously been found in horses, and thus the presence of related bacteria in parasites makes biological sense. Finally, morphological structures identified as candidate bacteria were found in the cells of Parascaris spp. female gonad sections, indicating that there are also possibly endosymbionts associated with these parasites. In summary, the overarching goal of this research was met. A common core microbiota was established for Parascaris spp., diversity metrics were compared for different life stages and organs, and the female gonad was explored in more detail. This research lays the groundwork for future studies involving the Parascaris spp. microbiome and provides more data to the effort to understand parasite microbiomes

Host microbiota and infection outcomes in thermally extreme environments

Hosts, their collection of commensal and pathogenic microbes, and the environment are all interlinked in a disease pyramid. Their interactions contribute to host fitness, particularly relevant in the context of a changing world. Climate change is leading to higher average temperatures as well as shifting the global distribution of infectious diseases, posing great risk to wildlife, ecosystem biodiversity, and human health. Host microbiomes are increasingly regarded as conferring extended phenotypes, potentially buffering host organisms against abiotic and biotic stressors. In this thesis, I combine meta-analytical and experimental approaches to explore the impact of warming temperatures and infection on ecological dynamics (i.e., microbiomes, infection disease outcomes) and evolutionary dynamics (i.e., host gene-based resistance). Firstly, I used a meta-analytic approach to investigate whether and how experimental temperatures altered host microbiome structures, across a wide range of host species. I found that experimental warming and cooling drove microbiome diversity loss, with the magnitude affected more by host habitat and experimental protocols, rather than host biological traits. I then extended this work to empirically include a biotic factor – pathogen infection – to investigate changes in Caenorhabditis elegans microbiome composition. I revealed that warming and infection could destabilize microbiome communities within hosts, but their effects were not additive. Focusing more on the impact of warming to host-pathogen interactions, I subsequently used a meta-analytic approach to tackle the relationship between warming and disease outcomes across ectothermic animals. I found that experimental warming drove higher mortality of infected hosts, with larger temperature increases associated with more host deaths. The magnitude of these effects varied by pathogen taxonomy and their evolutionary history within the host. Lastly, I zoomed out to empirically capture the possible host evolutionary paths for resistance to pathogens due to warming. I competed two C. elegans genotypes (susceptible wild-type vs. resistant mutant) across 10 host generations, varying in pathogen presence and the timing of warming during their development. I detected a loss of genetic-based resistance under periodic warming despite infection. I revealed that such host evolutionary trajectories could be driven by the combination of fitness constraints on genetic-based resistance, temperature-mediated host protection, infection severity, as well as the dilution of pathogen cells by resistant hosts. Work in this thesis is a timely contribution to our understanding of the diversity of consequences of warming to host-microbe interactions across the mutualist-parasite continuum. Biologists seeking to refine predictions of biodiversity amidst climate change should strongly consider the relationships of animals with their resident and attacking microbes