MSc Thesis - The effect of wastewater effluent on the gut microbiome of rainbow darter / The microbiome plays an important role in host physiology and can be influenced by species, diet, and environment. Municipal wastewater effluent contains a mixture of chemicals including antibiotics and antimicrobials that may affect the gut microbiome of fish living downstream of these discharges. Thus, this study examines the effect of wastewater treatment plant (WWTP) effluent on the gut microbiome of wild rainbow darter (Etheostoma cearuleum), and examines how the gut microbiome of wild fish changes in the lab. Fish were collected from sites upstream and downstream of 2 major WWTPs along the central Grand River and gut contents were aseptically sampled. After extracting gDNA, nested PCR of the V3-V4 region of the 16S rRNA gene, and Illumina sequencing were performed. The gut microbiome of exposed fish had increased bacterial diversity and was dominated by Proteobacteria, which has been linked to altered health outcomes in mammals. Next, rainbow darters were collected from a reference site on the Grand River. Fish were sampled in the field, after a 14 day lab acclimation, and after a 28 day exposure to environmental stressors (WWTP effluent or triclosan, an antimicrobial found in WWTP effluent). Surprisingly, there were no changes in the microbiome after exposure to environmental stressors. Major changes were observed between the field and laboratory fish suggesting that environment and diet are important factors influencing the gut microbiome. Changes in the gut microbiome continued up to 42 days in the lab, indicating longer acclimation periods may be needed. This study showed that effluents altered the gut microbiome of fish in the field, but not in the laboratory for unknown reasons. Laboratory studies indicated that transitioning to a new environment may require greater than 14 days before achieving a stable microbiome. / Thesis / Master of Science (MSc) / Wastewater is the largest source of pollution affecting Canada’s aquatic ecosystems; effluents contain antibiotics and antimicrobials that can affect fish and other aquatic life. The gut microbiome of fish is influenced by host species, its diet, and the environment, and thus contaminants released via wastewater effluents may alter the gut microbiome of fishes in receiving waters. This study found that the gut microbiota of rainbow darter fish exposed to wastewater effluents in the central Grand River (Waterloo/Kitchener, Ontario) were dominated by Proteobacteria and had increased diversity. Wild fish transitioned to the lab were dominated by Firmicutes and had decreased bacterial diversity in the gut compared to those in the wild. Altogether, these results suggest that wild fish exposed to wastewater effluents had altered gut microbiomes; transitions to new environments and laboratory acclimation periods are important considerations when studying the fish gut microbiome.

Necrotic enteritis (NE) is a major enteric disease in commercial poultry and manifests itself in clinical and subclinical forms. Despite years of research, NE is still among the leading diseases with the greatest economic impact on poultry production. Subclinical forms lead to poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio) but usually occurs with low mortality rates. The use of antibiotic growth promoters (AGPs) is proving to be an effective tool in maintaining gut health and modifying gut microbiota, thus improving broiler performance and reducing NE. Removal of AGPs has led to an increase in NE occurrence, particularly the subclinical forms. The lack of alternative strategies to control NE is mainly due to limited insight into the relationship between NE pathogenesis, the host microbiome and its immune responses. Therefore, key to overcoming NE is to define the cellular and molecular mechanisms that are involved in the progression of the disease, especially with regard to mucosal immune responses and gut microbiome. Also, assessing the impact of these changes on gut cell metabolism and function is of great importance. This information would be a valuable guide to prevent the onset or alleviate the negative impact of NE on bird's health and performance. In a series of experiments conducted for this project, the effect of single or multi-strain probiotics as well as multi-component additives were tested during NE challenge in order to define the cellular and molecular mechanisms that are involved in the progression of the disease. Results of these experiments revealed that challenging broilers with a 'naturally occurring' NE led to differential expression of tight junction (TJ) proteins in the jejunum compared to non-challenged birds. Supplementation of certain additives reduced NE lesion scores, improved performance and increased mRNA abundance of claudin-3, a key epithelial TJ protein. Multi-strain probiotics and multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) were more effective than single-strain probiotics or prebiotics. The aforementioned additives were also more effective in modulating immune responses to NE, especially by decreasing the mRNA abundance of IFN-γ and IL-10 in the jejunum. Furthermore, supplementation of these additives led to an increase in the expression of nutrient transporters (SGLT-1) and regulators of energy metabolism (PGC-1α, mTOR and AMPK); thus, improving nutrients absorption and metabolism. Microbial profiling using 16S rRNA sequencing showed that supplementation of each specific additive led to a signature-like microbiome in the ileal scrapings of broilers. However, supplementation of multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) modified the ileal microbiome in association with lower NE lesion scores, better performance and modulated immune responses. These additives reduced the relative abundance of bacteria such as ASF356, Bacteroides, Clostridium sensu stricto 1, Faecalibaculum, Lachnospiraceae UCG-001, Muribaculum, Oscillibacter, Parabacteroides, Rikenellaceae RC9 gut group, Ruminococcaceae UCG-014, and Ruminiclostridium 9 and increased the relative abundance of Lactobacillus compared to NE challenged birds. Collectively, these data indicate that during a subclinical naturally occurring NE, the use of multi-strain probiotics or multi-component additives, compared to single-strain probiotics or prebiotics, would be a more promising strategy in alleviating the effect of this enteric disease. / Doctor of Philosophy / Necrotic enteritis, an enteric disease, is one of the major diseases that negatively impacts the poultry industry and producers' profitability. The growing ban on the use of antibiotics that were used to prevent this disease has increased the number of necrotic enteritis outbreaks worldwide. Having a better understanding of the cellular and molecular mechanisms that are involved in the onset of this disease is of crucial importance and could lead to finding more effective ways to control this disease without drugs. The gut is the site of digestion and absorption of nutrients so any damage would lead to poor bird performance. In a series of experiments conducted for this project, several combinations of beneficial bacteria and nutrient sources that help bacterial growth in the gut (prebiotics) improved gut health leading to better performance during the grow-out period (days 0-42) when birds reach market age. These supplements protected the gut lining and reduced damages due to necrotic enteritis with less severe lesions. Barrier function of the gut was also improved by supplementing the diet with combination of beneficial bacteria and nutrients that help their growth in the gut. There are special types of proteins (called tight junctions) that seal up the space between intestinal cells (enterocytes) and prevent pathogens in the gut lumen from entering the body, thus preventing inflammation and disease. This helps the body to use the absorbed nutrients for growth rather than spending energy to fight pathogens, which collectively results in better growth performance. Concurrent supplementation of beneficial bacteria plus nutrients that help their growth balanced the immune responses in the gut by increasing the copy number of cytokines. Cytokines are proteins that orchestrate immune responses that the host mounts against pathogens. Certain cytokines regulate such responses by preventing the immune system from overreacting and mounting unnecessary reactions, thus preserving energy and nutrients for growth while reducing inflammation. Nutrient uptake from the gut lumen is facilitated by nutrient transporter proteins that reside on intestinal cells (enterocytes). Birds concurrently supplemented with beneficial bacteria and nutrients that help their growth in the gut increased the abundance of these proteins, resulting in improved nutrient uptake and performance compared to the control birds. Co-supplementation of beneficial bacteria and nutrient sources that help their growth modified the type and number of bacteria that are present in the gut lumen. The modified bacterial community were able to produce metabolites such as butyrate and propionate, which are beneficial for the health and growth of the intestinal cells, thus improving the bird's health and its performance. Overall, compared to beneficial bacteria alone, co-supplementation of beneficial bacteria with the nutrients that help their growth in the gut significantly reduced intestinal lesions and improved performance of broiler chickens during the production period. Moreover, dietary addition of these supplements improved gut barrier function by regulating the gene expression of tight junction proteins and gut mucosal immune responses as well as modifying the bacterial community of the gut. Therefore, such combination supplements hold promise in controlling necrotic enteritis in poultry and sustain good overall performance that translates into higher profitability to producers.

This thesis presents the first randomised controlled trial (RCT) to investigate whether supplemented probiotic bacteria-"live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" (Sanders, 2008)-affect mood and other psychological outcomes in people presenting with low mood. Seventy-nine participants with at least moderate symptoms of depression were randomised in a double-blind manner to receive either a probiotic preparation containing Lactobacillus helveticus and Bifidobacterium longum or a matched placebo for eight weeks. The RCT phase was followed by an open label extension in which all participants were offered the active study product for a further 8 weeks. Participants were followed up at 6 months post-study. Based on the existing evidence from gut-brain axis research, and on models linking depression with inflammation, immune activation, low vitamin D levels, and the gut microbiota (outlined in Chapters 1 and 2), it was hypothesised that: the overall sample would have elevated levels of inflammatory biomarkers and low levels of vitamin D at baseline, and that this would be associated with scores on psychological and irritable bowel syndrome (IBS) outcome measures; that group differences (active treatment versus placebo) would be observed in scores on psychological outcome measures after eight weeks of probiotic intervention; that group differences would also be observed in blood levels of proinflammatory cytokines, hsCRP, vitamin D and BDNF, and scores on a measure of gut function/IBS, and that levels of these variables may predict or impact on treatment response; and that group differences would be observed on outcome measures at the point of the 6-month follow-up between those who continued to take the probiotic and those who discontinued probiotic use. In total, 58 of the 77 participants who provided baseline blood samples (75%) had at least one marker of inflammation elevated outside the normal reference range at baseline. Baseline vitamin D was approaching the deficient level, displayed a seasonal pattern, and was associated with severity on one measure of cognition. No significant differences were found between the active treatment and placebo groups on any psychological outcome measure, the measure of gut function or in the level of any blood-based biomarker in the randomised phase. Baseline vitamin D level was found to moderate treatment effect on several outcome measures. The results of the open label extension supported the lack of efficacy observed in the randomised phase, and also allowed for the comparison of efficacy over intervention periods of varying durations. The results of the follow-up at 6 months post-trial indicated that, while mean scores on psychological outcome measures remained lower than baseline, there was regression on some outcome measures after the study. When the participants who replied to the 6 month follow-up questionnaire were divided into groups based on their self-reported dominant treatment since the trial (probiotics/nutrition, standard treatment or no treatment) there was no difference in mood or other psychological outcomes among the groups at 6 months. The current trial found no evidence that this probiotic formulation is effective in treating the symptoms of depression or IBS, or in moderating the levels of inflammatory and other biomarkers in a sample recruited with moderate depression. This finding does not support the theory proposed in several narrative reviews which suggests probiotics as a possible intervention for depression and other mental health outcomes, but is supported by the systematic review of human probiotics studies presented in Chapter 3 which found overall limited evidence of probiotic efficacy for psychological outcomes. Future studies in the area should attempt to further broaden this field, in particular by recruiting samples with mild and/or non-chronic depression for interventional studies, or by approaching probiotics as a preventative or adjuvant treatment strategy for depression.

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of similar to 64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor beta (TGF-beta) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-beta signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated - omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-beta signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

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Various disorders closely linked to gut dysbiosis have been associated with poor dietary patterns. Dietary prebiotic fibers play an essential role in modulating the gut microbiome by enhancing the abundance of beneficial microorganisms and improving the production of short-chain fatty acids. Arabinoxylan (AX) is a major component of most dietary fibers and has been shown to exhibit potential prebiotic properties and modulate gut microbiome composition. This study aimed to investigate the in vitro impact of bioengineered wheat arabinoxylan on depression-inversely linked gut microbes and human gut microbiome diversity and metabolism. This study demonstrates the ability of bioengineered AX to stimulate the growth of depression-inversely linked gut bacterial species (Faecalibacterium prausnitzii and Lacticaseibacillus rhamnosus LGG). On the microbiome composition, the bioengineered AX induced an increased abundance of beneficial bacterial taxa (Bacteroides, Bifidobacterium, Anaerofustis, and Eubacterium) compared to the control and native AX. These effects on microbes translated into significant metabolic activity and produced primary SCFAs (acetate, butyrate, and propionate). The findings from this study suggest that bioengineered wheat arabinoxylan could be considered a promising strategy for fostering next-generation prebiotics targeting depression-inversely linked gut microbes and also supports the structure-function relationship between AX and the human gut microbiome.

<p dir="ltr">Dietary fibers have been observed to modulate the gut microbiome in ways that prevent and moderate human diseases and confer health benefits onto their human host. How dietary fibers do this is through their structure; gut microbes are equipped with a variety of differ- ent carbohydrate-active enzymes (CAZymes) that allow some to hydrolyze glycosidic bonds, thereby utilizing the dietary fiber. The more complex the dietary fiber, the more diverse the maintained gut microbiota may be, as specialist species may be required for complete hydrol- ysis. Therefore, increasing structural complexity of dietary fibers may increase gut microbial diversity and help prevent diseases. To understand if structural features impact the gut mi- crobiome, a set of resistant glucans varying in structures, including mixed-linkage -glucans, resistant maltodextrins (similar to type IV resistant starch) and polydextroses, which are comprised entirely of glucose, were used as substrates in an in vitro sequential batch fermen- tation using fecal microbiota form three healthy donors as inocula. I measured metabolic outputs, growth curves, and community structures by 16S rRNA amplicon sequencing, which I analyzed for through alpha and beta diversity differences and taxa that overrepresented and increased in each treatment. My results show that, depending on the donor and the resistant glucan, structure does significantly impact the concentrations of short-chain fatty acids (SCFAs) and other metabolites that are produced. Resistant glucan structure also impacts alpha and beta diversity to a degree and linear discriminant analysis (by LEfSe) results also support that specific species have preference towards substrates as well. Next, resistant glucans were supplemented into a high-fat diet, and compared these diets to a low- fat diet (LFD), high-fat diet with cellulose (HFD), and high-fat without cellulose (HWC) in a mouse study using C57BL/6J mice over 4 weeks. Increasing microbial diversity will not only increase diversity in the gut microbiome, but it will also provide protective effects in behavior such as helping to prevent anxiety. I measured weight, metabolic outputs, 16S community structure, changes in alpha and beta diversity, and differential abundances of OTUs and taxa by discriminant analysis Effect Size (LEfSe) and Metastats, and anxiety behaviors using open field and light/dark box tests. Microbial community structure was significantly different in treatment groups from controls. Anxiety for mice in tapioca dextrin 01 (TD01), tapioca dextrin 03 (TD03), and resistant maltodextrin (RMF) treatment groups were gen- erally increased, suggesting that the chemical structure of these resistant dextrins may alter the gut microbiome in ways that may influence behavior.</p><p dir="ltr">My overall results support the hypothesis that the fine structural features of dietary fibers do significantly impact the gut microbiome by selecting for specific microbiota, and may even impact cognition and behavior.</p>

[ES] En esta tesis, se realizaron análisis genómicos, metagenómicos y metabolómicos en líneas de conejo seleccionadas de forma divergente para alta y baja VE del tamaño de la camada (TC). Estos animales mostraron diferencias en su potencial de resiliencia. Por ello, estas poblaciones divergentes son un excelente material biológico para estudiar la resiliencia animal a través de la VE. Se realizaron estudios de asociación del genoma (GWAS) utilizando la regresión de un solo marcador y la regresión bayesiana de múltiples marcadores. Cuatro regiones genómicas se asociaron con la VE en el cromosoma 3 de Oryctolagus cuniculus (OCU), OCU7, OCU10 y OCU14, explicando el 8,6% de la varianza genética total para la VE. Además, el estudio de huellas de selección (SS) identificó 134 regiones genómicas que podrían estar bajo selección para la VE. El solapamiento entre ambos estudios se identificó en el OCU3, donde también se encontraron mutaciones funcionales para los genes DOCK2, INSYN2B y FOXI1. Los genes candidatos de GWAS y SS fueron aquellos con mutaciones funcionales identificadas mediante el análisis de secuenciación del genoma completo (WGS) con pools de ADN. Los genes candidatos destacados mostraron funciones biológicas relacionadas con el desarrollo de estructuras sensoriales, la respuesta inmunitaria, la respuesta al estrés y el sistema nervioso. Todas ellas son funciones relevantes para modular la resiliencia de los animales. Por otra parte, los estudios metagenómicos y metabolómicos mostraron que la selección para la VE modificó el microbioma intestinal y la composición de su metaboloma. Las especies microbianas beneficiosas como Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus y Limosilactobacillus fermentum eran más abundantes en la población resiliente. En cambio, las especies microbianas nocivas, como Acetatifactor muris y Eggerthella sp, fueron más abundantes en los animales no resistentes. Los genes relacionados con la formación de biofilms, el metabolismo de aminoácidos aromáticos (fenilalanina, triptófano y tirosina) y el metabolismo del glutamato también se expresaron de forma diferencial entre las poblaciones de conejos. Además, se identificaron 15 metabolitos intestinales como potenciales biomarcadores para discriminar y predecir adecuadamente entre las poblaciones de conejos resistentes y no resistentes. Cinco de ellos, el equol, el 3-(4-hidroxifenil)lactato, el 5-aminovalerato, la N6-acetilisina y la serina son metabolitos de origen microbiano. Este es el primer estudio que desvela importantes mecanismos biológicos de la resiliencia animal generada por la selección de la VE de TC. El genoma y el microbioma intestinal y la composición del metaboloma se modificaron a lo largo del proceso de selección, afectando a la respuesta inmunitaria y al estrés. Se encontraron resultados coincidentes entre los estudios metagenómicos y del metaboloma. Por otro lado, en esta tesis desarrollamos por primera vez una herramienta flexible para simular la coevolución del genoma y el microbioma a través de un proceso de selección. La clave de esta herramienta fue la implementación de la herencia del microbioma. Está construida en R y basada en AlphaSimR para que el usuario pueda modificar el código e implementar diferentes escenarios. Esta tesis es el primer paso para desarrollar futuras estrategias y nuevas investigaciones para mejorar la resiliencia de los animales. Una selección que combine información genómica y metagenómica puede mejorar la respuesta de selección. Además, los metabolitos derivados del intestino con evidencia de crosstalk pueden utilizarse como biomarcadores para identificar animales resilientes por plasma, evitando la extracción de muestras fecales para determinar la composición del microbioma. Si estos estudios tienen éxito, estas estrategias podrían mejorar la resiliencia de los animales con el objetivo de buscar un sistema ganadero más sostenible. / [CA] En aquesta tesi, es van realitzar anàlisis genòmiques, metagenòmiques i metabolòmiques en línies de conill seleccionades de manera divergent per a alta i baixa VE de la grandària de la ventrada (GV). Aquests animals van mostrar diferències en el seu potencial de resiliència. Per això, aquestes poblacions divergents són un excel·lent material biològic per a estudiar la resiliència animal a través de la VE. Es van realitzar estudis d'associació del genoma (GWAS) utilitzant la regressió d'un solo marcador i la regressió bayesiana de múltiples marcadors. Quatre regions genòmiques es van associar amb la VE en el cromosoma 3 de Oryctolagus cuniculus (OCU), OCU7, OCU10 i OCU14, explicant el 8,6% de la variància genètica total per a la VE. A més, l'estudi de petjades de selecció (SS) va identificar 134 regions genòmiques que podrien estar sota selecció per a la VE. El solapament entre tots dos estudis es va identificar en l'OCU3, on també es van trobar mutacions funcionals per als gens DOCK2, INSYN2B i FOXI1. Els gens candidats de GWAS i SS van ser aquells amb mutacions funcionals identificades mitjançant l'anàlisi de seqüenciació del genoma complet (WGS) amb pools d'ADN. Els gens candidats destacats van mostrar funcions biològiques relacionades amb el desenvolupament d'estructures sensorials, la resposta immunitària, la resposta a l'estrés i el sistema nerviós. Totes elles són funcions rellevants per a modular la resiliència dels animals. D'altra banda, els estudis metagenòmiques i *metabolòmiques van mostrar que la selecció per a la VE va modificar el microbioma intestinal i la composició de la seua metaboloma. Les espècies microbianes beneficioses com Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus i Limosilactobacillus fermentum eren més abundants en la població resilient. En canvi, les espècies microbianes nocives, com Acetatifactor muris i Eggerthella sp, van ser més abundants en els animals no resistents. Els gens relacionats amb la formació de biofilms, el metabolisme d'aminoàcids aromàtics (fenilalanina, triptòfan i tirosina) i el metabolisme del glutamat també es van expressar de manera diferencial entre les poblacions de conills. A més, es van identificar 15 metabòlits intestinals com a potencials biomarcadores per a discriminar i predir adequadament entre les poblacions de conills resistents i no resistents. Cinc d'ells, el equol, el 3-(4-hidroxifenil)lactat, el 5-aminovalerato, la N6-acetilisina i la serina són metabòlits d'origen microbià. Aquest és el primer estudi que revela importants mecanismes biològics de la resiliència animal generada per la selecció de la VE de GC. El genoma i el microbioma intestinal i la composició del metaboloma es van modificar al llarg del procés de selecció, afectant la resposta immunitària i a l'estrés. Es van trobar resultats coincidents entre els estudis metagenòmiques i del metaboloma. D'altra banda, en aquesta tesi desenvolupem per primera vegada una eina flexible per a simular la coevolució del genoma i el microbioma a través d'un procés de selecció. La clau d'aquesta eina va ser la implementació de l'herència del microbioma. Està construïda en R i basada en AlphaSimR perquè l'usuari puga modificar el codi i implementar diferents escenaris. Aquesta tesi és el primer pas per a desenvolupar futures estratègies i noves investigacions per a millorar la resiliència dels animals. Una selecció que combine informació genòmica i metagenòmique pot millorar la resposta de selecció. A més, els metabòlits derivats de l'intestí amb evidència de crosstalk poden utilitzar-se com biomarcadores per a identificar animals resilients per plasma, evitant l'extracció de mostres fecals per a determinar la composició del microbioma. Si aquests estudis tenen èxit, aquestes estratègies podrien millorar la resiliència dels animals amb l'objectiu de buscar un sistema ramader més sostenible. / [EN] Disclosing the biological mechanisms of the VE can help to gain some insight into the biological basics of animal resilience. In this thesis, genomic, metagenomic, and metabolomic analyses were performed on rabbit lines divergently selected for high and low VE of litter size (LS). These animals showed differences in their resilience potential. Thus, these divergent populations are an excellent biological material for studying animal resilience through the VE. Genome-wide association studies (GWAS) were performed using single marker regression, and Bayesian multiple marker regression approaches. Four genomic regions were associated with the VE in the Oryctolagus cuniculus chromosome (OCU) 3, OCU7, OCU10, and OCU14, explaining 8.6% of the total genetic variance for the VE. In addition, the signature of selection (SS) study identified 134 genomic regions which could be under selection for VE. Overlapping between both studies was placed in the OCU3, where functional mutations for the DOCK2, INSYN2B and FOXI1 genes were also found. Candidate genes from GWAS and SS were those with functional mutations identified using whole genome sequencing (WGS) analysis with pools of DNA. Highlighted candidate genes showed biological functions related to the development of sensory structures, the immune response, the stress response, and the nervous system. All of them are relevant functions to modulate animal resilience. On the other hand, metagenomic and metabolomic studies showed that the selection for VE modified the gut microbiome and metabolome composition. Beneficial microbial species such as Alistipes prutedinis, Alistipes shahii, Odoribacter splanchnicus and Limosilactobacillus fermentum were more abundant in the resilient population. In contrast, harmful microbial species such as Acetatifactor muris and Eggerthella sp were more abundant in the non-resilient animals. Genes related to biofilm formation, aromatic amino acid metabolism (Phenylalanine, tryptophan, and tyrosine), and glutamate metabolism were also differentially expressed between the rabbit populations. Furthermore, 15 gut metabolites were identified as potential biomarkers to properly discriminate and predict between the resilient and non-resilient rabbit populations. Five of them, the equol, 3-(4-hydroxyphenyl)lactate, 5-aminovalerate, N6-acetyllisine, and serine were microbial-derived metabolites. This is the first study unravelling important biological mechanisms under the animal resilience generated by VE of LS selection. Genome and gut microbiome and metabolome composition were modified throughout the selection process, affecting the immune and stress response. Overlapping results were found between the metagenomic and metabolome studies. On the other hand, in this thesis, we developed a flexible tool for simulating the coevolution of the genome and microbiome across a selection process for the first time. The key of this tool was the implementation of the microbiome inheritance. It is constructed in R and based on AlphaSimR so the user can modify the code and implement different scenarios. This thesis is the first step to develop future strategies and further research to improve animal resilience. A selection combining genomic and metagenomic information may improve the selection response. Moreover, gut-derived metabolites with evidence of crosstalk can be used as biomarkers to identify resilient animals by plasma, avoiding the extraction of faecal samples to determine the microbiome composition. If these studies suceed, these strategies could improve animal resilience with the aim of search a more sustainable livestock system. Lastly, the simulation tool developed could help unravel the microbiome's implications in animal breeding programs. / This study was supported by projects AGL2014-5592, C2-1-P and C2-2-P, and AGL2017-86083, C2-1-P and C2-2-P, funded by the Spanish Ministerio de Ciencia e Innovación (MIC)-Agencia Estatal de Investigación (AEI) and the European Regional Development Fund (FEDER). Projects PID2020-115558GB-C21, funded by the Spanish Ministerio de Ciencia e Innovación (MIC)-Agencia Estatal de Investigación (AEI) and the European Regional Development Funds (FEDER) FPU17/01196 scholarship from the Spanish Ministry of Science, Innovation and Universities. / Casto Rebollo, C. (2023). Unravelling the Environmental Variance of Litter Size Through the Genome and Gut Microbiome [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192460

The composition of gut microbes affects host weight, immune function, and disease status, and is sensitive to diet, environment, and pharmaceutical exposure. The gut microbiome modulates the toxicity and bioavailability of chemical stressors, however the effects of chemicals on the gut microbiome of aquatic biota are largely unknown. The Waterloo and Kitchener wastewater treatment plants (WWTPs) release effluents containing antibiotics, pharmaceuticals, and other contaminants into the Grand River (ON) that may negatively affect the gut microbiome of downstream organisms. In this study done in Fall 2018, I collected freshwater mussels (Lasmigona costata), several species of insect larvae, and riparian spiders (Tetragnathidae) from sites upstream and downstream of these WWTPs. The gut microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16S rRNA genetic barcode. Changes in the relative abundance of major gut microbiome phyla were observed in all targeted aquatic organisms downstream of WWTPs except Hydropsychidae. Shannon alpha diversity, a measure of bacterial abundance and evenness, differed significantly among sites for mussels (one-way ANOVA: F=7.894, p=0.001), spiders (F=4.788, p=0.01), Perlidae (F=3.1, p=0.0056), Hydropsychidae (F=3.674, p=0.0014), and Heptageniidae (F=2.715, p=0.0143), but not for Baetidae and Ephemerellidae. In sites downstream of the Waterloo WWTP, alpha diversity decreased in spiders, while in sites downstream of the Kitchener WWTP diversity decreased in mussels and Perlidae, while increasing for spiders. Bray-Curtis beta diversity, a measure of dissimilarity between bacterial communities, was significantly dissimilar among sites in all invertebrate taxa (Permanova: p<0.02). Upstream sites differed from downstream Waterloo sites in spiders, Perlidae, and Hydropsychidae (Adonis pairwise: p<0.05), while upstream mussels, spiders, Perlidae, and Hydropsychidae differed from downstream Kitchener sites (p<0.05). Additionally, effluent-derived bacteria were found in the microbiomes of aquatic invertebrates downstream of the WWTPs and not upstream. Taxa was also a significant driver of bacterial composition and diversity in invertebrates. These results indicate that the gut microbiome of downstream organisms differed from the bacterial composition observed in the same invertebrate taxa upstream of the WWTPs, potentially leading to altered host health. This adds to our understanding of how chemical stressors impact the gut microbiome of aquatic and riparian biota; however, future studies are needed to investigate linkages between the gut microbiome and health of these species. / Thesis / Master of Science (MSc)