Microbiota en salmón del atlántico: Evaluación del rol de la comunidad microbiana en peces de cultivo y su relación con la salud durante el ciclo productivo.
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Date
2022
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Universidad de Concepción.
Abstract
Existe una creciente preocupación en garantizar el bienestar animal en la acuicultura. A tal efecto, la microbiota intestinal es un campo prometedor para evaluar y mejorar este bienestar. Lo anterior debido a que posee una gran relevancia en el hospedero influenciando, por ejemplo, el sistema digestivo e inmune. En ese sentido, además de la taxonomía, ha crecido el interés por conocer la funcionalidad metabólica de las comunidades microbianas, debido a que ofrece
ventajas al momento de evaluar el efecto sobre los cambios en el entorno. El objetivo de esta tesis fue establecer si la estructura taxonómica y funcional de la comunidad microbiana en salmón del Atlántico brinda herramientas para evaluar el bienestar animal durante el proceso productivo. Para este trabajo, se utilizó la secuenciación de la región V1-V9 del gen del ARNr 16S mediante Nanopore MinION. Posterior a ello, la asignaciones taxonómicas se realizaron mediante la plataforma “Epi2Me de Oxford Nanopore” o por la herramienta “NanoCLUST”. Además, para el cálculo de diversidad taxonómica se utilizó el paquete “Vegan” en el programa “R studio”, con el cual se calculó la diversidad alfa y la estimación de uniformidad por el índice de Pielou. La estructura de la comunidad microbiana se analizó utilizando Bray-Curtis con un análisis de coordenadas principales (PCoA). Por último, la predicción funcional del metagenoma se realizó mediante PICRUSt2 utilizando la bases de datos MetaCyc. Para el primer análisis, se determinó la
taxonomía de muestras de intestino de salmón del Atlántico con distención abdominal (DA), sin distención abdominal (no_DA) y muestras de agua de la Bocatoma (Boca), decantador (Deca), entradas y salidas del biofiltro (BF) de un sistema de recirculación acuícola (RAS) de agua dulce. Esto para identificar la microbiota en peces afectados y enfermos durante un brote epidémico posterior a un evento de vacunación. Las muestras de intestino de los peces con (DA) fueron
las que menores índices de diversidad presentaron, junto con una dominancia de Proteobacteria. Además, Aliivibrio wodanis es el principal patógeno presente en los peces con DA y un probable agente etiológico de los peces enfermos. Por otro lado, la microbiota de los peces no_DA poseen una mayor contribución en los pathway relacionados con metabolismo de aminoácidos y fermentación de ácidos grasos de cadena corta, lo que es indicativo de una mejor salud con respecto a los peces con DA. Adicionalmente, se determinó la contribución al metabolismo de compuestos inorgánicos utilizando la base de datos de “KEGG Ortology” para la construcción de
mapas metabólicos con la herramienta “KEGG Mapper” enfocados en el metabolismo del nitrógeno y sulfuro. Como resultado, encontramos que el metabolismo del sulfuro es el más representado en peces DA. Además, los procesos de desnitrificación y nitrificación se encuentran favorecidos en la muestra de agua Deca, por sobre las otras, lo que tiene estrecha relación con el rol que posee. Posterior a esto, se evaluó la taxonomía de smolts durante su transferencia
al agua de mar bajo diferentes tratamientos y se analizó la metagenómica funcional de la microbiota del intestino de los peces. Esto incluyó un grupo de muestreo sometido a la alimentación mediante una dieta funcional (FD) previo al proceso de transferencia al agua de mar. Todo lo anterior, con el objetivo de evaluar si un cambio gradual en la salinidad (GSC) previo a la transferencia de agua de mar favorece la diversidad de la microbiota por sobre un shock osmótico (SS). Como resultado, encontramos que la riqueza y diversidad microbiana fueron mayores en los peces sometidos a GSC, lo que sugiere una asociación positiva entre la comunidad microbiana y la salud de los peces. Además, se hacen presentes bacterias del género Vibrio en las muestras de intestino de los peces en agua de mar que no estaban presentes en agua dulce ni durante el GSC. Además, los peces alimentados con FD previo a un choque salino, presentan menos abundancia de este género. En cuanto a la funcionalidad metabólica, la biosíntesis de aminoácidos como valina, leucina e isoleucina se encuentran favorecidos en los peces previamente alimentados con FD. Por otro lado, la degradación de aminoácidos esenciales está favorecida en los peces alimentados con FD y en los sometidos a un SS. Por otro lado, la degradación de drogas, contaminantes y otros compuestos aromáticos se encuentran favorecidos en los peces alimentados con FD y en los peces sometidos GSC. Para finalizar, se estudió la microbiota de Caligus rogercresseyi de diferentes zonas del sur de Chile, como potencial reservorio de patógenos que pueden afectar al salmón del Atlántico. Para ello, de los resultados
taxonómicos, se filtraron aquellos que corresponden a bacterias patógenas de peces y se determinaron los índices de diversidad. Se identificaron patógenos pertenecientes al género Tenacibaculum. Adicionalmente, la abundancia y diversidad de patógenos está directamente relacionada con la biomasa de salmón producida en cada una de las zonas. En resumen, el conocimiento de la estructura taxonómica y funcional de la microbiota del intestino nos permite evaluar que peces poseen un mejor estado de salud con respecto a otros. Brindándonos una
herramienta para reconocer signos de enfermedad. Además, nos permite evaluar la calidad microbiológica del agua y nos brinda información sobre la microbiota de patógenos potenciales totales, tanto en el agua como en otros parásitos como C. rogercresseyi.
There is a growing concern to ensure this animal welfare in aquaculture. To this effect, intestinal microbiota is a promising field to evaluate and improve welfare. This is because it has great relevance in the host influencing, for example, the digestive and immune systems. In this sense, in addition to taxonomy, there has been a growing interest in knowing the metabolic functionality of microbial communities, because it offers advantages when evaluating the effect of changes in the environment. The objective of this thesis was to establish whether the taxonomic and functional structure of the microbial community in Atlantic salmon provides us with tools to evaluate animal welfare during the production process. For this work, the sequencing of the V1-V9 16S rRNA region using Nanopore MinION was used. Subsequently, taxonomic assignments were made using the "Epi2Me de Oxford Nanopore" platform or the "NanoCLUST" tool. In addition, the "Vegan" package in the "R studio" program was used to calculate the alpha diversity and the estimation of uniformity by the Pielou index. The microbial community structure was analyzed using Bray-Curtis with a principal coordinates analysis (PCoA). Finally, functional prediction of the metagenome was performed by PICRUSt2 using the MetaCyc database. For the first analysis, the taxonomy of gut samples from Atlantic salmon with abdominal distension (AD), without abdominal distension (no_AD) and water samples from the intake (Mouth), decanter (Deca), inlets and outlets of the biofilter (BF) of a freshwater aquaculture recirculation system (RAS) was determined. This was to identify the microbiota in affected and diseased fish during an epidemic outbreak following a vaccination event. Gut samples from fish with (DA) yielded the lowest diversity indices, along with a dominance of Proteobacteria. In addition, Aliivibrio wodanis is the main pathogen present in DA fish and a probable etiological agent of diseased fish. On the other hand, the microbiota of non-AD fish have a higher contribution in pathways related to amino acid metabolism and short-chain fatty acid fermentation, which is indicative of better health compared to AD fish. Additionally, the contribution to the metabolism of inorganic compounds was determined using the "KEGG Orthology" database for the construction of metabolic maps with the "KEGG Mapper" tool focused on nitrogen and sulfur metabolism. As a result, we found that sulfide metabolism is the most represented in DA weights. In addition, denitrification and nitrification processes are found to be favored in the DECA water sample over the others. This is closely related to the role it plays. Subsequently, the taxonomy of smolts during their transfer to seawater under different treatments was evaluated and, the functional metagenomics of the gut microbiota of the fish was analyzed. This included a sampling group subjected to feeding with a functional diet (FD) prior to the transfer process to seawater. The objective was to evaluate whether a gradual change in salinity (GSC) prior to seawater transfer favors microbiota diversity over an osmotic shock (SS). As a result, we found that microbial richness and diversity were higher in fish subjected to GSC, suggesting a positive association between microbial community and fish health. In addition, bacteria of the genus Vibrio are present in the gut samples of fish in seawater that were not present in freshwater or during GSC. In addition, fish feeding with a FD prior to salt shock show less abundance of this genus. In terms of metabolic functionality, the biosynthesis of amino acids such as valine, leucine and isoleucine are found to be favored in fish previously feeding with a FD. On the other hand, the degradation of essential amino acids is favored feeding with a FD and SS fish. On the other hand, the degradation of drugs, contaminants and other aromatic compounds is favored in FD-fed fish and in fish subjected to GSC. Finally, the microbiota of Caligus rogercresseyi from different areas of southern Chile was studied as a potential reservoir of pathogens that can affect Atlantic salmon. For this purpose, from the taxonomic results, those that correspond to fish pathogenic bacteria were filtered and the diversity indexes were determined. As a result, pathogens belonging to the genus Tenacibaculum were identified. Additionally, the abundance and diversity of pathogens is directly related to the biomass of salmon produced in each of the zones. In summary, knowledge of the taxonomic and functional structure of the gut microbiota allows us to evaluate which fish are healthier than others. This provides us with a tool to recognize signs of disease. It also allows us to evaluate the microbiological quality of the water and provides us with information on the microbiota of potential total pathogens, both in the water and in other parasites such as Caligus rogercresseyi.
There is a growing concern to ensure this animal welfare in aquaculture. To this effect, intestinal microbiota is a promising field to evaluate and improve welfare. This is because it has great relevance in the host influencing, for example, the digestive and immune systems. In this sense, in addition to taxonomy, there has been a growing interest in knowing the metabolic functionality of microbial communities, because it offers advantages when evaluating the effect of changes in the environment. The objective of this thesis was to establish whether the taxonomic and functional structure of the microbial community in Atlantic salmon provides us with tools to evaluate animal welfare during the production process. For this work, the sequencing of the V1-V9 16S rRNA region using Nanopore MinION was used. Subsequently, taxonomic assignments were made using the "Epi2Me de Oxford Nanopore" platform or the "NanoCLUST" tool. In addition, the "Vegan" package in the "R studio" program was used to calculate the alpha diversity and the estimation of uniformity by the Pielou index. The microbial community structure was analyzed using Bray-Curtis with a principal coordinates analysis (PCoA). Finally, functional prediction of the metagenome was performed by PICRUSt2 using the MetaCyc database. For the first analysis, the taxonomy of gut samples from Atlantic salmon with abdominal distension (AD), without abdominal distension (no_AD) and water samples from the intake (Mouth), decanter (Deca), inlets and outlets of the biofilter (BF) of a freshwater aquaculture recirculation system (RAS) was determined. This was to identify the microbiota in affected and diseased fish during an epidemic outbreak following a vaccination event. Gut samples from fish with (DA) yielded the lowest diversity indices, along with a dominance of Proteobacteria. In addition, Aliivibrio wodanis is the main pathogen present in DA fish and a probable etiological agent of diseased fish. On the other hand, the microbiota of non-AD fish have a higher contribution in pathways related to amino acid metabolism and short-chain fatty acid fermentation, which is indicative of better health compared to AD fish. Additionally, the contribution to the metabolism of inorganic compounds was determined using the "KEGG Orthology" database for the construction of metabolic maps with the "KEGG Mapper" tool focused on nitrogen and sulfur metabolism. As a result, we found that sulfide metabolism is the most represented in DA weights. In addition, denitrification and nitrification processes are found to be favored in the DECA water sample over the others. This is closely related to the role it plays. Subsequently, the taxonomy of smolts during their transfer to seawater under different treatments was evaluated and, the functional metagenomics of the gut microbiota of the fish was analyzed. This included a sampling group subjected to feeding with a functional diet (FD) prior to the transfer process to seawater. The objective was to evaluate whether a gradual change in salinity (GSC) prior to seawater transfer favors microbiota diversity over an osmotic shock (SS). As a result, we found that microbial richness and diversity were higher in fish subjected to GSC, suggesting a positive association between microbial community and fish health. In addition, bacteria of the genus Vibrio are present in the gut samples of fish in seawater that were not present in freshwater or during GSC. In addition, fish feeding with a FD prior to salt shock show less abundance of this genus. In terms of metabolic functionality, the biosynthesis of amino acids such as valine, leucine and isoleucine are found to be favored in fish previously feeding with a FD. On the other hand, the degradation of essential amino acids is favored feeding with a FD and SS fish. On the other hand, the degradation of drugs, contaminants and other aromatic compounds is favored in FD-fed fish and in fish subjected to GSC. Finally, the microbiota of Caligus rogercresseyi from different areas of southern Chile was studied as a potential reservoir of pathogens that can affect Atlantic salmon. For this purpose, from the taxonomic results, those that correspond to fish pathogenic bacteria were filtered and the diversity indexes were determined. As a result, pathogens belonging to the genus Tenacibaculum were identified. Additionally, the abundance and diversity of pathogens is directly related to the biomass of salmon produced in each of the zones. In summary, knowledge of the taxonomic and functional structure of the gut microbiota allows us to evaluate which fish are healthier than others. This provides us with a tool to recognize signs of disease. It also allows us to evaluate the microbiological quality of the water and provides us with information on the microbiota of potential total pathogens, both in the water and in other parasites such as Caligus rogercresseyi.
Description
Tesis para optar al Grado de Doctor en Ciencias con Mención en Microbiología.
Keywords
Microbiología, Fauna marina, Industria salmonera, Piscicultura