The Use of Bioinformatic Tools in Symbiosis and Co-Evolution Studies

Through millions of years, the multicellular organisms have coexisted and coevolved with the surrounding microorganisms, in an almost symbiotic relationship forming a complex entity known as holobiont. The composition and functions of these microbial communities were limited during many years to only a mere fraction, due to the use of culture-based techniques. The advent of molecular-based techniques allowed the identification of uncultured organisms in a culture-free manner. In recent years, the development of next generation sequencing techniques have allowed the high-throughput study of microbial communities allowing the identification and classification of otherwise uncultured microorganisms in a given environment, tissue or host through metagenomics. The next generation sequencing techniques have been used in the functional study of microbial assemblages and were able to identify the role of the microorganisms in biogeochemical cycles, pathogenic processes, metabolism and development, through metatranscriptomics. Taken together, the next generation sequencing based-studies have shown the existence of a complex metabolic network in different hosts and environments, with the microbial communities. This chapter will focus in different available bioinformatic tools that are suitable to study symbiosis and coevolution processes in a given sample

RNA-Seq Analysis on the Microbiota Associated with the White Shrimp (Litopenaeus vannamei) in Different Stages of Development

White leg shrimp (Litopenaeus vannamei) is a widely cultured species along the Pacific coast and is one of the most important crustaceans in world aquaculture. The microbiome composition of L. vannamei has been previously studied in different developmental stages, but there is limited information regarding the functional role of the microbiome during the development of L. vannamei. In this study the metatranscriptome in different developmental stages of L. vannamei (larvae, juvenile and adult) were generated using next generation sequencing techniques. The bacterial phyla found throughout all the stages of development belonged to the Proteobacteria, Firmicutes and Actinobacteria, these bacterial phyla are present in the digestive tract and are capable of producing several hydrolytic enzymes, which agrees with high representation of the primary metabolism and energy production, in both host and the microbiome. In this sense, functional changes were observed as the development progressed, in both host and the microbiome, in stages of larvae the most represented metabolic functions were associated with biomass production; while in juvenile and adult stages a higher proportion of metabolic functions associated to biotic and abiotic stress in L. vannamei and the microbiome were shown. This study provides evidence of the interaction of the microbiome with L. vannamei, and how the stage of development and the culture conditions of this species influences the gene expression and the microbiome composition, which suggests a complex metabolic network present throughout the life cycle of L. vannamei

RNA-Seq Analysis on the Microbiota Associated with the White Shrimp (<i>Litopenaeus vannamei</i>) in Different Stages of Development

White leg shrimp (Litopenaeus vannamei) is a widely cultured species along the Pacific coast and is one of the most important crustaceans in world aquaculture. The microbiome composition of L. vannamei has been previously studied in different developmental stages, but there is limited information regarding the functional role of the microbiome during the development of L. vannamei. In this study the metatranscriptome in different developmental stages of L. vannamei (larvae, juvenile and adult) were generated using next generation sequencing techniques. The bacterial phyla found throughout all the stages of development belonged to the Proteobacteria, Firmicutes and Actinobacteria, these bacterial phyla are present in the digestive tract and are capable of producing several hydrolytic enzymes, which agrees with high representation of the primary metabolism and energy production, in both host and the microbiome. In this sense, functional changes were observed as the development progressed, in both host and the microbiome, in stages of larvae the most represented metabolic functions were associated with biomass production; while in juvenile and adult stages a higher proportion of metabolic functions associated to biotic and abiotic stress in L. vannamei and the microbiome were shown. This study provides evidence of the interaction of the microbiome with L. vannamei, and how the stage of development and the culture conditions of this species influences the gene expression and the microbiome composition, which suggests a complex metabolic network present throughout the life cycle of L. vannamei

Identification of Sorghum (<i>Sorghum bicolor</i> (L.) Moench) Genotypes with Potential for Hydric and Heat Stress Tolerance in Northeastern Mexico

Sorghum (Sorghum bicolor (L.) Moench) is cultivated in regions with frequent drought periods and high temperatures, conditions that have intensified in the last decades. One of the most important photosynthetic components, sensible to hydric stress, is maximum quantum yield for photosystem II (PSII, or Fv/Fm). The objective of the present study was to identify sorghum genotypes with tolerance to hydric and heat stress. The treatments were hydric status (hydric stress or non-hydric stress (irrigation)), the plant’s developmental stages (pre or post-anthesis), and six genotypes. The response variables were Fv/Fm; photosynthetic rate (PN); stomatal conductance (gs); transpiration rate (E); relative water content (RWC); damage to cell membrane (DCM) at temperatures of 40 and 45 °C; and agronomic variables. The experiment was conducted in pots in open sky in Marín, N.L., in the dry and hot northeast Mexico. The treatment design was a split–split plot design, with three factors. Hydric stress diminished the functioning of the photosynthetic apparatus by 63%, due to damage caused to PSII. Pre-anthesis was the most vulnerable stage to hydric stress as it decreased the weight of grains per panicle (85%), number of grains per panicle (69%), and weight of 100 grains (46%). Genotypes LER 1 and LER 2 were identified as tolerant to hydric stress, as they had lower damage to PSII; LER 1 and LEB 2 for their superior RWC; and LER 1 as a thermo tolerant genotype, due to its lower DCM at 45 °C. It was concluded that LER 1 could have the potential for both hydric and heat stress tolerance in the arid northeast Mexico

A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity

Plants have evolved defense mechanisms to suppress viral transcription and replication by transcriptional and post-transcriptional gene silencing mediated by virus-derived small interfering RNAs (vsiRNAs). Based on this response, virus-induced gene silencing (VIGS)-based technology has been developed to silence target genes on either host plants or insect pests. This mechanism could also be used for the silencing of genes of interest in the medical field. We used the VIGS vector pEuMV-YP:Krt18, which was obtained by inserting the Mus musculus (M. musculus) Krt18 sequence into pEuMV-YP:&Delta;AV1. The objective was to evaluate the capacity of pEuMV-YP:Krt18 to induce Nicotiana benthamiana (N. benthamiana) production of vsiRNAs of a specific sequence that belongs to neither the plant genome nor the wild virus genome, which were used to induce cross-kingdom gene silencing between plants and mammals. The percentage of vsiRNA for each viral gene was calculated from an sRNA library of N. benthamiana plants infected by pEuMV-YP: Krt18. When the vsiRNAs were characterized, it was found that they corresponded to all the genes of the pEuMV-YP:Krt18 vector. These vsiRNAs induced the silencing of the Krt18 gene in M. musculus macrophages, supporting the ability to use VIGS vectors in plants as biofactories for the production of sRNAs that induce gene silencing in mammals