Estructura, composición y potencial genético para degradar celulosa de la microbiota intestinal delescarabajo veturiussp. (coleoptera: passalidae)

The global carbon cycle is responsible for maintaining the carbon dioxide and methane concentration in the atmosphere, influencing the weather and ocean acidification. Herbivores play an important role in the carbon cycle. However, the microorganisms that inhabit their guts are the ones responsible for the cellulose breakdown and consequent release of greenhouse gases such as CO2 and methane. Here we studied the gut microbiome of the Passalid beetle Veturius sp., from Braulio Carrillo National Park, Costa Rica. Veturius sp. only feed on decay wood and presents a subsocial behavior that may lead to the acquisition and sharing of microbial symbionts for efficient biomass and energy production. Family groups from different logs in the forest were sampled and the gut microbiome of larvae and adults as well as the woody gallery material (substrate) in which they resided was analyzed. The structure and composition of the communities was determined using amplicon sequencing of 16S rRNA genes. These results were then used in order to select the samples to perform metagenomic sequencing for further functional analysis and genome reconstruction. The results showed that adult, larvae and gallery material harbor significantly different communities, sharing less than 3% of total OTUs (total OTUs in the system= 11 712), with the gallery woody substrate having the higher diversity (3178 observed species) and richness (chao=6714). Firmicutes and Euryarchaeota were the dominant phyla in adults and larvae gut. The most abundant families of Firmicutes included Clostridiaceae, Lachnospiraceae and Ruminococcaceae, all known for its cellulose degradation capacity. A total of 766 partial genomes were reconstructed using the metagenomic sequences from adult, larvae and substrate; 101 of were classified as metagenome assembled genomes (MAG). Larvae and adults are enriched in microorganism with genomes having a myriad of glycosyl hydrolases, and other functions related to carbon metabolism; furthermore, methanogenesis markers were found in the larval partial genomes, suggesting that those performed the final steps of cellulose decomposition. Finally, the Veturius sp. metagenomes were compared with datasets from other cellulolytic systems, the results showed evidence of convergent evolution of functions between the larvae and cow rumen, suggesting that Veturius sp. and other beetle larvae are unexplored, yet important contributors to the carbon cycle and the biotic production of the greenhouse gas methane. The Passalid microbiome is important to the ecology and physiology of these beetles.UCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Salud::Maestría Académica en Microbiologí

The genomic basis of army ant chemosensory adaptations

The evolution of mass raiding has allowed army ants to become dominant arthropod predators in the tropics. Although a century of research has led to many discoveries about behavioural, morphological and physiological adaptations in army ants, almost nothing is known about the molecular basis of army ant biology. Here we report the genome of the iconic New World army ant Eciton burchellii, and show that it is unusu-ally compact, with a reduced gene complement relative to other ants. In contrast to this overall reduction, a particular gene subfamily (9-exon ORs) expressed predomi-nantly in female antennae is expanded. This subfamily has previously been linked to the recognition of hydrocarbons, key olfactory cues used in insect communication and prey discrimination. Confocal microscopy of the brain showed a correspond-ing expansion in a putative hydrocarbon response centre within the antennal lobe, while scanning electron microscopy of the antenna revealed a particularly high den-sity of hydrocarbon-sensitive sensory hairs. E. burchellii shares these features with its predatory and more cryptic relative, the clonal raider ant. By integrating genomic, transcriptomic and anatomical analyses in a comparative context, our work thus pro-vides evidence that army ants and their relatives possess a suite of modifications in the chemosensory system that may be involved in behavioural coordination and prey selection during social predation. It also lays the groundwork for future studies of army ant biology at the molecular level.National Science Foundation/[NSF IOS 1916995]/NSF/Estados UnidosNational Science Foundation/[NSF DEB 1900357]/NSF/Estados UnidosUniversity of Wisconsin-Madison/[BE 5177/4-1]//Estados UnidosUniversidad de Costa Rica/[810-B3-273]/UCR/Costa RicaNational Institutes of Health/[GM066699]/NIH/Estados UnidosMarie Skłodowska-Curie Individual Fellowship/[ID 797969]/MSCA IF/BélgicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Molecular and Environmental Controls on Aerobic Anoxygenic Phototrophy

Aerobic anoxygenic phototrophy (AAP) is a metabolic process found in diverse aerobic proteobacteria across aquatic environments. Unlike classical anoxygenic photosynthetic bacteria, the bacteria that perform AAP are often obligate aerobes and are thought to use this pathway to supplement their primarily heterotrophic metabolism. The environmental and molecular factors that control AAP, however, are poorly understood. Using the model marine organism Erythrobacter longus, and the recently isolated freshwater strain Porphyrobacter LM6, we investigated the metabolic pathways and regulatory mechanisms that interact with AAP. First, we constructed deletion mutants of E. longus for several genes involved in light harvesting and the glyoxylate shunt pathway. By comparing the growth of wild type and mutants we demonstrate that light enhanced the growth of wild-type E. longus on pyruvate, glucose and butyrate minimal medium, but not in rich medium; and that the enhanced growth was product of the absorption of energy from light. We discarded that the glyoxylate shunt as the metabolic pathway responsible for light enhanced growth in E. longus, yet we confirmed that the shunt is the only for acetate metabolism in this strain and possibly other AAP strains from the order Sphingomonadales. Next, we used global transposon mutagenesis to assess gene fitness under varying nutritional conditions in Porphyrobacter LM6. The mutant libraries were grown on two different carbon sources (glucose and butyrate) in two different light regimes: 24h dark, and 14h:10h light:dark cycles. As expected, genes in central carbon metabolism had differential fitness effects in butyrate vs. glucose. Notably, the glyoxylate shunt genes isocitrate lyase and malate synthase, along with the anaplerotic carbon assimilation gene malic enzyme, appear to be important on butyrate but not glucose. We next examined the role of phototrophy. Light provided a growth advantage to wild-type cells grown in glucose but had no effect in butyrate. Consistent with this, genes encoding pigment biosynthesis and photosystem machinery were not important for fitness in butyrate, but had strong fitness effects in cells grown in the light with glucose. We determined that the anapleortic reactions performed by phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase facilitate the light enhanced growth observed in glucose. Catalase/peroxidase and the oxidative stress response regulator oxyR had strong fitness effects in both butyrate and glucose, implying that ROS are a strong selective pressure for this organism. We also demonstrated that ppsR is a key regulator of phototrophy using a targeted gene knockout. These results suggest that the regulation of carbon metabolism and phototrophy are intertwined, and that, surprisingly, phototrophy is advantageous on glucose but not butyrate. Further, ROS detoxification appears to be a key pathway for survival of AAP bacteria. To further explore the impact of ROS in the physiology of AAP, we used another transposon library in E. longus. The production of bacteriochlorophyll-a (Bchla) in the presence of oxygen produces reactive oxygen species (ROS) that are detrimental for their survival in the environment. Yet, the mechanisms used by these bacteria to regulate phototrophic metabolism and overcome the effects of oxidative stress are not fully understood. As expected, we found that superoxide dismutase and catalase are important enzymes against reactive oxygen species (ROS). Mutants deficient in carotenoid biosynthesis also had low fitness under increased oxidative stress, confirming their photoprotective role in AAP bacteria. Glutathione-based systems for repairing ROS damage are vital for the survival of E. longus, as the enzymes glutathione synthase and glutathione peroxidase are required for growth. Mutants of the transcriptional regulator oxyR presented some of the lowest fitness suggesting its role as major regulator in response to oxidative stress. The mutants of catalase and glutathione reductases showed similar fitness patterns to oxyR regulon suggesting that these could be part of its regulon in E. longus. Taken together, our results demonstrate that E. longus, and likely other AAP strains, use a combination of enzymatic mechanisms and photoprotective carotenoids against reactive oxygen species (ROS). Together, our genetics and physiology results shed new light on a widespread and ecologically important metabolic strategy in aquatic systems

Tolerance evaluation of celery commercial cultivars and genetic variability of Fusarium oxysporum f. sp. apii

Celery is affected by several plant diseases such as Fusarium oxysporum f. sp. apii (Foa). Four races have been found in the US. Our goal was to find which races are present in Costa Rica using a multigene phylogenetic analysis and to quantify the tolerance of the imported commercial cul-tivars of celery. Isolates from 125 celery symptomatic plants from three different geographical locations were analyzed and 65 isolates were selected to perform the phylogenetic analysis. All isolates present the five nucleotides which differentiate Foa race 3 from the other races in the IGS rDNA region. Three different haplotypes close to race 3 were found, all of them were highly virulent, produced high losses, and affected all cultivars of imported commercial celery, which are genetically improved for resistance to races 2 and 4. Additionally, five different cultivars of commercial celery were evaluated against seven pathogen isolates identified as race 3 in green-house conditions. Two of the cultivars showed significantly less chlorosis, wilting, mortality, and higher fresh weight. Most of the Foa isolates significantly increased chlorosis, wilting and mor-tality compared to non-inoculated control. Celery producers in Costa Rica lack a seed that could offer total resistance to the Foa race 3 present in the country.Universidad de Costa Rica/[813-B1-006]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro de Investigación en Protección de Cultivos (CIPROC)UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomí

Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components

<div><p>Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus <i>Streptomyces</i> (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (<i>Anthostomella</i> sp.), two <i>Streptomyces</i> and one <i>Bacillus</i> bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.</p></div

Distribution of enzymatic activities of microbial isolates.

a<p>Lignin peroxidase activity in MEA-RBBR.</p>b<p>Cellulase activity in CMC agar.</p>c<p>Laccase activity on ABTS test.</p>d<p>Glucanase activity.</p>e<p>Xylanase activity.</p>f<p>Cellobiase activity.</p><p>*Taxonomic identification was not possible with the molecular markers available.</p><p>Distribution of enzymatic activities of microbial isolates.</p

Pattern of enzymatic degradation of plant cell wall components for the fungal and bacterial isolates obtained from Passalid beetles.

<p>Heat map of the enzymatic activity of fungi, eubacteria and actinomycetes by sample type (blue represents the lower activity whereas red represents higher activity on the assay). Numbers correspond to sample number. RBBR: Lignin peroxidase activity in MEA-RBBR. CMC: Cellulase activity in CMC agar. LAC: Laccase activity on ABTS test. G: Glucanase activity. X: Xylanase activity. C: Cellobiase activity.</p

Bayesian phylogenetic tree of the ITS region sequences of fungi isolated from the gut of Costa Rican beetles.

<p>Numbers above branches represent their Bayesian-calculated posterior probabilities (two million generations, chain temperature  = 0.2, standard deviation <0.02). Numbers between brackets represent the number of isolates that shared the same sequence. Compress branches are colored in green. Red diamonds indicates positive cellulolytic activity.</p

Bayesian phylogenetic tree of the rDNA 16S gene sequences of actinomycetes isolated from the gut of Costa Rican beetles.

<p>Numbers above branches represent their Bayesian-calculated posterior probabilities (two million generations, chain temperature  = 0.2, standard desviation <0.02). Compress branches are colored in green. Red diamonds indicates positive cellulolytic activity.</p