Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.Biological and Environmental Research/[DE-FC02-07ER64494]/BER/Estados UnidosNational Science Foundation/[DGE-1256259]/NSF/Estados UnidosNational Science Foundation/[DEB-0747002]/NSF/Estados UnidosNational Science Foundation/[MCB-0702025]/NSF/Estados UnidosNational Institutes of Health/[T32 GM07215]/NIH/Estados UnidosUniversidad de Costa Rica/[]/UCR/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaUniversity of Wisconsin-Madison's Hilldale Undergraduate Faculty Research Fellowship/[]//Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Detection of Rickettsia spp. in ticks of wildlife fauna from Costa Rica: First report of Rickettsia rhipicephali in Central America

In the past two decades, new species of Rickettsia have been detected and described worldwide, some of them considered pathogenic for humans. Although Costa Rica is considered a biodiversity hotspot, the knowledge about rickettsiae in sylvatic ecosystems and wild animals is scarce. The aim of this preliminary study was to detect and identify species of Rickettsia in ticks collected from wild animals in Costa Rica. A total 119 ticks were collected from 16 animal host species belonging to diverse vertebrate families (Didelphidae, Procyonidae, Felidae, Choloepodidae, Bradypodidae, Myrmecophagidae, Tayassuidae, Tapiridae, Phyllostomidae, Bufonidae, Geoemydidae, Boidae, Colubridae), and they were grouped into 43 pools to detect the presence of Rickettsia spp. DNA by PCR targeting the gltA gene. In positive pools, amplicons of the ompA, sca5 (ompB), and/or htrA genes were also amplified to identify the species present. The identification of some ticks was also confirmed by molecular methods. Four species of Rickettsia were detected in eight (19%) tick pools: Rickettsia amblyommatis in four pools of Amblyomma geayi (host: Caluromys derbianus) and one pool of Amblyomma cf. parvum (host: Nasua narica), Rickettsia rhipicephali in one pool of Dermacentor latus (host: Tayassu pecari), ‘Candidatus Rickettsia colombianensi’ in one pool of Amblyomma sp. nymphs (host: Boa constrictor), and Rickettsia sp. genotype IbR/CRC in one pool of Ixodes cf. boliviensis (host: Puma concolor). This is the first molecular detection of R. rhipicephali in Central America, and of ‘Candidatus R. colombianensi’ in Costa Rica. Results show that diverse wild animals and their ticks are associated with several species of rickettsiae in Costa Rica, which may come in contact with humans and other domestic animals in sylvatic environments

Evidence for Widespread Associations between Neotropical Hymenopteran Insects and Actinobacteria

The evolutionary success of hymenopteran insects has been associated with complex physiological and behavioral defense mechanisms against pathogens and parasites. Among these strategies are symbiotic associations between Hymenoptera and antibiotic-producing Actinobacteria, which provide protection to insect hosts. Herein, we examine associations between culturable Actinobacteria and 29 species of tropical hymenopteran insects that span five families, including Apidae (bees), Vespidae (wasps), and Formicidae (ants). In total, 197 Actinobacteria isolates were obtained from 22 of the 29 different insect species sampled. Through 16S rRNA gene sequences of 161 isolates, we show that 91% of the symbionts correspond to members of the genus Streptomyces with less common isolates belonging to Pseudonocardia and Amycolatopsis. Electron microscopy revealed the presence of filamentous bacteria with Streptomyces morphology in brood chambers of two different species of the eusocial wasps. Four fungal strains in the family Ophiocordycipitacea (Hypocreales) known to be specialized insect parasites were also isolated. Bioassay challenges between the Actinobacteria and their possible targeted pathogenic antagonist (both obtained from the same insect at the genus or species level) provide evidence that different Actinobacteria isolates produced antifungal activity, supporting the hypothesis of a defensive association between the insects and these microbe species. Finally, phylogenetic analysis of 16S rRNA and gyrB demonstrate the presence of five Streptomyces lineages associated with a broad range of insect species. Particularly our Clade I is of much interest as it is composed of one 16S rRNA phylotype repeatedly isolated from different insect groups in our sample. This phylotype corresponds to a previously described lineage of host-associated Streptomyces. These results suggest Streptomyces Clade I is a Hymenoptera host-associated lineage spanning several new insect taxa and ranging from the American temperate to the Neotropical region. Our work thus provides important insights into the widespread distribution of Actinobacteria and hymenopteran insects associations, while also pointing at novel resources that could be targeted for the discovery of active natural products with great potential in medical and biotechnological applications

New records and phylogenetic position of Ornithodoros knoxjonesi (Ixodida: Argasidae)

Larvae of Ornithodoros knoxjonesi collected at five localities in three countries were studied using morphologicaland molecular methods to confirm this species? taxonomic validity. The larva of O. knoxjonesi is characterized ashaving 14 pairs of dorsal setae, eight pairs of ventral setae, plus a posteromedian seta; an elongate dorsal plate,tapered anteriorly; and a hypostome that is narrower near its midlength, with posteriorly projecting denticles.Although the larvae of O. knoxjonesi and Ornithodoros peropteryx are morphologically quite similar, the larva ofO. knoxjonesi is characterized as having dorsal setae that are wider at the tip than at the base, while in O.peropteryx these setae are narrower at the tip than at the base; moreover, the dorsal setae are shorter in O.knoxjonesi (Al 0.037?0.065; Pl 0.035?0.059) than in O. peropteryx (Al 0.120−0.132; Pl 0.080−0.096). Thesespecies also differ in that O. knoxjonesi possesses only the Al seta on tarsus I, whereas O. peropteryx has both Aland Pl setae. And while both species have two setae on coxae I-III, in O. knoxjonesi the anterior seta is taperingand smooth and the posterior is fringed, while both setae are fringed in O. peropteryx. At the molecular level,based on a maximum likelihood analysis using approximately 400 bp of the mitochondrial 16S rDNA gene, O.knoxjonesi appears as an independent lineage, separated from O. peropteryx by a genetic distance of 16.28 %.Balantiopteryx plicata is a common host of O. knoxjonesi; however, in this work we report Pteronotus personatusand Pteronotus gymnonotus as new hosts of this tick species.Fil: Guzmán-Cornejo, Carmen. Consejo Nacional de Ciencia y Tecnología; MéxicoFil: Rebollo-Hernández, Andrea. Consejo Nacional de Ciencia y Tecnología; MéxicoFil: Troyo, Adriana. Universidad de Costa Rica; Costa RicaFil: Moreira-Soto, Rolando D.. Universidad de Costa Rica; Costa RicaFil: Hernández, Ligia V.. Universidad de Costa Rica; Costa RicaFil: Muñoz-Leal, Sebastián. Universidade de Sao Paulo; BrasilFil: Labruna, Marcelo B.. Universidade de Sao Paulo; BrasilFil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Venzal, José M.. Universidad de la Republica, Salto

PCoA clustering of Morisita-Horn Diversity Index.

<p>Sample shape indicates colony. Sample color indicates degradation (<b>A</b>) or layer (<b>B</b>). Panel <b>C</b> shows the correlation analysis. The vectors indicate the correlation of each OTU and the percentage of cellulose degradation with the principal coordinates shown.</p

Morisita-Horn Beta Diversity Clustering of Samples.

<p>The corresponding percentage of cellulose degradation, colony, layer, and taxonomic classification of OTUs are shown for each sample.</p

Electron microscopy of leaf-cutter ant refuse dumps.

<p><b>(A and B)</b> Scanning electron microscopy shows the ultrastructure of refuse dump leaf material and different bacterial morphologies. <b>(C)</b> Transmission electron microscopy shows leaf cells and surrounding bacteria. Red boxes indicate degraded plant cell wall and abnormal, clumped internal cell structure. <b>Photo credits:</b> Rolando Moreira Soto.</p

Correlation between number of reads and cellulose degradation by the community for OTU1 (<i>Acidovorax</i>) and OTU9 (<i>Ferruginibacter</i>) across sequenced samples.

<p>Correlation between number of reads and cellulose degradation by the community for OTU1 (<i>Acidovorax</i>) and OTU9 (<i>Ferruginibacter</i>) across sequenced samples.</p

Comparison of degradation ability across colonies and layers.

<p><b>(A and B</b>) Qualitative Assay Data. Test tubes containing carbon-free minimal media and a strip of cellulosic filter paper were used to enrich for cellulolytic communities. Failure plots, indicating when the filter paper broke apart in each culture, were fit with Kaplan Meier curves and analyzed using the Wilcoxon method to determine significant differences among colonies (indicated by letters A-C) and layers (no significant differences). (<b>C and D)</b> Quantitative Assay Data. Pre-weighed, submerged cellulosic filter paper allowed quantification of cellulose degradation after 10 days. Samples are grouped by colony or dump layer. Error bars represent one standard error from the mean. Significant differences were determined using Tukey’s HSD test and are indicated above the data. <b>Photo credits:</b> Gina Lewin (A, C).</p