Microbiome and plant cell transformation trigger insect gall induction in cassava.

Several specialised insects can manipulate normal plant development to induce a highly organised structure known as a gall, which represents one of the most complex interactions between insects and plants. Thus far, the mechanism for insect-induced plant galls has remained elusive. To study the induction mechanism of insect galls, we selected the gall induced by Iatrophobia brasiliensis (Diptera: Cecidomyiidae) in cassava (Euphorbiaceae: Manihot esculenta Crantz) as our model. PCR-based molecular markers and deep metagenomic sequencing data were employed to analyse the gall microbiome and to test the hypothesis that gall cells are genetically transformed by insect vectored bacteria. A shotgun sequencing discrimination approach was implemented to selectively discriminate between foreign DNA and the reference host plant genome. Several known candidate insertion sequences were identified, the most significant being DNA sequences found in bacterial genes related to the transcription regulatory factor CadR, cadmium-transporting ATPase encoded by the cadA gene, nitrate transport permease protein (nrtB gene), and arsenical pump ATPase (arsA gene). In addition, a DNA fragment associated with ubiquitin-like gene E2 was identified as a potential accessory genetic element involved in gall induction mechanism. Furthermore, our results suggest that the increased quality and rapid development of gall tissue are mostly driven by microbiome enrichment and the acquisition of critical endophytes. An initial gall-like structure was experimentally obtained in M. esculenta cultured tissues through inoculation assays using a Rhodococcus bacterial strain that originated from the inducing insect, which we related to the gall induction process. We provide evidence that the modification of the endophytic microbiome and the genetic transformation of plant cells in M. esculenta are two essential requirements for insect-induced gall formation. Based on these findings and having observed the same potential DNA marker in galls from other plant species (ubiquitin-like gene E2), we speculate that bacterially mediated genetic transformation of plant cells may represent a more widespread gall induction mechanism found in nature.

Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized - genetically and morphologically - several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas, Streptomyces, Enterobacter, Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Streptomyces sp. M54: an actinobacteria associated with a neotropical social wasp with high potential for antibiotic production.

Streptomyces symbionts in insects have shown to be a valuable source of new antibiotics. Here, we report the genome sequence and the potential for antibiotic production of "Streptomyces sp. M54", an Actinobacteria associated with the eusocial wasp, Polybia plebeja. The Streptomyces sp. M54 genome is composed of a chromosome (7.96 Mb), and a plasmid (1.91 Kb) and harbors 30 biosynthetic gene clusters for secondary metabolites, of which only one third has been previously characterized. Growth inhibition bioassays show that this bacterium produces antimicrobial compounds that are active against Hirsutella citriformis, a natural fungal enemy of its host, and the human pathogens Staphylococcus aureus and Candida albicans. Analyses through TLC-bioautography, LC-MS/MS and NMR allowed the identification of five macrocyclic ionophore antibiotics, with previously reported antibacterial, antitumor and antiviral properties. Phylogenetic analyses placed Streptomyces sp. M54 in a clade of other host-associated strains taxonomically related to Streptomyces griseus. Pangenomic and ANI analyses confirm the identity of one of its closest relatives as Streptomyces sp. LaPpAH-199, a strain isolated from an ant-plant symbiosis in Africa. In summary, our results suggest an insect-microbe association in distant geographic areas and showcase the potential of Streptomyces sp. M54 and related strains for the discovery of novel antibiotics.

Plant galls recorded from Guanacaste Conservation Area-Costa Rica as an integrated concept of a biological database / Registro de galhas em plantas da Área de Conservação Guanacaste, Costa Rica, como conceito integrado de um banco de dados biológico

Abstract: Galling insects are specialist herbivorous that have the ability of manipulating plant tissue to form complex biological structures called galls. Even though different organisms have the ability to induce galls in plants, insect galls have the highest degree of structural complexity. The main goal of this study was to obtain a preliminary systematic record of plant gall morphotypes from the Guanacaste Conservation Area in Costa Rica and integrate the information into a biological database. Plant gall morphotypes were recorded, characterized and deposited into a specialized herbarium established as a reference for the inventory. Moreover, organisms associated with gall morphotypes were included in the inventory when it was possible to obtain and identify them. Galls were collected in the rainy season over a period of three years. In total, we recorded forty-four families, seventy genera, and eighty-seven host plant species. One hundred thirty-one morphotypes of plant galls were identified in the Guanacaste Conservation Area. The family with the highest number of gall morphotypes was Fabaceae (8.4%). Leaves were the organ with the largest number of galls (71%), followed by stems (17.6%), and apical buds (6.9%). The predominant gall shape was globular (25.2%), followed by discoid (18.3%). Fifty-nine percent of the galls had a glabrous texture, which was most common on leaves, with 77%. One hundred twenty of our field records (91.6%) of plant galls were new morphotypes not only for Costa Rica but also the world. As a consequence of this research and considering the prospect of future increases in new gall records (and associated organisms), we proposed having the biological entities resulting from the inventory placed in a cecidiarium. This repository represents a standardized and comprehensive way to manage the data and biological materials associated with the plant galls. We also suggest a nomenclature for standardizing gall morphotype registries and identifications. This work is the first and most detailed inventory of plant galls carried out thus far in the Guanacaste Conservation Area.

Resumo: Os insetos galhadores são herbívoros especializados, que têm a habilidade de manipular os tecidos vegetais, formando uma complexa estrutura biológica. Diferentes organismos têm a capacidade de induzir galhas, porém as de insetos têm maior grau de complexidade estrutural. O principal objetivo desse estudo foi realizar um levantamento sistemático preliminar das galhas de insetos na Área de Conservação Guanacaste, na Costa Rica, e inserir as informações em uma base de dados biológicos. Os morfotipos de galhas foram registrados, caraterizados e depositados em um herbário estabelecido como base de referência deste inventário. Além disso, quando foi possível obter e identificar os organismos associados a cada morfotipo de galha, eles foram incluídos no inventário. As amostras de galhas foram coletadas na estação chuvosa, durante três anos. No total, foram registradas 44 famílias, 70 gêneros e 87 espécies de plantas hospedeiras. Cento e trinta e um morfotipos de galhas foram identificados na Área de Conservação de Guanacaste. A família com o maior número de morfotipos de galhas foi Fabaceae (8.4%). Os órgãos vegetais com o maior número de galhas foram as folhas (71,0%), seguidas dos caules (17,6%), e das gemas apicais com 6,9%. A forma predominante das galhas foi globoide (25,2%), seguida da lenticular (18,3%) e 59% das galhas apresentaram textura glabra, observada mais frequentemente folhas (77%). Cento e vinte morfotipos de galhas (91.6%) representaram novos registros não só na Costa Rica como também no mundo. Como consequência desta pesquisa e considerando as perspectivas de aumentos futuros de novos registros de galhas (e organismos associados), nós propomos que as entidades biológicas resultantes deste inventário sejam depositadas em um cecidiário. Este repositório representa uma maneira padronizada e abrangente de gerenciar e integrar os dados e os materiais biológicos associados às galhas das plantas. Também sugerimos uma terminologia para padronizar os registros e identificações dos morfotipos de galhas. Este inventário de galhas em plantas é o primeiro e o mais detalhado já realizado na Área de Conservação Guanacaste.

Phylogenetic analyses of antibiotic-producing Streptomyces sp. isolates obtained from the stingless-bee Tetragonisca angustula (Apidae: Meliponini).

Many insects have been associated with actinobacteria in protective symbiosis where antimicrobial metabolites inhibit host pathogens. However, the microbiota of neotropical insects such as the stingless-bee Tetragonisca angustula is poorly explored. T. angustula is a meliponid bee widely distributed in Latin America, its honey is traditionally exploited because of its ethno-pharmacological properties and its antimicrobial activity has been demonstrated. Also, the well-structured nest of this species allows exploration of the microbiota of its different components. Even though Streptomyces spp. have been cultured from stingless-bees, little is known about their role in this insect-microbe relationship. In this study, we examined the association between culturable actinobacteria and T. angustula, and evaluated the isolates' potential as antimicrobial producers. We isolated 51 actinobacteria from adult bees and different substrates of the hive of T. angustula (pollen and honey storage, garbage pellets and cerumen). We then performed a 16S rRNA phylogenetic analysis that clusters the bacteria to previously described lineages of host-associated Streptomyces. In addition, all the isolates were classified according to their antibacterial activity against human pathogens, measured by a growth inhibition test based on diffusion in agar. More than 50 % of our isolates exhibit antimicrobial activity, mainly to Gram-positive bacteria and fungi and only two against Gram-negative bacteria. Additionally, we obtained electron micrographs of adult bees with what appears to be patches of hyphae with Streptomyces-like cell morphology on their body surface. Our results suggest that T. angustula possibly uptakes and transfers actinobacteria from the environment, acting as vectors for these potentially beneficial organisms. This research provides new insights regarding the microbiota associated with T. angustula and justify future studies exploring the full diversity of the microbial community associated with the hive and the possible exchange of microbes with the crops they pollinate.

Assessment of Bacterial Communities Associated With the Skin of Costa Rican Amphibians at La Selva Biological Station.

Amphibian skin is a suitable environment for rich communities of microorganisms, both beneficial and detrimental to the host. The amphibian cutaneous microbiota has been hypothesized to play an important role as symbionts, protecting their hosts against disease. Costa Rica has one of the most diverse assemblages of amphibians in the world and we know very little about the microbiota of these tropical animals. For comparison with other studies, we explore the diversity of the skin bacterial communities employing16S rRNA amplicon sequencing of swab samples from twelve species of frogs at La Selva Biological Station in Sarapiquí, Heredia province. The predominant phylum detected in our studies was Proteobacteria, followed by Bacteroidetes and Actinobacteria, with these three phyla representing 89.9% of the total bacterial taxa. At the family level, Sphingobacteriaceae and Comamonadaceae were highly represented among samples. Our results suggest that host species and host family are significant predictors of the variation in microbiota composition. This study helps set the foundation for future research about microbiota composition and resilience to unfavorable conditions, leading to improvement in managing strategies for endangered amphibian species.

Detection, ultrastructure and phylogeny of Sclerorhabditis neotropicalis (Nematoda: Rhabditidae) nematodes associated with the Azteca ant-Cecropia tree symbiosis / Detección, ultraestructura y filogenia de los nemátodos Sclerorhabditis neotropicalis (Nematoda: Rhabditidae) asociados con la simbiosis de la hormiga Azteca y el árbol

Abstract The nematodes involved in ant-plant symbioses have been poorly studied, creating an area of research that is now receiving increasing attention. One example is Sclerorhabditis neotropicalis that inhabit the nests of Azteca ants in Cecropia trees. The goal of the present study is to increase our knowledge about these organisms and their possible symbiotic relationship with the ants and their host plant. The samples were collected during 2011-2013 in different locations in Costa Rica: Parque Nacional Carara (Puntarenas province), San Pedro de Montes de Oca and Highway 27 (San Jose) and La Selva Biological Station (Sarapiquí, Heredia). We examined 576 internodes from 23 plants, comprising four species of Cecropia (including one non-myrmecophyte) and five Azteca species. S. neotropicalis was found in all but one of the Azteca/Cecropia combinations examined. Queen and worker ants were placed in water in order to dislodge nematodes and the results were largely positive, suggesting that alate queens carry S. neotropicalis between trees and that workers carry them between internodes. In addition to transport, preliminary observations suggest that the ants provide masses of scraped parenchyma (possibly fertilized with feces) as a substrate for bacteria upon which the nematodes presumably feed. We also found S. neotropicalis associated with Azteca in another myrmecophyte, Cordia alliodora, suggesting that this ant/nematode association is not restricted to Cecropia. SEM photographs of S. neotropicalis were provided to supplement the original description of this species, and molecular phylogenetic analyses employing three different markers suggest that Sclerorhabditis forms a well-supported clade of bacteria-feeding nematodes associated with Azteca. Indirect evidence suggests that this is a mutualistic association, but the benefits to the ants remain unknown. Rev. Biol. Trop. 66(1): 368-380. Epub 2018 March 01.

Resumen Cecropia. Los nemátodos asociados a la simbiosis entre plantas y hormigas han sido poco estudiados, creando un área de investigación que está recibiendo cada vez más atención. Un ejemplo de esto es Sclerorhabditis neotropicalis, que habita en los nidos de las hormigas Azteca asociadas a árboles de Cecropia. El objetivo de este trabajo es incrementar el conocimiento sobre esta especie de nemátodo y su relación con Azteca-Cecropia. Las muestras fueron recolectadas durante 2011-2013 en diferentes localidades de Costa Rica: Parque Nacional Carara (Puntarenas), San Pedro de Montes de Oca y Ruta 27 (San José) y la Estación Biológica La Selva (Sarapiquí, Heredia). Se examinaron 576 entrenudos de 23 plantas, comprendiendo cuatro especies de Cecropia (incluyendo una especie no mirmecófita) y cinco especies de Azteca. Se encontraron individuos de S. neotropicalis en todas las combinaciones de Azteca/Cecropia examinadas. Las hormigas reinas y trabajadoras fueron colocadas en agua para observar los nematodos y los resultados fueron en gran medida positivos, lo cual sugiere que las reinas aladas transportan S. neotropicalis hacia nuevos árboles y que las trabajadoras los transportan entre los entrenudos. Además del transporte, las observaciones preliminares insinúan que las hormigas proporcionan una masa de parénquima raspado (posiblemente fertilizado con heces) como sustrato para bacterias que luego servirán de alimento a los nematodos. Adicionalmente, encontramos S. neotropicalis asociado con Azteca en otro tipo de árbol, Cordia alliodora, sugiriendo que esta asociación de hormigas/nematodos no se limita a Cecropia. Las fotografías de microscopía electrónica de S. neotropicalis obtenidas en este trabajo complementan la descripción original de esta especie. Por otra parte, los análisis filogenéticos moleculares sugieren que Sclerorhabditis forma un clado bien definido de nematodos bacteriófagos asociados con Azteca. La evidencia indirecta apunta a que se trata de una asociación mutualista, pero los beneficios para las hormigas siguen siendo desconocidos.

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.

Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies.

Leaf-cutter ants (Atta and Acromyrmex) use fresh leaves to cultivate a mutualistic fungus (Leucoagaricus gongylophorus) for food in underground gardens. A new ant queen propagates the cultivar by taking a small fragment of fungus from her parent colony on her nuptial flight and uses it to begin her own colony. Recent research has shown that the ants' fungus gardens are colonized by symbiotic bacteria that perform important functions related to nitrogen fixation and have been implicated in contributing to plant biomass degradation. Here, we combine bacterial culturing in several media for counts and identification using the 16S rRNA gene with electron microscopy to investigate the process of cellulose degradation in the fungus garden and refuse dumps, and to assess the potential role of symbiotic bacteria. We show through electron microscopy that plant cell walls are visibly degraded in the bottom section of fungus gardens and refuse dumps, and that bacteria are more abundant in these sections. We also consistently isolated cellulolytic bacteria from all sections of fungus gardens. Finally, we show by culture-dependent and electron microscopy analysis that the fungus garden pellets carried by recently mated queens are colonized by fungus garden-associated bacteria. Taken together, our results indicate that cellulose is degraded in fungus gardens, and that fungus garden bacteria that may contribute to this deconstruction are vertically transmitted by new queens.

Characterization of the Skin Microbiota of the Cane Toad Rhinella cf. marina in Puerto Rico and Costa Rica.

Rhinella marina is a toad native to South America that has been introduced in the Antilles, likely carrying high loads of microorganisms, potentially impacting local community diversity. The amphibian skin is involved in pathogen defense and its microbiota has been relatively well studied, however, research focusing on the cane toad microbiota is lacking. We hypothesize that the skin microbial communities will differ between toads inhabiting different geographical regions in Central America and the Caribbean. To test our hypothesis, we compared the microbiota of three populations of R. cf. marina toads, two from Costa Rican (native) and one Puerto Rican (exotic) locations. In Costa Rica, we collected 11 toads, 7 in Sarapiquí and 4 from Turrialba while in Puerto Rico, 10 animals were collected in Santa Ana. Separate swab samples were collected from the dorsal and ventral sites resulting in 42 samples. We found significant differences in the structure of the microbial communities between Puerto Rico and Costa Rica. We detected as much as 35 different phyla; however, communities were dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Alpha diversity and richness were significantly higher in toads from Puerto Rico and betadiversity revealed significant differences between the microbiota samples from the two countries. At the genus level, we found in Santa Ana, Puerto Rico, a high dominance of Kokuria, Niabella, and Rhodobacteraceae, while in Costa Rica we found Halomonas and Pseudomonas in Sarapiquí, and Acinetobacter and Citrobacter in Turrialba. This is the first report of Niabella associated with the amphibian skin. The core microbiome represented 128 Operational Taxonomic Units (OTUs) mainly from five genera shared among all samples, which may represent the symbiotic Rhinella's skin. These results provide insights into the habitat-induced microbial changes facing this amphibian species. The differences in the microbial diversity in Puerto Rican toads compared to those in Costa Rica provide additional evidence of the geographically induced patterns in the amphibian skin microbiome, and highlight the importance of discussing the microbial tradeoffs in the colonization of new ecosystems.

Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants.

Bacteria-mediated acquisition of atmospheric N2 serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N2 fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N2-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.