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.

Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets.

As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects.

Characterization of true-branching cyanobacteria from geothermal sites and hot springs of Costa Rica.

Costa Rica is at the centre of the Mesoamerican biodiversity hotspot. Little is known about cyanobacteria from this region so far. Here, four isolates of the order Stigonematales (section V) were characterized in a polyphasic approach. All strains were isolated from geothermal sites and hot springs of Costa Rica. However, one of them, identified as Westiellopsis sp. Ar73, did not grow at more than 40 degrees C. Based on its identical 16S rRNA to several previously isolated Westiellopsis sp. and Fischerella muscicola strains, a ubiquitous distribution throughout tropical and subtropical regions can be implied. In contrast, the isolates MV9, MV11 and RV14 grew well up to 50-55 degrees C. Based on morphologic, ultrastructural, molecular and physiologic data, MV9, MV11 and RV14 were identified to belong to the genus Fischerella. Two distinct intergenic transcribed spacer (ITS) types, with or without tRNA genes, were detected for all Stigonematales analysed here, indicating ITS polymorphism as a characteristic feature of heterocystous cyanobacteria. In phylogenetic trees, these Fischerella spp. formed a new and distinct clade within the wider lineage of thermophilic Fischerella (Mastigocladus cf. laminosus), which might represent a geographic lineage. Thus, geographic isolation may be an underestimated aspect of microbial evolution. The strains presented here are suitable as new models to study this group of cyanobacteria.

Steinernema costaricense n. sp. and S. puntauvense n. sp. (Rhabditida: Steinernematidae), two new entomopathogenic nematodes from Costa Rica.

Steinernema costaricense n. sp. and S. puntauvense n. sp. were recovered during a survey for native entomopathogenic nematodes in Costa Rica. Morphological data, molecular (28S rDNA sequence data) studies and cross-hybridisation tests were used for diagnostic and identification purposes. Additionally, 28S rDNA sequence data were used to assess the evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. costaricense n. sp. include: the body size of the infective juvenile (av. 1,696); the presence of protruding 'horn-like' cephalic papillae; the position of the excretory pore in the infective juvenile (av. 77 microm) and the first generation male (av. 117 microm); the D% value of the infective juvenile (av. 53) and the first generation male (av. 56); the E% value of the infective juvenile (av. 85); and the morphology of the spicules and gubernaculum of the first generation male. Diagnostic traits for S. puntauvense n. sp. are: the position of the excretory pore in the infective juveniles (av. 25 microm); the shape and size of the spicules and gubernaculum of the first generation male; and the shape of the tail of the first generation female. In addition to these traits, 28S rDNA sequences analysis and hybridisation tests showed that both new Steinernema species are distinct and unique entities.

Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae)

Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae). The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts. Rev. Biol. Trop. 55 (2): 401-415. Epub 2007 June, 29.

El tracto digestivo de orugas constituye un microambiente extremo, debido a su elevado pH y presencia de sustancias antimicrobianas secretadas por el insecto o derivadas del tejido vegetal ingerido. Al mismo tiempo, el intestino alberga gran cantidad de alimento, por el cual los microorganismos presentes podrían competir entre sí y con su hospedero. Existe poca información sobre la microbiota asociada con el intestino de orugas tropicales, aunque se ha demostrado la presencia de bacterias de diversos géneros tanto en el intestino como en el interior de pupas. El estudio de las actividades enzimáticas de estos microorganismos constituye un punto de partida en la comprensión de la posible relación metabólica y fisiológica que establecen con sus hospederos, a la vez que permite investigar enzimas con potenciales aplicaciones biotecnológicas. En este trabajo se evaluó la presencia de actividades gelatinolítica, caseinolítica, esterásica, lipolítica, quitinolítica, amilásica, celulolítica y xilanolítica en dos colecciones de aislamientos bacterianos provenientes de tractos digestivos de orugas y de pupas de los lepidópteros Automeris zugana y Rothschildia lebeau. Se utilizaron ensayos bioquímicos tradicionales para detectar enzimas secretadas en condiciones aerobias, en las que ambas colecciones exhibieron un comportamiento enzimático similar. Ningún aislamiento produjo un resultado positivo en todas las pruebas, pero como conjunto ambas colecciones fueron capaces de utilizar todos los sustratos evaluados. Los aislamientos obtenidos de pupas presentaron prácticamente las mismas actividades que sus homólogos provenientes de intestinos. En ambas colecciones fue posible agrupar los aislamientos de acuerdo con su patrón de producción de enzimas. Además de un conjunto heterogéneo de aislamientos poco activos (dos o menos actividades), se destacan dos grupos muy activos (al menos cinco actividades), que manifiestan una aparente especialización en los sustratos que utilizan. El primero de ellos está constituido exclusivamente por miembros de la familia Enterobacteriaceae, los cuales exhibieron un alto porcentaje de positividad en actividades lipolítica y quitinolítica, pero no demostraron la expresión de las actividades amilásica, celulolítica ni xilanolítica. El segundo grupo, formado en su gran mayoría por bacilos Gram-positivos, presenta la situación opuesta: alta positividad en actividades amilásica, celulolítica y xilanolítica, no detección de actividad quitinolítica y pocos aislamientos con actividad lipolítica. Este trabajo pretende ser la base de futuras investigaciones que exploren el potencial biotecnológico de aislamientos bacterianos provenientes del tracto digestivo de orugas.

[Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae)]. / Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae).

The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts.

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica.

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.

[A new species of Euglena (Euglenozoa: Euglenales) isolated from extreme environments in "boiling mudflats" of Rincón de la Vieja volcano, Costa Rica]. / Una nueva especie de Euglena (Euglenozoa: Euglenales) aislada de ambientes extremófilos en las Pailas de Barro del Volcán Rincón de la Vieja, Costa Rica.

A new species of euglena isolated from a hot and acid mud pool located in Las Pailas de Barro, Volcán Rincón de la Vieja, Costa Rica is described. This species inhabits hot and acid environments. Euglena pailasensis sp. nov. main features are: the absence of flagella, the presence filaments like "pilis", the presence of chloroplasts with pyrenoids crossed by several tylakoids, and acid and heat tolerance. Molecular phylogeny studies using 18S rDNA and Gap C genes indicated that the new species is related to E. mutabilis. Its taxonomic characters based on morphology, biology and sequence of the 18S rDNA and Gap C genes are discussed and compared with other closely related species of the genus.

[Ultrastructural description of Euglena pailasensis (Euglenozoa) from Rincón de la Vieja volcano, Guanacaste, Costa Rica]. / Descripción ultraestructural de Euglena pailasensis (Euglenozoa) del Volcán Rincón de la Vieja, Guanacaste, Costa Rica.

The euglenoids are unicellular eukaryotic flagellates living in a diversity of soils and aquatic environments and ecosystems. This study describes the ultrastructure of an euglenoid isolated from the surface of a boiling mud pool with temperatures ranging from 38 to 98 degrees C and pH 2 - 4. The hot mud pool is located in Area de Pailas de Barro, Las Pailas, Rincón de la Vieja Volcano, Guanacaste, Costa Rica. The morphological characterization of the Euglena pailasensis was performed by SEM and TEM. It was determined that, although the euglenoid was obtained from an extreme volcanic environment, the general morphology corresponds to that of a typical member of Euglena of 30-45 microm long and 8-10 microm wide, with membrane, pellicle, chloroplasts, mitochondria, nucleus, pigments and other cytoplasmic organelles. E. pailasensis is delimited by a membrane and by 40 to 90 pellicle strips. It was observed up to 5 elongated chloroplasts per cell. The chloroplast contains several osmiophilic globules and a pyrenoid penetrated by few thylakoid pairs. The nutritious material is reserved in numerous small paramylon grains located at the center of the cell, mitocondria are characterized by the presence of crests in radial disposition toward the interior of the lumen. It was also observed around the external surface "pili" like filaments originating from the pellicle strips. There is no evidence for the presence of flagella in the ampulla (reservoir/canal area), a fact confirmed by negative staining, and a difference regarding other species of Euglena. The observed ultrastructural characteristics are not sufficient to explain the adaptation of this species to acid and hot environments.

Does a polyphagous caterpillar have the same gut microbiota when feeding on different species of food plants?

We used classical culture techniques to explore gut bacteria and changes associated with dietary change in the highly polyphagous, tropical caterpillar Automeris zugana (Saturniidae). Fifty-five third instar wild-caught sibs feeding on Annona purpurea (Annonaceae) in the Area de Conservación Guanacaste (ACG) in northwestern Costa Rica were divided into eight groups. Each of seven groups was reared to the ultimate instar on another species of food plant normally used by A. zugana. Some pupae were also analyzed for the presence of bacteria. Aerobic bacterial cultures were obtained from all 33 caterpillar guts and the eight pupae inventoried. There was no clear pattern in species composition of cultivated bacteria among the eight diets, and each caterpillar on a given food plant carried only a small fraction of the total set of species isolated from the set of caterpillars feeding on that food plant. Taken as a whole, the larvae and pupae contained 22 species of cultivable bacteria in 12 genera. Enterobacter, present in 81.8% of the samples, was the genus most frequently isolated from the caterpillars, followed by Micrococcus and Bacillus. Bacillus thuringiensis was isolated from 30.3% of the dissected caterpillars, but found in caterpillars feeding on only half of the species of food plants.

Does a polyphagous caterpillar have the same gut microbiota when feeding on different species of food plants

We used classical culture techniques to explore gut bacteria and changes associated with dietary change in the highly polyphagous, tropical caterpillar Automeris zugana (Saturniidae). Fifty-five third instar wild-caught sibs feeding on Annona purpurea (Annonaceae) in the Area de Conservación Guanacaste (ACG) in northwestern Costa Rica were divided into eight groups. Each of seven groups was reared to the ultimate instar on another species of food plant normally used by A. zugana. Some pupae were also analyzed for the presence of bacteria. Aerobic bacterial cultures were obtained from all 33 caterpillar guts and the eight pupae inventoried. There was no clear pattern in species composition of cultivated bacteria among the eight diets, and each caterpillar on a given food plant carried only a small fraction of the total set of species isolated from the set of caterpillars feeding on that food plant. Taken as a whole, the larvae and pupae contained 22 species of cultivable bacteria in 12 genera. Enterobacter, present in 81.8 of the samples, was the genus most frequently isolated from the caterpillars, followed by Micrococcus and Bacillus. Bacillus thuringiensis was isolated from 30.3 of the dissected caterpillars, but found in caterpillars feeding on only half of the species of food plants.

Characterization of a photosynthetic Euglena strain isolated from an acidic hot mud pool of a volcanic area of Costa Rica.

Abstract Conspicuous green patches on the surface of an acidic hot mud pool located near the Rincón de la Vieja volcano (northwestern Costa Rica) consisted of apparently unialgal populations of a chloroplast-bearing euglenoid. Morphological and physiological studies showed that it is a non-flagellated photosynthetic Euglena strain able to grow in defined mineral media at temperatures up to 40 degrees C and exhibiting higher thermotolerance than Euglena gracilis SAG 5/15 in photosynthetic activity analyses. Molecular phylogeny studies using 18S rDNA and GapC genes indicated that this strain is closely related to Euglena mutabilis, another acid-tolerant photosynthetic euglenoid, forming a clade deeply rooted in the Euglenales lineage. To our knowledge this is the most thermotolerant euglenoid described so far and the first Euglenozoan strain reported to inhabit acidic hot aquatic habitats.