How conspecific and allospecific eggs and larvae drive oviposition preference in Drosophila.

Where to lay the eggs is a crucial decision for females as it influences the success of their offspring. Female flies prefer to lay eggs on food already occupied and consumed by larvae, which facilitates social feeding, but potentially could also lead to detrimental interactions between species. Whether females can modulate their attraction to cues associated with different species is unknown. Here, we analyzed the chemical profiles of eggs and larvae of 16 Drosophila species, and tested whether Drosophila flies would be attracted to larvae-treated food or food with eggs from 6 different Drosophila species. The chemical analyses revealed that larval profiles from different species are strongly overlapping, while egg profiles exhibit significant species specificity. Correspondingly, female flies preferred to lay eggs where they detected whatever species' larval cues, while we found a significant oviposition preference only for eggs of some species but not others. Our findings suggest that both larval and egg cues present at a given substrate can drive oviposition preference in female flies.

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.

Bacteria Contribute to Plant Secondary Compound Degradation in a Generalist Herbivore System.

Herbivores must overcome a variety of plant defenses, including coping with plant secondary compounds (PSCs). To help detoxify these defensive chemicals, several insect herbivores are known to harbor gut microbiota with the metabolic capacity to degrade PSCs. Leaf-cutter ants are generalist herbivores, obtaining sustenance from specialized fungus gardens that act as external digestive systems and which degrade the diverse collection of plants foraged by the ants. There is in vitro evidence that certain PSCs harm Leucoagaricus gongylophorus, the fungal cultivar of leaf-cutter ants, suggesting a role for the Proteobacteria-dominant bacterial community present within fungus gardens. In this study, we investigated the ability of symbiotic bacteria present within fungus gardens of leaf-cutter ants to degrade PSCs. We cultured fungus garden bacteria, sequenced the genomes of 42 isolates, and identified genes involved in PSC degradation, including genes encoding cytochrome P450 enzymes and genes in geraniol, cumate, cinnamate, and α-pinene/limonene degradation pathways. Using metatranscriptomic analysis, we showed that some of these degradation genes are expressed in situ Most of the bacterial isolates grew unhindered in the presence of PSCs and, using gas chromatography-mass spectrometry (GC-MS), we determined that isolates from the genera Bacillus, Burkholderia, Enterobacter, Klebsiella, and Pseudomonas degrade α-pinene, ß-caryophyllene, or linalool. Using a headspace sampler, we show that subcolonies of fungus gardens reduced α-pinene and linalool over a 36-h period, while L. gongylophorus strains alone reduced only linalool. Overall, our results reveal that the bacterial communities in fungus gardens play a pivotal role in alleviating the effect of PSCs on the leaf-cutter ant system.IMPORTANCE Leaf-cutter ants are dominant neotropical herbivores capable of deriving energy from a wide range of plant substrates. The success of leaf-cutter ants is largely due to their external gut, composed of key microbial symbionts, specifically, the fungal mutualist L. gongylophorus and a consistent bacterial community. Both symbionts are known to have critical roles in extracting energy from plant material, yet comparatively little is known about their roles in the detoxification of plant secondary compounds. In this study, we assessed if the bacterial communities associated with leaf-cutter ant fungus gardens can degrade harmful plant chemicals. We identify plant secondary compound detoxification in leaf-cutter ant gardens as a process that depends on the degradative potential of both the bacterial community and L. gongylophorus Our findings suggest that the fungus garden and its associated microbial community influence the generalist foraging abilities of the ants, underscoring the importance of microbial symbionts in plant substrate suitability for herbivores.

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

Larvae of Ornithodoros knoxjonesi collected at five localities in three countries were studied using morphological and molecular methods to confirm this species' taxonomic validity. The larva of O. knoxjonesi is characterized as having 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 of O. 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). These species also differ in that O. knoxjonesi possesses only the Al seta on tarsus I, whereas O. peropteryx has both Al and Pl setae. And while both species have two setae on coxae I-III, in O. knoxjonesi the anterior seta is tapering and 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 personatus and Pteronotus gymnonotus as new hosts of this tick species.

Papel potencial de Aedes albopictus Skuse en la transmisión de virus dengue (DENV) en una zona de actividad piñera de Costa Rica

RESUMEN Objetivo: Evaluar, bajo una perspectiva ecológica, la presencia de Aedes albopictus y su infección natural por virus dengue (DENV) en una zona de actividad piñera de Costa Rica. Método: Se colectaron mosquitos adultos en galerías forestales colindantes con piñeras, viviendas en proximidad a cultivos (<1 km) y viviendas en lejanía (110 km). Se empleó el índice de Shannon-Wiener para estimar biodiversidad. La infestación larvaria se evaluó en plantas de piña y viviendas y se calcularon índices aédicos de viviendas (IV) y de contenedores (IC). La detección de DENV en adultos (cuerpos y cabezas) y en larvas de Ae. albopictus se efectuó mediante RT-PCR y secuenciación. Resultados: Se colectaron 1376 adultos en total: Ae. albopictus (5,81%), Anopheles apicimacula (5,01%), Culex coronator (11,55%), Cx. inflictus (6,1%), Cx. nigripalpus (48,11%), Cx. quinquefasciatus (23,34%) y Limatus durhamii (0,07%). El índice de biodiversidad fue mayor en galerías forestales. Ae. albopictus adultos fueron colectados principalmente en el área de piñeras (73/80), aunque sólo dos larvas en las plantas de piña. Los índices aédicos en proximidad (IV: 40,7%, IC: 26,9%) y en lejanía (IV: 51,7%, IC: 29,6%) no mostraron diferencias significativas (IV Z=0,56, p=0,58; IC Z=0,16, p=0,87). Se detectó DENV-2 y DENV-3 en 2/20 grupos de cabezas y DENV-1 en 2/74 grupos de larvas de Ae. albopictus. Discusión: Las galerías forestales próximas a cultivos de piña podrían considerarse "islas ecológicas" adecuadas para el refugio de Ae. albopictus. La presencia de DENV en adultos y larvas sugiere un papel activo de Ae. albopictus en la transmisión de virus en este ecosistema.

ABSTRACT Objective To evaluate, under an ecological perspective, the presence of Aedes albopictus and the wild infection by dengue viruses (DENV) in an area of pineapple activity in Costa Rica. Materials and methods Adult mosquitoes were collected in forest galleries limiting pineapple plantations, houses adjacent to plantations (<1 km), and distant houses (1-10 km). Shannon-Wiener index was used to estimate biodiversity. Larval infestation was evaluated in pineapple plants and houses, and aedic house (HI) and container (CI) indices were calculated. Detection of DENV in Ae. albopictus adults (bodies and heads) and larvae was performed by RT-PCR and sequencing. Results A total 1376 adult mosquitoes were collected: Ae. albopictus (5.81%), Anopheles apicimacula (5.01%), Culex coronator (11.55%), Cx. inflictus (6.1%), Cx. nigripalpus (48.11%), Cx. quinquefasciatus (23.34%), and Limatus durhamii (0.07%). Biodiversity index was higher in forest galleries. Most adult Ae. albopictus were collected in forests close to pineapple fields (73/80), although only 2 larvae were detected in pineapple plants. Larval indices in adjacent houses (HI: 40.7%, CI: 26.9%) and distant houses (HI: 51.7%, CI: 29.6%) were similar (HI Z=0.56, p=0.58; CI Z=0.16, p=0.87). DENV-2 and DENV-3 were detected in 2/20 "pools" of Ae. albopictus heads and DENV-1 in 2/74 "pools" of larvae. Conclusion Forest galleries that are in proximity to pineapple plantations could be considered "ecological islands" that are suitable for refuge of Ae. albopictus. Presence of DENV in adults and larvae suggests an active role for Ae. albopictus in virus transmission within this ecosystem.

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.

'Candidatus Rickettsia nicoyana': A novel Rickettsia species isolated from Ornithodoros knoxjonesi in Costa Rica.

Rickettsiae are intracellular bacteria commonly associated with hematophagous arthropods. Most of them have been described in hard ticks, but some have been found in soft ticks. Here we report the detection and isolation of a new Rickettsia from Ornithodoros knoxjonesi larvae collected from Balantiopteryx plicata (Emballonuridae) in Nicoya, Costa Rica. Two ticks were processed to detect Rickettsia spp. genes gltA, ompA, ompB, and htrA by PCR. Part of the macerate was also inoculated into Vero E6 and C6/36 cell lines, and cells were evaluated by Giménez stain, indirect immunofluorescence assay (IFA), and PCR. Both ticks were positive by PCR and rickettsial growth was successful in Vero E6 cells. Amplification and sequencing of near full length rrs, gltA, sca4 genes, and fragments of ompA and ompB showed that the Rickettsia sp. was different from described species. The highest homologies were with 'Candidatus Rickettsia wissemanii' and Rickettsia peacockii: 99.70% (1321/1325) with both sequences for rrs, 99.58% (1172/1177) and 99.76% (1246/1249) for gltA, 99.26% with both sequences (2948/2970 and 2957/2979) for sca4, 98.78% (485/491) and 98.39% (2069/2115) for ompA, and 98.58 (1453/1474) and 98.92% (1459/1475) for ompB; respectively. Bat blood, spleen, liver, and lung samples analyzed for Rickettsia detection were negative. Results demonstrate that the Rickettsia isolated from O. knoxjonesi is probably an undescribed species that belongs to the spotted fever group, for which 'Candidatus Rickettsia nicoyana' is proposed. Considering that B. plicata inhabits areas where contact with humans may occur and that human parasitism by Ornithodoros has been reported in the country, it will be important to continue with the characterization of this species and its pathogenic potential.

Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses.

Outbreaks of spotted fevers have been reported in Costa Rica since the 1950s, although vectors responsible for transmission to humans have not been directly identified. In this study, species of Rickettsia were detected in ectoparasites from Costa Rica, mostly from five study sites where cases of spotted fevers have been reported. Ticks and fleas were collected using drag cloths or directly from domestic and wild animals and pooled according to species, host, and location. Pools were analyzed initially by PCR to detect a fragment of Rickettsia spp. specific gltA gene, and those positive were confirmed by detection of htrA and/or ompA gene fragments. Partial sequences of the gltA gene were obtained, as well as at least one ompA and/or ompB partial sequence of each species. Rickettsia spp. were confirmed in 119 of 497 (23.9%) pools of ticks and fleas analyzed. Rickettsia rickettsii was identified in one nymph of Amblyomma mixtum and one nymph of Amblyomma varium. Other rickettsiae present were 'Candidatus Rickettsia amblyommii' in A. mixtum, Amblyomma ovale, Dermacentor nitens, and Rhipicephalus sanguineus s. l.; Rickettsia bellii in Amblyomma sabanerae; Rickettsia felis in Ctenocephalides felis; and Rickettsia sp. similar to 'Candidatus R. asemboensis' in C. felis, Pulex simulans, A. ovale, and Rhipicephalus microplus. Results show the presence of rickettsiae in vectors that may be responsible for transmission to humans in Costa Rica, and evidence suggests exposure to rickettsial organisms in the human environment may be common. This is the first study to report R. rickettsii in A. varium and in A. mixtum in Costa Rica.