In this study, we evaluated the efficacy of various primers for the purpose of DNA barcoding old, pinned museum specimens of blackflies (Diptera: Simuliidae). We analysed 271 pinned specimens representing two genera and at least 36 species. Due to the age of our material, we targeted overlapping DNA fragments ranging in size from 94 to 407 bp. We were able to recover valid sequences from 215 specimens, of which 18% had 500- to 658-bp barcodes, 36% had 201- to 499-bp barcodes and 46% had 65- to 200-bp barcodes. Our study demonstrates the importance of choosing suitable primers when dealing with older specimens and shows that even very short sequences can be diagnostically informative provided that an appropriate gene region is used. Our study also highlights the lack of knowledge surrounding blackfly taxonomy, and we briefly discuss the need for further phylogenetic studies in this socioeconomically important family of insects.

Several studies have shown Dengue Virus (DENV) nucleic acids and/or antibodies present in Neotropical wildlife including bats, suggesting that some bat species may be susceptible to DENV infection. Here we aim to elucidate the role of house-roosting bats in the DENV transmission cycle. Bats were sampled in households located in high and low dengue incidence regions during rainy and dry seasons in Costa Rica. We captured 318 bats from 12 different species in 29 households. Necropsies were performed in 205 bats to analyze virus presence in heart, lung, spleen, liver, intestine, kidney, and brain tissue. Histopathology studies from all organs showed no significant findings of disease or infection. Sera were analyzed by PRNT90 for a seroprevalence of 21.2% (51/241), and by PCR for 8.8% (28/318) positive bats for DENV RNA. From these 28 bats, 11 intestine samples were analyzed by RT-PCR. Two intestines were DENV RNA positive for the same dengue serotype detected in blood. Viral isolation from all positive organs or blood was unsuccessful. Additionally, viral load analyses in positive blood samples by qRT-PCR showed virus concentrations under the minimal dose required for mosquito infection. Simultaneously, 651 mosquitoes were collected using EVS-CO2 traps and analyzed for DENV and feeding preferences (bat cytochrome b). Only three mosquitoes were found DENV positive and none was positive for bat cytochrome b. Our results suggest an accidental presence of DENV in bats probably caused from oral ingestion of infected mosquitoes. Phylogenetic analyses suggest also a spillover event from humans to bats. Therefore, we conclude that bats in these urban environments do not sustain DENV amplification, they do not have a role as reservoirs, but function as epidemiological dead end hosts for this virus.

Mosquito sampling using efficient traps that can assess species diversity and/or presence of dominant vectors is important for understanding the entomological risk of mosquito-borne disease transmission. Here, we present results from a survey of mosquito species sampled with ovitraps in a neotropical rainforest of Costa Rica. We found the method to be an efficient sampling tool. With a total sampling effort of 29 traps, we collected 157 fourth-instar larvae and three pupae belonging to eight mosquito taxonomic units (seven species and individuals from a homogenous taxonomic unit identified to the genus level). In our samples, we found two medically important species, Sabethes chloropterus (Humboldt) and Trichoprosopon digitatum (Rondani). The former is a proven vector of Yellow Fever in sylvatic environments and the later has been found infected with several arboviruses. We also found that mosquito species abundance and diversity increased with canopy cover and in environments where leaf litter dominated the ground cover. Finally, our results suggest that ovitraps have a great potential for systematic sampling in longitudinal and cross-sectional ecological "semi-field" studies in neotropical settings.

Some details on the biology, behavior and laboratory mass rearing of Anastrepha obliqua are offered. Information on larvaI diets and oviposition substrates are discussed. Eggs of A. obliqua are, very succeptible to dehydratation and they collapse just few minutes after oviposition, if substrate for oviposition is not near 100% R.H. When using fruits as oviposition substrate, smallerfruit species, Spanish plums (Spondias spp.) offer higher yields. Bigger fruits (mango) loose large amounts of water and it accumulates in the sand substrate drowning mature larvae. After analyzing the biological cycle, the weaker part seems to be the 20 minute period in which mature larvae abandon the fallen fruit substrate and crawl few a centimeters on the floor seeking for an appropiate place for ovipositing. In this period large amounts of predators easily diminish larval population. Once pupation takes place, adult forms hatch in small groups, after eleven days, when atmosferic relativehumidity reaches 70%. Another observations are also included.

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