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We searched for evidence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) mosquitoes from Panama. Conventional PCR was performed on 469 Ae. aegypti and 349 Ae. albopictus. We did not discover kdr mutations in Ae. albopictus, but 2 nonsynonymous kdr mutations, V1016I (found in 101 mosquitoes) and F1534C (found in 29 of the mosquitoes with the V1016I), were detected in Ae. aegypti. These kdr mutations were present in all specimens that were successfully sequenced for both IIS5-S6 and IIIS6 regions, which included samples collected from 8 of the 10 provinces of Panama. No other kdr mutations were found in Ae. aegypti, including V1016G, which has already been reported in Panama. Findings suggest that the V1016I-F1534C variant is prevalent in Panama, which might be related to the introduction and passive movement of mosquitoes as part of the used-tire trade. However, we cannot rule out the possibility that selection on de novo replacement of kdr mutations also partially explains the widespread distribution pattern of these mutations. These 2 ecological and evolutionary processes are not mutually exclusive, though, as they can occur in tandem. Research in Panama needs to calculate the genotypic and allelic frequencies of kdr alleles in local Ae. aegypti populations and to test whether some combinations confer phenotypic resistance or not. Finally, future studies will have to track the introduction and spreading of new kdr mutations in both Aedes species., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. While research on COVID-19 has mainly focused on its epidemiology, pathogenesis, and treatment, studies on the naso-oropharyngeal microbiota have emerged in the last few years as an overlooked area of research. Here, we analyzed the bacterial community composition of the naso-oropharynx in 50 suspected SARS-CoV-2 cases (43 detected, 7 not detected) from Veraguas province (Panama) distributed across five age categories. Statistical analysis revealed no significant differences ( p < 0.05) in bacterial alpha and beta diversities between the groups categorized by SARS-CoV-2 test results, age, or patient status. The genera Corynebacterium , Staphylococcus , Prevotella , Streptococcus, and Tepidiphilus were the most abundant in both detected and not-detected SARS-CoV-2 group. The linear discriminant analysis effect size (LEfSe) for biomarker exploration indicated that Veillonella and Prevotella were enriched in detected and hospitalized patients with SARS-CoV-2 relative to non-detected patients, while Thermoanaerobacterium and Haemophilus were enriched in non-detected patients with SARS-CoV-2. The results also indicated that the genus Corynebacterium was found to decrease in patients with detected SARS-CoV-2 relative to those with non-detected SARS-CoV-2. Understanding the naso-oropharyngeal microbiota provides insights into the diversity, composition, and resilience of the microbial community in patients with SARS-CoV-2.

Vector-borne diseases are a major source of morbidity in Panama. Herein, we describe historical usage patterns of synthetic insecticides to control arthropod disease vectors in this country. We examine the influence of interventions by vector control programs on the emergence of insecticide resistance. Chemical control has traditionally focused on two mosquito species: Anopheles albimanus, a major regional malaria vector, and Aedes aegypti, a historical vector of yellow fever, and current vector of dengue, chikungunya, and Zika. Countrywide populations of An. albimanus depict hyperirritability to organochlorine insecticides administered by indoor residual spraying, although they appear susceptible to these insecticides in bioassays settings, as well as to organophosphate and carbamate insecticides in field tests. Populations of Ae. aegypti show resistance to pyrethroids, particularly in areas near Panama City, but the spread of resistance remains unknown in Ae. aegypti and Aedes albopictus. A One Health approach is needed in Panama to pinpoint the insecticide resistance mechanisms including the frequency of knockdown mutations and behavioral plasticity in populations of Anopheles and Aedes mosquitoes. This information is necessary to guide the sustainable implementation of chemical control strategies and the use of modern vector control technologies such as genetically modified mosquitoes, and endosymbiont Wolbachia-based biological control., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Centers for Disease Control and Prevention., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Here, the main goal is to assess natural infections of Plasmodium spp. in anophelines in a forest reserve from the same region where we previously found a surprisingly high rate (5.2%) of plasmodia infections ( n = 25) in Kerteszia mosquitoes ( N = 480) on the slopes of Serra do Mar, Atlantic Forest, Brazil. The mosquito collection sampling was carried out at the Legado das Águas Forest Reserve using CDC light traps and Shannon traps at night (5-10 pm) in 3-day collections in November 2021 and March, April, May, and November 2022. The captured specimens were morphologically identified at the species level and had their genomic DNA extracted in pools of up to 10 mosquitoes/pool. Each pool was tested using 18S qPCR and cytb nested PCR plus sequencing. A total of 5301 mosquitoes, mostly belonging to the genus Kerteszia (99.7%), were sampled and sorted into 773 pools. Eight pools positive for Plasmodium spp. were identified: four for Plasmodium spp., one for P. vivax or P. simium , one for P. malariae or P. brasilianum , and two for the P. falciparum -like parasite. After Sanger sequencing, two results were further confirmed: P. vivax or P. simium and P. malariae or P. brasilianum . The minimum infection rate for Kerteszia mosquitoes was 0.15% (eight positive pools/5285 Kerteszia mosquitoes). The study reveals a lower-than-expected natural infection rate (expected = 5.2% vs. observed = 0.15%). This low rate relates to the absence of Alouatta monkeys as the main simian malaria reservoir in the studied region. Their absence was due to a significant population decline following the reemergence of yellow fever virus outbreaks in the Atlantic Forest from 2016 to 2019. However, this also indicates the existence of alternative reservoirs to infect Kerteszia mosquitoes. The found zoonotic species of Plasmodium , including the P . falciparum -like parasite, may represent a simian malaria risk and thus a challenge for malaria elimination in Brazil.

Sylvatic New World mosquitoes (e.g. Old-growth Forest species) can transmit viruses among non-human primates. This could be a continuous source of viral cycling and spillover events from animals to humans, particularly in changing environments. However, most species of Neotropical sylvatic mosquitoes (genera Aedes, Haemagogus, and Sabethes), which include vector and non-vector species, currently lack genomic resources because there is no reliable and accurate approach for creating de novo reference genomes for these insects. This is a major knowledge gap in the biology of these mosquitoes, restricting our ability to predict and mitigate the emergence and spread of novel arboviruses in Neotropical regions. We discuss recent advances and potential solutions for generating hybrid de novo assemblies from vector and non-vector species using pools of consanguineous offspring. We also discussed research opportunities likely to emerge from these genomic resources., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: This work aims to analyze the landscape of scientific publications on subjects related to One Health and infectious diseases in Panama. The research questions are: How does the One Health research landscape look like in Panama? Are historical research efforts aligned with the One Health concept? What infectious diseases have received more attention from the local scientific community since 1990?, Methods: Boolean searches on the Web of Science, SCOPUS and PubMed were undertaken to evaluate the main trends of publications related to One Health and infectious disease research in the country of Panama, between 1990 and 2019., Results: 4546 publications were identified since 1990, including 3564 peer-reviewed articles interconnected with One Health related descriptors, and 211 articles focused particularly on infectious diseases. A pattern of exponential growth in the number of publications with various contributions from Panamanian institutions was observed. The rate of multidisciplinary research was moderate, whereas those of interinstitutional and intersectoral research ranged from low to very low. Research efforts have centered largely on protozoan, neglected and arthropod-borne diseases with a strong emphasis on malaria, Chagas and leishmaniasis., Conclusion: Panama has scientific capabilities on One Health to tackle future infectious disease threats, but the official collaboration schemes and strategic investment to develop further competencies need to be conciliated with modern times, aka the pandemics era. The main proposition here, addressed to the government of Panama, is to launch a One Health regional center to promote multidisciplinary, interinstitutional and intersectoral research activities in Panama and beyond., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

In the absence of entomological information, tools for predicting Anopheles spp. presence can help evaluate the entomological risk of malaria transmission. Here, we illustrate how species distribution models (SDM) could quantify potential dominant vector species presence in malaria elimination settings. We fitted a 250 m resolution ensemble SDM for Anopheles albimanus Wiedemann. The ensemble SDM included predictions based on seven different algorithms, 110 occurrence records and 70 model projections. SDM covariates included nine environmental variables that were selected based on their importance from an original set of 28 layers that included remotely and spatially interpolated locally measured variables for the land surface of Costa Rica. Goodness of fit for the ensemble SDM was very high, with a minimum AUC of 0.79. We used the resulting ensemble SDM to evaluate differences in habitat suitability (HS) between commercial plantations and surrounding landscapes, finding a higher HS in pineapple and oil palm plantations, suggestive of An. albimanus presence, than in surrounding landscapes. The ensemble SDM suggested a low HS for An. albimanus at the presumed epicenter of malaria transmission during 2018-2019 in Costa Rica, yet this vector was likely present at the two main towns also affected by the epidemic. Our results illustrate how ensemble SDMs in malaria elimination settings can provide information that could help to improve vector surveillance and control.

Local adaptation is important when predicting arthropod-borne disease risk because of its impacts on vector population fitness and persistence. However, the extent that vector populations are adapted to the environment generally remains unknown. Despite low population structure and high gene flow in Aedes aegypti mosquitoes across Panama, excepting the province of Bocas del Toro, we identified 128 candidate SNPs, clustered within 17 genes, which show a strong genomic signal of local environmental adaptation. This putatively adaptive variation occurred across fine geographical scales with the composition and frequency of candidate adaptive loci differing between populations in wet tropical environments along the Caribbean coast and dry tropical conditions typical of the Pacific coast. Temperature and vegetation were important predictors of adaptive genomic variation in Ae. aegypti with several potential areas of local adaptation identified. Our study lays the foundations of future work to understand whether environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and whether this could either aid or hinder efforts of population control., Competing Interests: The authors received funding from The Edward M. and Jeanne C. Kashian Family Foundation Inc., and Nicholas Logothetis of Chartwell Consulting. There are no patents, products in development or marketed products associated with this research to declare., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Monitoring the invasion process of the Asian tiger mosquito Aedes albopictus and its interaction with the contender Aedes aegypti, is critical to prevent and control the arthropod-borne viruses (i.e., Arboviruses) they transmit to humans. Generally, the superior ecological competitor Ae. albopictus displaces Ae. aegypti from most geographic areas, with the combining factors of biology and environment influencing the competitive outcome. Nonetheless, detailed studies asserting displacement come largely from sub-tropical areas, with relatively less effort being made in tropical environments, including no comprehensive research about Aedes biological interactions in Mesoamerica. Here, we examine contemporary and historical mosquito surveillance data to assess the role of shifting abiotic conditions in shaping the spatiotemporal distribution of competing Aedes species in the Republic of Panama. In accordance with prior studies, we show that Ae. albopictus has displaced Ae. aegypti under suboptimal wet tropical climate conditions and more vegetated environments within the southwestern Azuero Peninsula. Conversely, in the eastern Azuero Peninsula, Ae. aegypti persists with Ae. albopictus under optimal niche conditions in a dry and more seasonal tropical climate. While species displacement was stable over the course of two years, the presence of both species generally appears to fluctuate in tandem in areas of coexistence. Aedes albopictus was always more frequently found and abundant regardless of location and climatic season. The heterogenous environmental conditions of Panama shape the competitive outcome and micro-geographic distribution of Aedes mosquitoes, with potential consequences for the transmission dynamics of urban and sylvatic zoonotic diseases., Supplementary Information: The online version of this article (10.1007/s10530-021-02482-y)., Competing Interests: Conflict of interestThe authors received funding from The Edward M. and Jeanne C. Kashian Family Foundation Inc., and Nicholas Logothetis of Chartwell Consulting. There are no patents, products in development or marketed products associated with this research to declare., (© The Author(s) 2021.)

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens., Competing Interests: The authors have declared that no competing interests exist.

As the threat of arboviral diseases continues to escalate worldwide, the question of, "What types of human communities are at the greatest risk of infection?" persists as a key gap in the existing knowledge of arboviral diseases transmission dynamics. Here, we comprehensively review the existing literature on the socioeconomic drivers of the most common Aedes mosquito-borne diseases and Aedes mosquito presence/abundance. We reviewed a total of 182 studies on dengue viruses (DENV), chikungunya virus (CHIKV), yellow fever virus (YFVV), Zika virus (ZIKV), and presence of Aedes mosquito vectors. In general, associations between socioeconomic conditions and both Aedes -borne diseases and Aedes mosquitoes are highly variable and often location-specific. Although 50% to 60% of studies found greater presence or prevalence of disease or vectors in areas with lower socioeconomic status, approximately half of the remaining studies found either positive or null associations. We discuss the possible causes of this lack of conclusiveness as well as the implications it holds for future research and prevention efforts., Competing Interests: None. All authors contributed equally to this study., (© 2020 The Authors.)

The Republic of Panama has the second most unequally distributed wealth in Central America, has recently entered the list of countries affected by the COVID-19 pandemic, and has one of the largest testing rate per inhabitant in the region and consequently the highest incidence rate of COVID-19, making it an ideal location to discuss potential scenarios for assessing epidemic preparedness, and to outline research opportunities in the Region of the Americas. We address two timely important questions: What are the unique risks of COVID-19 in Panama that could help other countries in the Region be better prepared? And what kind of scientific knowledge can Panama contribute to the regional and global study of COVID-19? This paper provides suggestions about how the research community could support local health authorities plan for different scenarios and decrease public anxiety. It also presents basic scientific opportunities about emerging pandemic pathogens towards promoting global health from the perspective of a middle income country., Competing Interests: Conflicts of interest. None declared.

Competing Interests: The authors have declared that no competing interests exist.

We tested 700 serum samples collected throughout Panama from 2015 to 2016 for detecting antibodies and RNA of arboviruses. In convalescent specimens, microsphere immunoassay detected an antibody prevalence of 59.3% for dengue virus (DENV) and 30.3% for Zika virus (ZIKV), which included samples that were collected before the Panamanian surveillance system reported the first case of Zika in the country. For acute sera, the most common arbovirus was DENV with 18 positive samples (6%), followed by four (1.3%) of ZIKV and one (0.6%) of chikungunya virus (CHIKV). Our results indicate a change in the chronology of when ZIKV was first detected in Panama and stress the importance of integrating various approaches to enable improved surveillance of both endemic and emerging arboviruses.

We generated nine coding-complete chikungunya virus genome sequences from blood samples collected during the early 2015 outbreak in Bolivia. Relative to other publicly available chikungunya sequences, the Bolivian samples represent a monophyletic group, suggesting that a single lineage was widely circulating in the country between February and May 2015., (Copyright © 2020 França et al.)

The genus Alphavirus harbours mostly insect-transmitted viruses that cause severe disease in humans, livestock and wildlife. Thus far, only three alphaviruses with a host range restricted to insects have been found in mosquitoes from the Old World, namely Eilat virus (EILV), Taï Forest alphavirus (TALV) and Mwinilunga alphavirus (MWAV). In this study, we found a novel alphavirus in one Culex declarator mosquito sampled in Panama. The virus was isolated in C6/36 mosquito cells, and full genome sequencing revealed an 11 468 nt long genome with maximum pairwise nucleotide identity of 62.7 % to Sindbis virus. Phylogenetic analyses placed the virus as a solitary deep rooting lineage in a basal relationship to the Western equine encephalitis antigenic complex and to the clade comprising EILV, TALV and MWAV, indicating the detection of a novel alphavirus, tentatively named Agua Salud alphavirus (ASALV). No growth of ASALV was detected in vertebrate cell lines, including cell lines derived from ectothermic animals, and replication of ASALV was strongly impaired above 31 °C, suggesting that ASALV represents the first insect-restricted alphavirus of the New World.

Spatial isolation is one of the main drivers of allopatric speciation, but the extent to which spatially-segregated populations accumulate genetic differences relevant to speciation is not always clear. We used data from ultraconserved elements (UCEs) and whole mitochondrial genomes (i.e., mitogenomes) to explore genetic variation among allopatric populations of the weakly electric fish Sternopygus dariensis across the Isthmus of Panama. We found strong genetic divergence between eastern and western populations of S. dariensis. Over 77% of the UCE loci examined were differentially fixed between populations, and these loci appear to be distributed across the species' genome. Population divergence occurred within the last 1.1 million years, perhaps due to global glaciation oscillations during the Pleistocene. Our results are consistent with a pattern of genetic differentiation under strict geographic isolation, and suggest the presence of incipient allopatric species within S. dariensis. Genetic divergence in S. dariensis likely occurred in situ, long after the closure of the Isthmus of Panama. Our study highlights the contribution of spatial isolation and vicariance to promoting rapid diversification in Neotropical freshwater fishes. The study of spatially-segregated populations within the Isthmus of Panama could reveal how genetic differences accumulate as allopatric speciation proceeds.

Long term surveillance of vectors and arboviruses is an integral aspect of disease prevention and control systems in countries affected by increasing risk. Yet, little effort has been made to adjust space-time risk estimation by integrating disease case counts with vector surveillance data, which may result in inaccurate risk projection when several vector species are present, and when little is known about their likely role in local transmission. Here, we integrate 13 years of dengue case surveillance and associated Aedes occurrence data across 462 localities in 63 districts to estimate the risk of infection in the Republic of Panama. Our exploratory space-time modelling approach detected the presence of five clusters, which varied by duration, relative risk, and spatial extent after incorporating vector species as covariates. The Ae. aegypti model contained the highest number of districts with more dengue cases than would be expected given baseline population levels, followed by the model accounting for both Ae. aegypti and Ae. albopictus. This implies that arbovirus case surveillance coupled with entomological surveillance can affect cluster detection and risk estimation, potentially improving efforts to understand outbreak dynamics at national scales., Competing Interests: The authors have declared that no competing interests exist.

The microbiome plays a key role in the biology, ecology and evolution of arthropod vectors of human pathogens. Vector-bacterial interactions could alter disease transmission dynamics through modulating pathogen replication and/or vector fitness. Nonetheless, our understanding of the factors shaping the bacterial community in arthropod vectors is incomplete. Using large-scale 16S amplicon sequencing, we examine how habitat disturbance structures the bacterial assemblages of field-collected whole-body hematophagous arthropods that vector human pathogens including mosquitoes (Culicidae), sand flies (Psychodidae), biting midges (Ceratopogonidae) and hard ticks (Ixodidae). We found that all comparisons of the bacterial community among species yielded statistically significant differences, but a difference was not observed between adults and nymphs of the hard tick, Haemaphysalis juxtakochi. While Culicoides species had the most distinct bacterial community among dipterans, tick species were composed of entirely different bacterial OTU's. We observed differences in the proportions of some bacterial types between pristine and disturbed habitats for Coquillettidia mosquitoes, Culex mosquitoes, and Lutzomyia sand flies, but their associations differed within and among arthropod assemblages. In contrast, habitat quality was a poor predictor of differences in bacterial classes for Culicoides biting midges and hard tick species. In general, similarities in the bacterial communities among hematophagous arthropods could be explained by their phylogenetic relatedness, although intraspecific variation seems influenced by habitat disturbance., Competing Interests: The authors received funding from The Edward M. and Jeanne C. Kashian Family Foundation Inc., and Nicholas Logothetis of Chartwell Consulting. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.

Objective: To explore possible changes in the community attributes of haematophagous insects as a function of forest disturbance. We compare the patterns of diversity and abundance, plus the behavioural responses of three epidemiologically distinct vector assemblages across sites depicting various levels of forest cover., Methods: Over a 3-year period, we sampled mosquitoes, sandflies and biting-midges in forested habitats of central Panama. We placed CDC light traps in the forest canopy and in the understorey to gather blood-seeking females., Results: We collected 168 405 adult haematophagous dipterans in total, including 26 genera and 86 species. Pristine forest settings were always more taxonomically diverse than the disturbed forest sites, confirming that disturbance has a negative impact on species richness. Species of Phlebotominae and Culicoides were mainly classified as climax (i.e. forest specialist) or disturbance-generalist, which tend to decrease in abundance along with rising levels of disturbance. In contrast, a significant portion of mosquito species, including primary and secondary disease vectors, was classified as colonists (i.e. disturbed-areas specialists), which tend to increase in numbers towards more disturbed forest habitats. At pristine forest, the most prevalent species of Phlebotominae and Culicoides partitioned the vertical niche by being active at the forest canopy or in the understorey; yet this pattern was less clear in disturbed habitats. Most mosquito species were not vertically stratified in their habitat preference., Conclusion: We posit that entomological risk and related pathogen exposure to humans is higher in pristine forest scenarios for Culicoides and Phlebotominae transmitted diseases, whereas forest disturbance poses a higher entomological risk for mosquito-borne infections. This suggests that the Dilution Effect Hypothesis (DEH) does not apply in tropical rainforests where highly abundant, yet unrecognised insect vectors and neglected zoonotic diseases occur. Comprehensive, community level entomological surveillance is, therefore, the key for predicting potential disease spill over in scenarios of pristine forest intermixed with anthropogenic habitats. We suggest that changes in forest quality should also be considered when assessing arthropod-borne disease transmission risk., (© 2019 John Wiley & Sons Ltd.)

The genus Phlebovirus (order Bunyavirales, family Phenuiviridae) comprises 57 viruses that are grouped into nine species-complexes. Sandfly-transmitted phleboviruses are found in Europe, Africa and the Americas and are responsible for febrile illness and infections of the nervous system in humans. The aim of this study was to assess the genetic diversity of sandfly-transmitted phleboviruses in connected and isolated forest habitats throughout the Panama Canal area in Central Panama. In total, we collected 13 807 sandflies comprising eight phlebotomine species. We detected several strains pertaining to five previously unknown viruses showing maximum pairwise identities of 45-78 % to the RNA-dependent RNA polymerase genes of phleboviruses. Entire coding regions were directly sequenced from infected sandflies as virus isolation in cell culture was not successful. The viruses were tentatively named La Gloria virus (LAGV), Mona Grita virus (MOGV), Peña Blanca virus (PEBV), Tico virus (TICV) and Tres Almendras virus (TRAV). Inferred phylogenies and p-distance-based analyses revealed that PEBV groups with the Bujaru phlebovirus species-complex, TRAV with the Candiru phlebovirus species-complex and MOGV belongs to the proposed Icoarci phlebovirus species-complex, whereas LAGV and TICV seem to be distant members of the Bujaru phlebovirus species-complex. No specific vector or habitat association was found for any of the five viruses. Relative abundance of sandflies was similar over habitat types. Our study shows that blood-feeding insects originating from remote and biodiverse habitats harbour multiple previously unknown phleboviruses. These viruses should be included in future surveillance studies to assess their geographic distribution and to elucidate if these viruses cause symptoms of disease in animals or humans.

Background: The long-distance dispersal of the invasive disease vectors Aedes aegypti and Aedes albopictus has introduced arthropod-borne viruses into new geographical regions, causing a significant medical and economic burden. The used-tire industry is an effective means of Aedes dispersal, yet studies to determine Aedes occurrence and the factors influencing their distribution along local transport networks are lacking. To assess infestation along the primary transport network of Panama we documented all existing garages that trade used tires on the highway and surveyed a subset for Ae. aegypti and Ae. albopictus. We also assess the ability of a mass spectrometry approach to classify mosquito eggs by comparing our findings to those based on traditional larval surveillance., Results: Both Aedes species had a high infestation rate in garages trading used tires along the highways, providing a conduit for rapid dispersal across Panama. However, generalized linear models revealed that the presence of Ae. aegypti is associated with an increase in road density by a log-odds of 0.44 (0.73 ± 0.16; P = 0.002), while the presence of Ae. albopictus is associated with a decrease in road density by a log-odds of 0.36 (0.09 ± 0.63; P = 0.008). Identification of mosquito eggs by mass spectrometry depicted similar occurrence patterns for both Aedes species as that obtained with traditional rearing methods., Conclusions: Garages trading used tires along highways should be targeted for the surveillance and control of Aedes-mosquitoes and the diseases they transmit. The identification of mosquito eggs using mass spectrometry allows for the rapid evaluation of Aedes presence, affording time and cost advantages over traditional vector surveillance; this is of importance for disease risk assessment.

Background: Malaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles mosquito species, which makes identification of vectors of human Plasmodium challenging. Strategies by Panamanian health authorities to bring malaria under control targeting Anopheles vectors could be ineffective if they tackle a misidentified species., Methods: A rapid mass spectrometry identification procedure was developed to accurately and timely sort out field-collected Neotropical Anopheles mosquitoes into vector and non-vector species. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of highly-abundant proteins were generated from laboratory-reared mosquitoes using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouchers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field-collected Neotropical Anopheles mosquito species were classified using a combination of custom-made unsupervised (i.e., Principal component analysis-PCA) and supervised (i.e., Linear discriminant analysis-LDA) classification algorithms., Results: Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra-specific variability with enough well-preserved proteins to differentiate among distinct biological species, consistent with previous literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair. LDA demonstrated high discriminatory power and consistency, with validation and cross-validation positive identification rates above 93% at the species level., Conclusion: The selected sample processing procedure can be used to identify field-collected Anopheles species, including vectors of Plasmodium, in a short period of time, with a minimal amount of tissue and without the need of an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working without a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication efforts in Panama and other countries with large number of mosquito's species by improving vector surveillance in epidemic-prone sites such as indigenous Comarcas.

Electric fishes are a diverse group of freshwater organisms with the ability to generate electric organ discharges (EODs) that are used for communication and electrolocation. This group (ca. 200 species) has originated in South America, and six species colonized the Central American Isthmus. Here, we assembled the complete mitochondrial genomes (mitogenomes) for three Central American electric fishes (i.e. Sternopygus dariensis, Brachyhypopomus occidentalis, and Apteronotus rostratus), and, based on these data, explored their phylogenetic position among Gymnotiformes. The three mitogenomes show the same gene order, as reported for other fishes, with a size ranging from 16,631 to 17,093 bp. We uncovered a novel 60 bp intergenic spacer (IGS) located between the COII and tRNA Lys genes, which appears to be unique to the Apteronotidae. Furthermore, phylogenetic relationships supported the traditional monophyly of Gymnotiformes, with the three species positioned within their respective family. In addition, the genus Apteronotus belongs to the early diverging lineage of the order. Finally, we found high sequence divergence (13%) between our B. occidentalis specimen and a sequence previously reported in GenBank, suggesting that the prior mitogenome of B. occidentalis represents a different South American species. Indeed, phylogenetic analyses using Cytochrome b gene across the genus placed the previously reported individual within B. bennetti. Our study provides novel mitogenome resources that will advance our understanding of the diversity and phylogenetic history of Neotropical fishes., (Published by Elsevier B.V.)

Six Phlebotominae sand fly species are incriminated as biological vectors of human pathogens in Panama, but molecular corroboration is still needed. We aim at confirming the identity of Phlebotominae species documented as anthropophilic in Panama. Adult sandflies were collected from August 2010 to February 2012 in Central Panama using CDC light traps. Species confirmation was accomplished through molecular barcodes and allied sequences from GenBank. A total of 53,366 sand fly specimens representing 18 species were collected. Five species were validated molecularly as single phylogenetic clusters, but Psychodopygus thula depicted two genetically divergent lineages, which may be indicative of cryptic speciation.

Background: We sought to identify if socioeconomic and demographic factors play a role in resident knowledge, attitude, and practice regarding Dengue, Chikungunya, and Zika in order to inform effective management procedures for disease prevention in Panama, a middle-income tropical country in Central America. All three are arthropod-borne viruses transmitted by Aedes mosquito vectors present in the focal region of Panama City, the largest city in Central America and an urban region of extreme socioeconomic polarization., Methods: Between November 2017 and February 2018, we administered standardized, anonymous knowledge, attitude, and practice surveys to 263 residents split between two neighborhoods of high socioeconomic status (SES) and two neighborhoods of low SES. We then summed the knowledge, attitude, and practice scores respectively, and used linear and logistic regressions to quantify relationships with socioeconomic and demographic factors., Results: Low-SES neighborhoods with high proportions of low income residents, residents over 70 years old had lower mean knowledge scores compared to other groups. Furthermore, residents in neighborhoods of low SES reported more mosquito biting relative to residents in neighborhoods of high SES, yet comparably lower level of concerns for disease transmission. Additionally, knowledge was lower for the more novel emergent threats of Chikungunya and Zika, compared to the endemic Dengue., Conclusion: Findings suggest that low-SES neighborhoods with high proportions of low income, low education, and elderly residents should be targeted for outreach programs designed to prevent DENV, CHIKV, or ZIKV in Panama City. These outcomes support our initial hypotheses as lower relative knowledge and fewer practices related to the prevention of Dengue, Chikungunya, and Zika were found in low-SES neighborhoods. There is also a widespread lack of adequate knowledge regarding these diseases as well as low levels of concern in areas of highly reported mosquito biting. We provide suggestions for taking neighborhood socioeconomic status and specific aspects resident health literacy and attitude into account for creating more effective outreach campaigns as both endemic and novel arthropod-borne disease rates continue to increase throughout Latin America.

American trypanosomiasis, or Chagas disease, is a growing public health problem in Panama, and further forest degradation due to human population growth is expected to worsen the situation. Most people infected with the parasite Trypanosoma cruzi are silently ill, and their life expectancy is severely compromised, which contributes to further deterioration of living conditions in endemic regions. Here, we review the outcomes of nearly 100 years of ecological and epidemiological investigation about Chagas disease in Panama, in an attempt to highlight progress, identify needs, and re-orient future efforts. Rhodnius pallescens and Triatoma dimidiata are both primary vectors of T. cruzi in Panama, but R. pallescens seems more efficient in human-altered forest ecosystems due to a greater degree of association with Attalea butyracea. In contrast, T. dimidiata transmits T. cruzi efficiently under more sylvatic conditions (e.g. settlements inside old-growth or secondary forest patches), where its populations reach considerable numbers irrespective of the absence of A. butyracea. A trend of increasing forest degradation, suburbanization, and development of tourism in Panama favoring the establishment of A. butyracea and other palm tree species (Acrocomia sp.) suggests that a colonist species like R. pallescens will continue to play a more prominent role in the transmission of T. cruzi than a forest specialist like T. dimidiata. However, studies about the taxonomic status and ecology of these vectors are still needed in Panama to address their transmission potential fully. The implementation of an active surveillance system and education programs could greatly minimize the risk of Chagas disease transmission in Panama, preventing fatal infections in children from endemic areas.

The Intermediate Disturbance Hypothesis (IDH) is well-known in ecology providing an explanation for the role of disturbance in the coexistence of climax and colonist species. Here, we used the IDH as a framework to describe the role of forest disturbance in shaping the mosquito community structure, and to identify the ecological processes that increase the emergence of vector-borne disease. Mosquitoes were collected in central Panama at immature stages along linear transects in colonising, mixed and climax forest habitats, representing different levels of disturbance. Species were identified taxonomically and classified into functional categories (i.e., colonist, climax, disturbance-generalist, and rare). Using the Huisman-Olff-Fresco multi-model selection approach, IDH testing was done. We did not detect a unimodal relationship between species diversity and forest disturbance expected under the IDH; instead diversity peaked in old-growth forests. Habitat complexity and constraints are two mechanisms proposed to explain this alternative postulate. Moreover, colonist mosquito species were more likely to be involved in or capable of pathogen transmission than climax species. Vector species occurrence decreased notably in undisturbed forest settings. Old-growth forest conservation in tropical rainforests is therefore a highly-recommended solution for preventing new outbreaks of arboviral and parasitic diseases in anthropic environments.

RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We report the complete mitochondrial genome of two specimens of Orange-billed Sparrow Arremon aurantiirostris from Colón Province, in central Panama. The two specimens were collected on the same day, and at the same locality; however, they showed substantial divergence (6.3% average pairwise divergence among coding genes). A survey of ND2 sequence variation across Panama suggests that this divergence is the result of geographic differentiation and secondary contact. This high level of mitochondrial divergence among co-occurring individuals raises the possibility of multiple biological species in Orange-billed Sparrows. Our results are yet another demonstration that much remains to be discovered regarding avian biodiversity in Panama and throughout the Neotropics.

Background: Host specificity is a fundamental determinant of tick population and pathogen transmission dynamics, and therefore has important implications for human health. Tick host specificity is expected to be particularly high in the tropics, where communities of ticks, hosts and pathogens are most diverse. Yet the degree to which tropical tick species are host-specific remains poorly understood. Combining new field data with published records, we assessed the specificity of tick-host associations in Panama, a diverse Neotropical region., Methods: The resulting dataset includes 5,298 adult ticks belonging to 41 species of eight genera that were directly collected from 68 vertebrate host species of 17 orders. We considered three important aspects of tick host specificity: (i) the relative ecological importance of each host species (structural specificity); (ii) relatedness among host species (phylogenetic specificity); and (iii) spatial scale-dependence of tick-host relationships (geographical specificity). Applying quantitative network analyses and phylogenetic tools with null model comparisons, we assessed the structural and phylogenetic specificity across three spatial scales, ranging from central Panama to countrywide. Further, we tested whether species-rich tick genera parasitized a wider variety of hosts than species-poor genera, as expected when ticks specialize on different host species., Results: Most tick species showed high structural and/or phylogenetic specificity in the adult stage. However, after correcting for sampling effort, we found little support for geographical specificity. Across the three scales, adult ticks tended to be specific to a limited number of host species that were phylogenetically closely related. These host species in turn, were parasitized by tick species from distinct genera, suggesting switching among distantly related hosts is common at evolutionary timescales. Further, there was a strong positive relationship between the taxonomic richness of the tick genera and that of their hosts, consistent with distinct tick species being relatively specific to different host species., Conclusions: Our results indicate that in the adult stage, most ticks in the diverse Neotropical community studied are host specialists. This contrasts with earlier assessments, but agrees with findings from other host-parasite systems. High host specificity in adult ticks implies high susceptibility to local tick-host co-extirpation, limited ability to colonize new habitats and limited potential for interspecific pathogen transmission.

In the tropics, ticks parasitize many classes of vertebrate hosts. However, because many tropical tick species are only identifiable in the adult stage, and these adults usually parasitize mammals, most attention on the ecology of tick-host interactions has focused on mammalian hosts. In contrast, immature Neotropical ticks are often found on wild birds, yet difficulties in identifying immatures hinder studies of birds' role in tropical tick ecology and tick-borne disease transmission. In Panama, we found immature ticks on 227 out of 3,498 individually-sampled birds representing 93 host species (24% of the bird species sampled, and 13% of the Panamanian land bird fauna). Tick parasitism rates did not vary with rainfall or temperature, but did vary significantly with several host ecological traits. Likewise, Neotropical-Nearctic migratory birds were significantly less likely to be infested than resident species. Using a molecular library developed from morphologically-identified adult ticks specifically for this study, we identified eleven tick species parasitizing birds, indicating that a substantial portion of the Panamanian avian species pool is parasitized by a diversity of tick species. Tick species that most commonly parasitized birds had the widest diversity of avian hosts, suggesting that immature tick species are opportunistic bird parasites. Although certain avian ecological traits are positively associated with parasitism, we found no evidence that individual tick species show specificity to particular avian host ecological traits. Finally, our data suggest that the four principal vectors of Rocky Mountain Spotted Fever in the Neotropics rarely, if ever, parasitize Panamanian birds. However, other tick species that harbor newly-discovered rickettsial parasites of unknown pathogenicity are frequently found on these birds. Given our discovery of broad interaction between Panamanian tick and avian biodiversity, future work on tick ecology and the dynamics of emerging tropical tick-borne pathogens should explicitly consider wild bird as hosts.

From 2002-2005, Panama experienced a malaria epidemic that has been associated with El Niño Southern Oscillation weather patterns, decreased funding for malaria control, and landscape modification. Case numbers quickly decreased afterward, and Panama is now in the pre-elimination stage of malaria eradication. To achieve this new goal, the characterization of epidemiological risk factors, foci of transmission, and important anopheline vectors is needed. Of the 24,681 reported cases in these analyses (2000-2014), ~62% occurred in epidemic years and ~44% in indigenous comarcas (5.9% of Panama's population). Sub-analyses comparing overall numbers of cases in epidemic and non-epidemic years identified females, comarcas and some 5-year age categories as those disproportionately affected by malaria during epidemic years. Annual parasites indices (APIs; number of cases per 1,000 persons) for Plasmodium vivax were higher in comarcas compared to provinces for all study years, though P. falciparum APIs were only higher in comarcas during epidemic years. Interestingly, two comarcas report increasing numbers of cases annually, despite national annual decreases. Inclusion of these comarcas within identified foci of malaria transmission confirmed their roles in continued transmission. Comparison of species distribution models for two important anophelines with Plasmodium case distribution suggest An. albimanus is the primary malaria vector in Panama, confirmed by identification of nine P. vivax-infected specimen pools. Future malaria eradication strategies in Panama should focus on indigenous comarcas and include both active surveillance for cases and comprehensive anopheline vector surveys.

Landscape changes occurring in Panama, a country whose geographic location and climate have historically supported arbovirus transmission, prompted the hypothesis that arbovirus prevalence increases with degradation of tropical forest habitats. Investigations at four variably degraded sites revealed a diverse array of potential mosquito vectors, several of which are known vectors of arbovirus pathogens. Overall, 675 pools consisting of 25,787 mosquitoes and representing 29 species from nine genera (collected at ground and canopy height across all habitats) were screened for cytopathic viruses on Vero cells. We detected four isolates of Gamboa virus (family:Bunyaviridae; genus:Orthobunyavirus) from pools of Aedeomyia squamipennis captured at canopy level in November 2012. Phylogenetic characterization of complete genome sequences shows the new isolates to be closely related to each other with strong evidence of reassortment among the M segment of Panamanian Gamboa isolates and several other viruses of this group. At the site yielding viruses, Soberanía National Park in central Panama, 18 mosquito species were identified, and the predominant taxa included A. squamipennis,Coquillettidia nigricans, and Mansonia titillans., (© The American Society of Tropical Medicine and Hygiene.)

Prior studies of mitochondrial variation in White-breasted Wood-Wrens (Henicorhina leucosticta) have suggested that populations in South American and Mesoamerica might represent multiple species. Here we report the complete mitochondrial genomes from two individuals of H. leucosticta, representing the Panamanian subspecies pittieri and alexandri. The two sequences were 16,721 and 16,726 base pairs in size with both genomes comprised of the usual 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes, and one displacement loop region in the standard avian order. Uncorrected pairwise divergence between mitogenome features was high, with the highest divergence occurring in protein-coding genes (average = 8.2%), followed by control region (6.7%). RNA features had lower pairwise divergences (average tRNA = 4.3%, average rRNA = 2.3%). The protein-coding ATPase 6 gene had a different stop codon between these two specimens. The high level of sequence variation between these subspecies suggests that Mesoamerican H. leucosticta might be comprised of multiple species. We urge a full phylogeographic survey of this widespread Neotropical forest bird.

We sequenced and compared the mitogenome organization of two specimens of suboscine tyrant flycatcher Mionectes oleagineus from western and eastern Panama, representing distinct mtDNA clades. These samples show identical gene arrangement and vary in size by less than 5 base pairs. Both depict a non-standard avian gene order with an extra non-coding region (e.g. the remnant CR2), which differs in one base pair between them. Small size differences are also found on the control region and the 16S rRNA. Average uncorrected pairwise divergence among protein-coding genes (PCGs) was 2.8, ranging from 1.9% for COXIII and ND6 to 3.2% for ND2 and ATP6, respectively. These mitogenomes may be useful for understanding the evolutionary dynamics of gene order in bird mitochondrial genomes.

The New World jacanas, Jacana spinosa (Mexico to Panama and also the West Indies) and Jacana jacana (Panama and South America), are polyandrous freshwater waders that are common throughout the Neotropics. These two species hybridize narrowly at their contact zone in Panama, and as part of a study of the hybrid zone dynamics, we present complete, annotated mitochondrial genomes for both species. The two species have very similar mitochondrial genomes, showing identical gene orders, and differing in size in only two RNA features and the control region, and among protein-coding genes, the two genomes had average uncorrected pairwise divergence of 1.8%, ranging from 0.7% for ND4L and 3.6% for ATP8. However, control region divergence is high (∼ 16%). These mitochondrial genome sequences may be useful tools for understanding jacana hybridization dynamics, especially regarding potential mitonuclear incompatibilities.

We review existing information on the epidemiology of American Cutaneous Leishmaniasis (ACL) in Panama, with emphasis on the bionomics of anthropophilic Lutzomyia sand fly species. Evidence from Panamanian studies suggests that there are six anthropophilic species in the country: Lutzomyia trapidoi, Lu. panamensis, Lu. gomezi, Lu. ylephiletor, Lu. sanguinaria and Lu. pessoana (Henceforth Lu. carrerai thula). In general, these taxa are abundant, widespread and feed opportunistically on their hosts, which make them potential transmitters of pathogens to a broad range of wildlife, domesticated animals and humans. Furthermore, nearly all man-biting species in Panama (with the exception of Lu. gomezi) expand demographically during the rainy season when transmission is likely higher due to elevated Leishmania infection rates in vector populations. Despite this, data on the distribution and prevalence of ACL suggest little influence of vector density on transmission intensity. Apart from Lu. trapidoi, anthropophilic species seem to be most active in the understory, but vertical stratification, as well as their opportunistic feeding behavior, could vary geographically. This in turn seems related to variation in host species composition and relative abundance across sites that have experienced different degrees of human alteration (e.g., deforestation) in leishmaniasis endemic regions of Panama.

Mosquito blood meals provide information about the feeding habits and host preference of potential arthropod-borne disease vectors. Although mosquito-borne diseases are ubiquitous in the Neotropics, few studies in this region have assessed patterns of mosquito-host interactions, especially during actual disease outbreaks. Based on collections made during and after an outbreak of equine viral encephalitis, we identified the source of 338 blood meals from 10 species of mosquitoes from Aruza Abajo, a location in Darien province in eastern Panama. A PCR based method targeting three distinct mitochondrial targets and subsequent DNA sequencing was used in an effort to delineate vector-host relationships. At Aruza Abajo, large domesticated mammals dominated the assemblage of mosquito blood meals while wild bird and mammal species represented only a small portion of the blood meal pool. Most mosquito species fed on a variety of hosts; foraging index analysis indicates that eight of nine mosquito species utilize hosts at similar proportions while a stochastic model suggests dietary overlap among species was greater than would be expected by chance. The results from our null-model analysis of mosquito diet overlap are consistent with the hypothesis that in landscapes where large domestic animals dominate the local biomass, many mosquito species show little host specificity, and feed upon hosts in proportion to their biomass, which may have implications for the role of livestocking patterns in vector-borne disease ecology.

Aedeomyia squamipennis and Culex (Melanoconion) ocossa, two ubiquitous Neotropical mosquito species, are likely involved in the transmission of several bird pathogens in Gamboa, central Panama. However, knowledge on their eco-epidemiological profiles is still incomplete. Our goal in this study was to investigate temporal trends of vector density and their relationship with avian plasmodia prevalence. This information is central to identifying the risk posed by each vector species to the avian community locally. We found that A. squamipennis maintains stable population size across climatic seasons and thus maybe a more efficient vector of avian malaria than C. ocossa. In contrast, C. ocossa, which undergoes considerable population expansion in the rainy season and contraction in the dry season, is likely only an important avian malaria vector during part of the year. This is consistent with the larger number of parasite isolations and Plasmodium cyt b lineages recovered from A. squamipennis than from C. ocossa and might be explained by marked differences in their seasonality and host-feeding preferences. More Plasmodium PCR testing in mosquito communities from other areas of Panama might reveal additional vectors of avian plasmodia.

Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni, have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3' COI), the Barcode region in the five prime end of the COI (5' COI), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3' COI depicted six highly supported molecular lineages (A-F), none of which was An. malefactor. In contrast, phylogenetic inference with the 5' COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3' COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Free-ranging ticks are widely known to be restricted to the ground level of vegetation. Here, we document the capture of the tick species Amblyomma tapirellum in light traps placed in the forest canopy of Barro Colorado Island, central Panama. A total of forty eight adults and three nymphs were removed from carbon dioxide-octenol baited CDC light traps suspended 20 meters above the ground during surveys for forest canopy mosquitoes. To our knowledge, this represents the first report of questing ticks from the canopy of tropical forests. Our finding suggests a novel ecological relationship between A. tapirellum and arboreal mammals, perhaps monkeys that come to the ground to drink or to feed on fallen fruits.

Free-ranging ticks are widely known to be restricted to the ground level of vegetation. Here, we document the capture of the tick species Amblyomma tapirellum in light traps placed in the forest canopy of Barro Colorado Island, central Panama. A total of forty eight adults and three nymphs were removed from carbon dioxide-octenol baited CDC light traps suspended 20 meters above the ground during surveys for forest canopy mosquitoes. To our knowledge, this represents the first report of questing ticks from the canopy of tropical forests. Our finding suggests a novel ecological relationship between A. tapirellum and arboreal mammals, perhaps monkeys that come to the ground to drink or to feed on fallen fruits.

Background: The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated., Methods: The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches., Results: Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis., Conclusion: Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.