The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmission.

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).

Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus.

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.

High infestation of invasive Aedes mosquitoes in used tires along the local transport network of Panama.

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.

Forest disturbance and vector transmitted diseases in the lowland tropical rainforest of central 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.

OBJECTIF: Explorer les changements possibles dans les attributs communautaires des insectes hématophages en fonction de la perturbation des forêts. Nous comparons les modèles de diversité et d'abondance, ainsi que les réponses comportementales de trois assemblages de vecteurs épidémiologiquement distincts sur des sites illustrant divers niveaux de couverture forestière. MÉTHODES: Au cours d'une période de trois ans, nous avons échantillonné des moustiques, des phlébotomes et des moucherons piqueurs dans les habitats forestiers du centre de Panama. Nous avons placé des pièges à lumière CDC dans la canopée de la forêt et dans le sous-étage pour recueillir les femelles en quête de sang. RÉSULTATS: Nous avons collecté un total de 168.405 diptères hématophages adultes, dont 26 genres et 86 espèces. Les environnements de forêt intacts étaient toujours plus diversifiés du point de vue taxonomique que les sites forestiers perturbés, confirmant que les perturbations avaient un impact négatif sur la richesse en espèces. Les espèces de phlébotome et Culicoïdes étaient principalement classés comme climax (spécialiste de la forêt) ou généralistes de perturbation, qui ont tendance à diminuer en abondance parallèlement aux niveaux croissants de perturbation. En revanche, une partie importante des espèces de moustiques, y compris les vecteurs primaires et secondaires de maladies, a été classée dans la catégorie des colons (c'est-à-dire spécialistes des zones perturbées), qui ont tendance à se multiplier vers des habitats forestiers plus perturbés. Dans la forêt vierge, les espèces de phlébotomes et Culicoïdes les plus répandues cloisonnaient la niche verticale en étant actives dans la canopée de la forêt ou dans le sous-étage; pourtant, cette tendance était moins nette dans les habitats perturbés. La plupart des espèces de moustiques n'étaient pas stratifiées verticalement dans leur préférence d'habitat. CONCLUSION: Nous estimons que le risque entomologique et l'exposition associée des agents pathogènes à l'homme est plus élevé dans les scénarios de forêt vierge pour les maladies transmises par les phlébotomes et Culicoïdes, alors que la perturbation des forêts pose un risque entomologique plus élevé pour les infections transmises par les moustiques. Cela suggère que l'hypothèse de l'effet de dilution ne s'applique pas dans les forêts tropicales humides où se reproduisent très abondamment les insectes vecteurs, mais non reconnus, et où des maladies zoonotiques négligées surviennent. Une surveillance entomologique approfondie au niveau de la communauté est donc la clé pour prédire le potentiel de propagation des maladies dans des scénarios de forêt vierge mélangée à des habitats anthropiques. Nous suggérons que les changements dans la qualité des forêts soient également pris en compte lors de l'évaluation du risque de propagation de maladies transmises par les arthropodes.

Maternal invasion history of Aedes aegypti and Aedes albopictus into the Isthmus of Panama: Implications for the control of emergent viral disease agents.

Despite an increase in dengue outbreaks and the arrival of chikungunya and Zika disease in Panama, studies on the demographic history of the invasive Aedes mosquitoes that are the principle vectors of these diseases are still lacking in this region. Here, we assess the genetic diversity of these mosquitoes in order to decipher their invasion histories into the Isthmus of Panama. DNA sequences from the mitochondrial cytochrome C oxidase I gene obtained from 30 localities in 10 provinces confirmed the presence of more than one mitochondrial haplogroup (i.e., maternal lineage) in each species. The invasion of Aedes albopictus was likely from temperate European countries, as the most frequent and widespread haplogroup in Panama harbored variants that are uncommon elsewhere in the Americas. Two infrequent and geographically restricted Ae. albopictus haplotypes appear to have subsequently invaded Panama from neighboring Costa Rica and the USA, respectively. In addition, we recovered two deeply divergent mitochondrial clades in Panamanian Aedes aegypti. The geographic origins of these clades is unknown, given that divergence in the mitochondrial genome is probably due to ancient population processes within the native range of Ae. aegypti, rather than due to its global expansion out of Africa. However, Panamanian Ae. aegypti mitochondrial sequences within the first clade were closely related to others from Colombia, Bolivia, Brazil, Mexico and the USA, suggesting two separate invasions from Western Hemisphere source populations. The pattern of increased genetic diversity in Aedes mosquitoes in Panama is likely facilitated by the numerous land and water inter-connections across the country, which allows them to enter via sea- and land-transportation from Europe, North, Central and South America. Our results here should be considered in disease mitigation programs if emergent arboviruses are to be effectively diminished in Panama through vector suppression.

Disturbance and mosquito diversity in the lowland tropical rainforest of central Panama.

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.

Epidemic and Non-Epidemic Hot Spots of Malaria Transmission Occur in Indigenous Comarcas of Panama.

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.

Novel genetic diversity within Anopheles punctimacula s.l.: phylogenetic discrepancy between the Barcode cytochrome c oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2).

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.

Amblyomma tapirellum (Acari: Ixodidae) collected from tropical forest canopy.

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.

[Evaluation of the toxic activity of the pyrethroid insecticides deltamethrin and lambdacyhalothrin in two Panamanian field populations of Rhodnius pallescens (Hemíptera: Reduviidae)]. / Evaluación de la actividad tóxica de los insecticidas piretroides deltametrina y lambdacihalotrina en dos poblaciones de campo de Rhodnius pallescens (Hemíptera: Reduviidae) de Panamá

INTRODUCTION: Systematic evaluation of the susceptibility of disease vectors to insecticides permits the detection of the development of insecticide resistance over time. This is necessary to evaluate the effectiveness of control methods and to plan management strategies of the resistance. OBJECTIVE: The baseline susceptibility was determined for I and V instar nymphs of Rhodnius pallescens to the active ingredients of the insecticides deltamethrin and lambdacyhalothrin. MATERIALS AND METHODS: The bioassays were applied to two field populations of R. pallescens collected in Chilibre and Cerro Cama, Panamá. A standard protocol for topical application was adapted from that developed for Rhodnius prolixus. Bioassays were performed using topical applications on the dorsal abdominal surface, with volumes of 0.1µl and 0.5µl acetone solution of insecticide for nymphs of stage I and V respectively, using 5µl and 25µl Hamilton microsyringes with a repeating dispenser. Ten nymphs were used for each insecticide concentration. Results. With the topical application of lambdacyhalothrin on first-instar nymphs from Chilibre and Cerro Cama, the LD50 values expressed in ng/insect were 0.13 and 0.11 respectively. In fifth-instar nymphs the LD50 values were 1.71 and 3.48, respectively. For deltamethrin, the topical application on first-instar nymphs resulted in LD50 values of 0.02 and 0.02, and in fifth-instar nymphs the LD50 values were 2.11 and 1.55, respectively. CONCLUSIONS: In I and V instar nymphs from the two R. pallescens populations, resistance factor values demonstrated no significant difference in the toxic effects of the two insecticides and indicated that the R. pallescens populations were susceptible them.

Evaluación de la actividad tóxica de los insecticidas piretroides deltametrina y lambdacihalotrina en dos poblaciones de campo de Rhodnius pallescens (Hemíptera: Reduviidae) de Panamá / Evaluation of the toxic activity of the pyrethroid insecticides deltamethrin and lambdacyhalothrin in two Panamanian field populations of Rhodnius pallescens (Hem¨ªptera: Reduviidae)

Introducción. La evaluación de la sensibilidad de las poblaciones de Rhodnius pallescens permite detectar y vigilar los grados de resistencia en el transcurso del tiempo, con el fin de evaluar los efectos de las estrategias de control y manejo de la resistencia. Objetivo. Determinar la línea base de sensibilidad de los principios activos deltametrina y lambdacihalotrina en ninfas de estadios I y V, en dos poblaciones de campo de R. pallescens. Materiales y métodos. Los bioensayos se realizaron tomando como referencia el protocolo estandarizado de aplicación tópica para R. prolixus y adaptado para R. pallescens. Resultados. Con la aplicación tópica del insecticida lambdacihalotrina en ninfas de estadio I en R. pallescens (Chilibre) y R. pallescens (Cerro Cama), los valores de la dosis letal 50 (DL50) expresados en nanogramos por insecto fueron de DL50=0,129 y DL50=0,109, respectivamente. En ninfas de estadio V los valores fueron de DL50=1,712 y DL50=3,478, respectivamente. Por su parte, la aplicación tópica de deltametrina en ninfas de estadio I, registró valores de DL50=0,022 y DL50=0,021 y, en ninfas de estadio V, de DL50=2,110 y DL50=1,548. Conclusiones. En ninfas de estadios I y V de ambas poblaciones de R. pallescens, se observó mediante los valores obtenidos de factor de resistencia que no existen ninguna diferencia significativa en el efecto tóxico de los insecticidas estudiados. Esto permite establecer que las dos cepas de R. pallescens son sensibles a los insecticidas deltametrina y lambdacihalotrina.

Introduction. Systematic evaluation of the susceptibility of disease vectors to insecticides permits the detection of the development of insecticide resistance over time. This is necessary to evaluate the effectiveness of control methods and to plan management strategies of the resistance.Objective. The baseline susceptibility was determined for I and V instar nymphs of Rhodnius pallescens to the active ingredients of the insecticides deltamethrin and lambdacyhalothrin.Materials and methods. The bioassays were applied to two field populations of R. pallescens collected in Chilibre and Cerro Cama, Panam¨¢. A standard protocol for topical application was adapted from that developed for Rhodnius prolixus. Bioassays were performed using topical applications on the dorsal abdominal surface, with volumes of 0.1 ¦Ìl and 0.5 ¦Ìl acetone solution of insecticide for nymphs of stage I and V respectively, using 5 ¦Ìl and 25 ¦Ìl Hamilton microsyringes with a repeating dispenser. Ten nymphs were used for each insecticide concentration.Results. With the topical application of lambdacyhalothrin on first-instar nymphs from Chilibre and Cerro Cama, the LD50 values expressed in ng/insect were 0.13 and 0.11 respectively. In fifth-instar nymphs the LD50 values were 1.71 and 3.48, respectively. For deltamethrin, the topical application on first-instar nymphs resulted in LD50 values of 0.02 and 0.02, and in fifth-instar nymphs the LD50 values were 2.11 and 1.55, respectively.Conclusions. In I and V instar nymphs from the two R. pallescens populations, resistance factor values demonstrated no significant difference in the toxic effects of the two insecticides and indicated that the R. pallescens populations were susceptible them.