Strong Positive Selection in Aedes aegypti and the Rapid Evolution of Insecticide Resistance.

Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti's biology and global population structure and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti, many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species and add to a growing body of evidence suggesting that Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

Diversity of cave Phlebotomines (Diptera: Psychodidae) from a Colombian cave.

Sandflies are vector species of Leishmania, among many other pathogens, with a global distribution and a variety of ecological niches. Previous samplings have found that karstic formations (i.e., caves, grottos, and folds formed by the erosion of limestone) serve as a natural habitat to sandfly species. The majority of samplings of cave sandfly diversity have occurred in Brazil and to date none have studied the species composition in a cave in the Northern Andes. We collected sandflies in the Cave "Los Guácharos", in the state of Antioquia, Colombia. The sampling was carried out during two consecutive nights in September 2019. CDC-type light traps were installed inside the cavern and in other surrounding karst systems (caves, rock-breaks, and folds). In total, we identified 17 species of sandfly from the cave and surrounding karst systems, including a new record for Colombia (Bichromomyia olmeca), and provide the first karstic reports for four other species (Lutzomyia gomezi, Lutzomyia hartmanni, Pintomyia ovallesi, and Psychodopygus panamensis). We then used the results of our survey and published literature to test two hypotheses. First, that sandfly diversity in Neotropical caves is richest nearer to the equator, and second that there is a phylogenetic signal of karstic habitat use in sandflies. Counter to our predictions, we found no evidence that diversity follows a latitudinal gradient. Further, we find no evidence of a phylogenetic signal of karstic habitat use, instead finding that the use of caves likely evolved multiple times across several genera. Our results highlight the importance of a wide sampling to understand the natural habitat of sandflies and other disease vectors.

Corrigendum to "Enzymatic, antimicrobial, and leishmanicidal bioactivity of gram-negative bacteria strains from the midgut of Lutzomyia evansi, an insect vector of leishmaniasis in Colombia" [Biotechnol. Rep. 24 (2019) 1-9].

[This corrects the article DOI: 10.1016/j.btre.2019.e00379.].

Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome.

Phlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human populations mainly located at developing countries. Their intestinal microbiota is involved in a wide range of biological and physiological processes, and could exclude or facilitate such transmission of pathogens. In this study, we investigated the Eubacterial microbiome from digestive tracts of Lu. evansi adults structure using 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq) obtained from digestive tracts of Lu. evansi adults. The samples were collected at two locations with high incidence of the disease in humans: peri-urban and forest ecosystems from the department of Sucre, Colombia. 289,068 quality-filtered reads of V4 region of 16S rRNA gene were obtained and clustered into 1,762 operational taxonomic units (OTUs) with 97% similarity. Regarding eubacterial diversity, 14 bacterial phyla and 2 new candidate phyla were found to be consistently associated with the gut microbiome content. Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla in all the samples and the core microbiome was particularly dominated by Methylobacterium genus. Methylobacterium species, are known to have mutualistic relationships with some plants and are involved in shaping the microbial community in the phyllosphere. As a remarkable feature, OTUs classified as Wolbachia spp. were found abundant on peri-urban ecosystem samples, in adult male (OTUs n = 776) and unfed female (OTUs n = 324). Furthermore, our results provide evidence of OTUs classified as Cardinium endosymbiont in relative abundance, notably higher with respect to Wolbachia. The variation in insect gut microbiota may be determined by the environment as also for the type of feeding. Our findings increase the richness of the microbiota associated with Lu. evansi. In this study, OTUs of Methylobacterium found in Lu. evansi was higher in engorged females, suggesting that there are interactions between microbes from plant sources, blood nutrients and the parasites they transmit during the blood intake.

Enzymatic, antimicrobial, and leishmanicidal bioactivity of gram-negative bacteria strains from the midgut of Lutzomyia evansi, an insect vector of leishmaniasis in Colombia.

Knowledge regarding new compounds, peptides, and/or secondary metabolites secreted by bacteria isolated from the intestine of phebotominae has the potential to control insect vectors and pathogens (viruses, bacteria, and parasites) transmitted by them. In this respect, twelve Gram-negative bacteria isolated from the intestine of Lutzomyia evansi were selected and screened for their enzymatic, antimicrobial, and leishmanicidal activity. E. cancerogenus, E. aerogenes, P. otitidis, E. cloacae, L. soli, and P. ananatis exhibited enzymatic activity. 83.3% of the isolates displayed lipolytic and nitrate reductase activity and 58.3% of the isolates displayed protease activity. Hemolytic activity (17%) was identified only in E. hormaechei, and P. ananatis. E. cancerogenus, A. calcoaceticus, and P. otitidis showed cellulolytic activity. A. gyllenbergii, P. aeruginosa, and E. hormaechei showed amylolytic activity. In general, the totality of methanolic extracts exhibited antimicrobial activity, where E. hormaechei, A. calcoaceticus, and E. cancerogenus presented the highest activity against the evaluated reference bacteria strains. Cell-free supernatants (CFSS) of the Gram-negative bacteria showed higher growth inhibitory activity against the reference Gram-positive bacteria. The CFS of A. gyllenbergii was the most active antimicrobial in this study, against S. aureus (AAODs = 95.12%) and E. faecalis (AAODs = 86.90%). The inhibition percentages of CFS against Gram-positive bacteria showed statistically significant differences (repeated measure ANOVA df= 2; F= 6.095; P= 0.007832). The E. hormaechei methanolic extract showed leishmanicidal activity (CE-50 µg/ml = 47.7 + 3.8) against metacyclic promastigotes of Leishmania braziliensis (UA301). Based on this finding, we discuss the possible implications of these bacteria in digestion and physiological processes in the Lu. evansi intestine. P. ananatis, E. cloacae, E. hormaechei, and P. otitidis were considered the most promising bacteria in this study and they could potentially be used for biological control.

Species of sand flies (Diptera: Psychodidae) collected from natural reserves in the Pacific and Darien regions of Colombia.

INTRODUCTION: The departments of Chocó and Antioquia in Colombia show climatic and vegetation conditions favoring the establishment of vector species of the genus Lutzomyia and the transmission of Leishmania spp. to human populations entering conserved forest environments. OBJECTIVE: To report the species of Phlebotomine sandflies present in three natural reserves in the Darien and Pacific regions of Colombia. MATERIALS AND METHODS: Sand flies were collected specifically in the natural reserves El Aguacate (Acandí, Chocó), Nabugá (Bahía Solano, Chocó) and Tulenapa (Carepa, Antioquia). Sand flies were collected with CDC light traps, active search in resting places and Shannon traps. The taxonomic determination of species was based on taxonomic keys. For some species of taxonomic interest, we evaluated the partial sequences of the 5' region of COI gene. RESULTS: A total of 611 adult sand flies were collected: 531 in Acandí, 45 in Carepa and 35 in Bahía Solano. Seventeen species of the genus Lutzomyia, three of the genus Brumptomyia and one of the genus Warileya were identified. The genetic distances (K2P) and grouping supported (>99%) in the neighbor joining dendrogram were consistent for most established molecular operational taxonomic units (MOTU) of the Aragaoi group and clearly confirmed the identity of Lu. coutinhoi. CONCLUSION: Species that have importance in the transmission of leishmaniasis in Acandí, Bahía Solano and Carepa were identified. The presence of Lu. coutinhoi was confirmed and consolidated in Colombia.

Especies de flebotomíneos (Diptera: Psychodidae) recolectados en reservas naturales de las regiones del Darién y del Pacífico en Colombia / Species of sand flies (Diptera: Psychodidae) collected from natural reserves in the Pacific and Darien regions of Colombia

Resumen Introducción. Los departamentos de Chocó y Antioquia en Colombia presentan condiciones climáticas y de vegetación que favorecen el establecimiento de especies de vectores del género Lutzomyia y la transmisión de Leishmania spp. a poblaciones humanas que ingresan a ambientes selváticos conservados. Objetivo. Reportar las especies de flebotomíneos presentes en tres reservas naturales de las regiones del Darién y del Pacífico en Colombia. Materiales y métodos. Los flebotomíneos se recolectaron en las reservas naturales El Aguacate (Acandí, Chocó), Nabugá (Bahía Solano, Chocó) y Tulenapa (Carepa, Antioquia). La recolección se hizo con trampas de luz CDC, mediante búsqueda activa en sitios de reposo y con trampas Shannon. La determinación taxonómica de especies se basó en las claves taxonómicas. En algunas especies de interés taxonómico, se evaluó el uso de secuencias parciales de la región 5' del gen COI. Resultados. Se recolectaron 611 flebotomíneos adultos: 531 en Acandí, 45 en Carepa y 35 en Bahía Solano. Se identificaron 17 especies del género Lutzomyia, tres del género Brumptomyia y una del género Warileya. Las distancias genéticas (K2P) y los soportes de agrupación (>99 %) en el dendrograma de neighbor joining correspondieron a la mayoría de unidades taxonómicas operacionales moleculares (Molecular Operational Taxonomic Units, MOTU) establecidas para el grupo Aragaoi y confirmaron claramente la identidad de Lu. coutinhoi. Conclusión. Se identificaron especies que tienen importancia en la transmisión de la leishmaniasis en Acandí, Bahía Solano y Carepa. Se confirmó la presencia de Lu. coutinhoi en Colombia.

Abstract Introduction: The departments of Chocó and Antioquia in Colombia show climatic and vegetation conditions favoring the establishment of vector species of the genus Lutzomyia and the transmission of Leishmania spp. to human populations entering conserved forest environments. Objective: To report the species of Phlebotomine sandflies present in three natural reserves in the Darien and Pacific regions of Colombia. Materials and methods: Sand flies were collected specifically in the natural reserves El Aguacate (Acandí, Chocó), Nabugá (Bahía Solano, Chocó) and Tulenapa (Carepa, Antioquia). Sand flies were collected with CDC light traps, active search in resting places and Shannon traps. The taxonomic determination of species was based on taxonomic keys. For some species of taxonomic interest, we evaluated the partial sequences of the 5' region of COI gene. Results: A total of 611 adult sand flies were collected: 531 in Acandí, 45 in Carepa and 35 in Bahía Solano. Seventeen species of the genus Lutzomyia, three of the genus Brumptomyia and one of the genus Warileya were identified. The genetic distances (K2P) and grouping supported (>99%) in the neighbor joining dendrogram were consistent for most established molecular operational taxonomic units (MOTU) of the Aragaoi group and clearly confirmed the identity of Lu. coutinhoi. Conclusion: Species that have importance in the transmission of leishmaniasis in Acandí, Bahía Solano and Carepa were identified. The presence of Lu. coutinhoi was confirmed and consolidated in Colombia.

DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia.

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.

Fauna de flebotmmíneos (diptera: psychodidae) en acandí (chocó, Colombia) / Fauna of Phlebotominae (Diptera: Psychodidae) in Acandí (Chocó, Colombia)

La región geográfica denominada Chocó-Darién-Caribe es uno de los ecosistemas de bosque húmedo tropical de gran diversidad, pero aún poco explorado. El presente estudio documenta algunas especies de flebotomíneos colectados en un área de transmisión de leishmaniasis cutánea. Se realizó un estudio entomológico en la reserva natural el Aguacate, municipio de Acandí, Chocó. La metodología incluyó el uso de trampas de luz CDC y búsqueda activa en raíces tabulares, colectando con aspiradores bucales. Se recolectaron 1.205 individuos, de los cuales sobresalen Lutzomyia panamensis, Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia sanguinaria, Lutzomyia olmeca bicolor y Lutzomyia hartmanni reconocidos como vectores potenciales de leishmaniasis cutánea en el nuevo mundo. Se relata el hallazgo de 16 especies del género Lutzomyia França y dos especies del género Brumptomyia Sherlock para la costa del Darién-Caribe colombiano, destacando la presencia de las especies Lutzomyia atroclavata (Knab) y Brumptomyia mesai (Sherlock) como nuevos registros para el departamento del Chocó. Este estudio aporta al conocimiento de la fauna de flebotomíneos del municipio de Acandí, Chocó.

The geographic region called Chocó-Darién-Caribe is a tropical forest ecosystem considered of great diversity but still a poorly known region. The current study focuses on some phlebotominae species collected in a geographic area where it has been documented cutaneous leishmaniasis transmission. An entomological study was conducted at the Natural Reserve El Aguacate, in Acandí municipality, Chocó. Sampling methods included light traps such as CDC and collections of adult sand flies in resting sites such as tree buttresses using mouth aspirators devices. The collection findings comprise a total of 1205 phlebotominae adults, with some species of note such as Lutzomyia panamensis, Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia sanguinaria, Lutzomyia olmeca bicolor and Lutzomyia hartmanni which are recognized as potential vectors of cutaneous leishmaniasis in the new world. Sixteen species of the genus Lutzomyia França and two species of the genus Brumptomyia Sherlock are recorded for the colombian Darién-Caribe region. The species Lutzomyia atroclavata (Knab) and Brumptomyia mesai (Sherlock) are new findings in Chocó. This study is a contribution to the phlebotominae species of Acandí municipality in Chocó.