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Background: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings., Methods: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F 1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2-5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed., Results: Results showed location-associated heterogeneity in both F 1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F 1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F 1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F 1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche., Conclusions: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F 1 progeny under normal laboratory conditions, but this may not last beyond the F 1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F 1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings., (© 2021. The Author(s).)

A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Following previously identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, the effects of pyrethroid exposure on the microbiota of F 1 progeny of field-collected Anopheles albimanus. Larval and adult mosquitoes were exposed to the pyrethroids alphacypermethrin (only adults), permethrin, and deltamethrin. While there were no significant differences in bacterial composition between insecticide-resistant and insecticide-susceptible mosquitoes, bacterial composition between insecticide-exposed and non-exposed mosquitoes was significantly different for alphacypermethrin and permethrin exposure. Along with other bacterial taxa not identified to species, Pantoea agglomerans (a known insecticide-degrading bacterial species) and Pseudomonas fragi were more abundant in insecticide-exposed compared to non-exposed adults, demonstrating that insecticide exposure can alter mosquito bacterial communities. We also show for the first time that the cuticle surfaces of both larval and adult An. albimanus harbor more diverse bacterial communities than their internal microbial niches. Together, these findings demonstrate how insecticide pressure could be selecting for certain bacteria within mosquitoes, especially insecticide-metabolizing bacteria, thus potentially contributing to insecticide resistance.

Background: Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America., Methods: Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed., Results: Primers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids., Conclusions: New molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

Decades of unmanaged insecticide use and routine exposure to agrochemicals have left many populations of malaria vectors in the Americas resistant to multiple classes of insecticides, including pyrethroids. The molecular basis of pyrethroid resistance is relatively uncharacterised in American malaria vectors, preventing the design of suitable resistance management strategies. Using whole transcriptome sequencing, we characterized the mechanisms of pyrethroid resistance in Anopheles albimanus from Peru and Guatemala. An. albimanus were phenotyped as either deltamethrin or alpha-cypermethrin resistant. RNA from 1) resistant, 2) unexposed, and 3) a susceptible laboratory strain of An. albimanus was sequenced and analyzed using RNA-Seq. Expression profiles of the three groups were compared based on the current annotation of the An. albimanus reference genome. Several candidate genes associated with pyrethroid resistance in other malaria vectors were found to be overexpressed in resistant An. albimanus. In addition, gene ontology terms related to serine-type endopeptidase activity, extracellular activity and chitin metabolic process were also commonly overexpressed in the field caught resistant and unexposed samples from both Peru and Guatemala when compared to the susceptible strain. The cytochrome P450 CYP9K1 was overexpressed 14x in deltamethrin and 8x in alpha-cypermethrin-resistant samples from Peru and 2x in deltamethrin-resistant samples from Guatemala, relative to the susceptible laboratory strain. CYP6P5 was overexpressed 68x in deltamethrin-resistant samples from Peru but not in deltamethrin-resistant samples from Guatemala. When comparing overexpressed genes between deltamethrin-resistant and alpha-cypermethrin-resistant samples from Peru, a single P450 gene, CYP4C26, was overexpressed 9.8x (p<0.05) in alpha-cypermethrin-resistant samples. In Peruvian deltamethrin-resistant samples, the knockdown resistance mutation (kdr) variant alleles at position 1014 were rare, with approximately 5% frequency, but in the alpha-cypermethrin-resistant samples, the frequency of these alleles was approximately 15-30%. Functional validation of the candidate genes and the kdr mutation as a resistance marker for alpha-cypermethrin will confirm the role of these mechanisms in conferring pyrethroid resistance., Competing Interests: The authors have declared that no competing interests exist.

Background: Anopheles albimanus is a key malaria vector in the northern neotropics. Current vector control measures in the region are based on mass distributions of long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) with pyrethroids. Resistance to pyrethroid insecticides can be mediated by increased esterase and/or multi-function oxidase activity and/or mutations in the voltage-gated sodium channel gene. The aim of this work was to characterize the homologous kdr region of the voltage-gated sodium channel gene in An. albimanus and to conduct a preliminary retrospective analysis of field samples collected in the 1990's, coinciding with a time of intense pyrethroid application related to agricultural and public health insect control in the region., Methods: Degenerate primers were designed to amplify the homologous kdr region in a pyrethroid-susceptible laboratory strain (Sanarate) of An. albimanus. Subsequently, a more specific primer pair was used to amplify and sequence the region that contains the 1014 codon associated with pyrethroid resistance in other Anopheles spp. (L1014F, L1014S or L1014C)., Results: Direct sequencing of the PCR products confirmed the presence of the susceptible kdr allele in the Sanarate strain (L1014) and the presence of homozygous-resistant kdr alleles in field-collected individuals from Mexico (L1014F), Nicaragua (L1014C) and Costa Rica (L1014C)., Conclusions: For the first time, the kdr region in An. albimanus is described. Furthermore, molecular evidence suggests the presence of kdr-type resistance in field-collected An. albimanus in Mesoamerica in the 1990s. Further research is needed to conclusively determine an association between the genotypes and resistant phenotypes, and to what extent they may compromise current vector control efforts.

Fifty microsatellite loci were identified in the malaria vector Anopheles albimanus. Markers segregating in F2 progeny of crosses between laboratory strains of An. albimanus were used to construct a preliminary genetic map. More than 300 progeny were genotyped, but the resolution of the map was limited by the lack of polymorphisms in the microsatellite alleles. A robust linkage map for chromosome 2 was established, and additional markers were assigned to the third and X chromosomes by linkage to morphological markers of known physical location. Additional non-informative microsatellite sequences are provided including some showing similarity to those of An. gambiae. This study significantly increases the number of genetic markers available for An. albimanus and provides useful tools for population genetics and genetic mapping studies in this important malaria vector.

Background: Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5-8 microsatellite loci., Results: We found high levels of polymorphism for all of the Amazonian populations (mean RS = 7.62, mean HO = 0.742), and low levels for the Belize and Guatemalan populations (mean RS = 4.3, mean HO = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (FST = 0.1859 - 0.3901, P < 0.05). Both the DA and FST distance-based trees illustrated the main division to be between Central America and Amazonia., Conclusion: We detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in Ne among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.

Background: The cost of mosquito repellents in Latin America has discouraged their wider use among the poor. To address this problem, a low-cost repellent was developed that reduces the level of expensive repellent actives by combining them with inexpensive fixatives that appear to slow repellent evaporation. The chosen actives were a mixture of para-menthane-diol (PMD) and lemongrass oil (LG)., Methods: To test the efficacy of the repellent, field trials were staged in Guatemala and Peru. Repellent efficacy was determined by human-landing catches on volunteers who wore the experimental repellents, control, or 15% DEET. The studies were conducted using a balanced Latin Square design with volunteers, treatments, and locations rotated each night., Results: In Guatemala, collections were performed for two hours, commencing three hours after repellent application. The repellent provided >98% protection for five hours after application, with a biting pressure of >100 landings per person/hour. The 15% DEET control provided lower protection at 92% (p < 0.0001). In Peru, collections were performed for four hours, commencing two hours after repellent application. The PMD/LG repellent provided 95% protection for six hours after application with a biting pressure of >46 landings per person/hour. The 20% DEET control provided significantly lower protection at 64% (p < 0.0001)., Conclusion: In both locations, the PMD/LG repellent provided excellent protection up to six hours after application against a wide range of disease vectors including Anopheles darlingi. The addition of fixatives to the repellent extended its longevity while enhancing efficacy and significantly reducing its cost to malaria-endemic communities.

Background: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings. Methods: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2–5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed. Results: Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche. Conclusions: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings. [ABSTRACT FROM AUTHOR]