Human-aided dispersal and population bottlenecks facilitate parasitism escape in the most invasive mosquito species.

During biological invasion process, species encounter new environments and partially escape some ecological constraints they faced in their native range, while they face new ones. The Asian tiger mosquito Aedes albopictus is one of the most iconic invasive species introduced in every inhabited continent due to international trade. It has also been shown to be infected by a prevalent yet disregarded microbial entomoparasite Ascogregarina taiwanensis. In this study, we aimed at deciphering the factors that shape the global dynamics of A. taiwanensis infection in natural A. albopictus populations. We showed that A. albopictus populations are highly colonized by several parasite genotypes but recently introduced ones are escaping it. We further performed experiments based on the invasion process to explain such pattern. To that end, we hypothesized that (i) mosquito passive dispersal (i.e. human-aided egg transportation) may affect the parasite infectiveness, (ii) founder effects (i.e. population establishment by a small number of mosquitoes) may influence the parasite dynamics, and (iii) unparasitized mosquitoes are more prompt to found new populations through active flight dispersal. The two first hypotheses were supported as we showed that parasite infection decreases over time when dry eggs are stored and that experimental increase in mosquitoes' density improves the parasite horizontal transmission to larvae. Surprisingly, parasitized mosquitoes tend to be more active than their unparasitized relatives. Finally, this study highlights the importance of global trade as a driver of biological invasion of the most invasive arthropod vector species.

Insecticide resistance intensity and efficacy of synergists with pyrethroids in Anopheles gambiae (Diptera: Culicidae) from Southern Togo.

BACKGROUND: This study was designed to provide insecticide resistance data for decision-making in terms of resistance management plans in Togo. METHODS: The susceptibility status of Anopheles gambiae sensu lato (s.l.) to insecticides used in public health was assessed using the WHO tube test protocol. Pyrethroid resistance intensity bioassays were performed following the CDC bottle test protocol. The activity of detoxification enzymes was tested using the synergists piperonyl butoxide, S.S.S-tributlyphosphorotrithioate and ethacrinic acid. Species-specific identification of An. gambiae s.l. and kdr mutation genotyping were performed using PCR techniques. RESULTS: Local populations of An. gambiae s.l. showed full susceptibility to pirimiphos methyl at Lomé, Kovié, Anié, and Kpèlè Toutou. At Baguida, mortality was 90%, indicating possible resistance to pirimiphos methyl. Resistance was recorded to DDT, bendiocarb, and propoxur at all sites. A high intensity of pyrethroid resistance was recorded and the detoxification enzymes contributing to resistance were oxidases, esterases, and glutathione-s-transferases based on the synergist tests. Anopheles gambiae sensu stricto (s.s.) and Anopheles coluzzii were the main species identified. High kdr L1014F and low kdr L1014S allele frequencies were detected at all localities. CONCLUSION: This study suggests the need to reinforce current insecticide-based malaria control interventions (IRS and LLINs) with complementary tools.

Evaluation of the durability of long-lasting insecticidal nets in Guatemala.

BACKGROUND: Insecticide-treated bed nets (ITNs) are widely used for the prevention and control of malaria. In Guatemala, since 2006, ITNs have been distributed free of charge in the highest risk malaria-endemic areas and constitute one of the primary vector control measures in the country. Despite relying on ITNs for almost 15 years, there is a lack of data to inform the timely replacement of ITNs whose effectiveness becomes diminished by routine use. METHODS: The survivorship, physical integrity, insecticide content and bio-efficacy of ITNs were assessed through cross-sectional surveys conducted at 18, 24 and 32 months after a 2012 distribution of PermaNet® 2.0 in a malaria focus in Guatemala. A working definition of 'LLIN providing adequate protection' was developed based on the combination of the previous parameters and usage of the net. A total of 988 ITNs were analysed (290 at 18 months, 349 at 24 months and 349 at 32 months). RESULTS: The functional survivorship of bed nets decreased over time, from 92% at 18 months, to 81% at 24 months and 69% at 32 months. Independent of the time of the survey, less than 80% of the bed nets that were still present in the household were reported to have been used the night before. The proportion of bed nets categorized as "in good condition" per World Health Organization (WHO) guidelines of the total hole surface area, diminished from 77% to 18 months to 58% at 32 months. The portion of ITNs with deltamethrin concentration less than 10 mg/m2 increased over time. Among the bed nets for which bioassays were conducted, the percentage that met WHO criteria for efficacy dropped from 90% to 18 months to 52% at 32 months. The proportion of long-lasting insecticidal nets (LLINs) providing adequate protection was 38% at 24 months and 21% at 32 months. CONCLUSIONS: At 32 months, only one in five of the LLINs distributed in the campaign provided adequate protection in terms of survivorship, physical integrity, bio-efficacy and usage. Efforts to encourage the community to retain, use, and properly care for the LLINs may improve their impact. Durability assessments should be included in future campaigns.

Evaluation of the durability and use of long-lasting insecticidal nets in Nicaragua.

BACKGROUND: Vector control for malaria prevention relies most often on the use of insecticide-treated bed net (ITNs) and indoor residual spraying. Little is known about the longevity of long-lasting insecticidal nets (LLINs) in the Americas. The physical integrity and insecticide retention of LLINs over time were monitored after a bed net distribution campaign to assess community practices around LLIN care and use in Waspam, northeastern Nicaragua. METHODS: At least 30 nets were collected at 6, 12, 24, and 36 months post distribution. Physical integrity was measured by counting holes and classifying nets into categories (good, damaged, and too torn) depending on a proportionate hole index (pHI). Insecticide bioefficacy was assessed using cone bioassays, and insecticide content measured using a cyanopyrethroid field test (CFT). RESULTS: At 6 months, 87.3 % of LLINs were in good physical condition, while by 36 months this decreased to 20.6 %, with 38.2 % considered 'too torn.' The median pHI increased from 7 at the 6-month time point to 480.5 by 36 months. After 36 months of use, median mortality in cone bioassays was 2 % (range: 0-6 %) compared to 16 % (range: 2-70 %) at 6 months. There was a decrease in the level of deltamethrin detected on the surface of the LLINs with 100 % of tested LLINs tested at 12 months and 24 months crossing the threshold for being considered a failed net by CFT. CONCLUSIONS: This first comprehensive analysis of LLIN durability in Central America revealed rapid loss of chemical bioefficacy and progressive physical damage over a 36-month period. Use of these findings to guide future LLIN interventions in malaria elimination settings in Nicaragua, and potentially elsewhere in the Americas, could help optimize the successful implementation of vector control strategies.

Insecticide resistance status of Aedes aegypti in Bangladesh.

BACKGROUND: Arboviral diseases, including dengue and chikungunya, are major public health concerns in Bangladesh where there have been unprecedented levels of transmission reported in recent years. The primary approach to control these diseases is to control the vector Aedes aegypti using pyrethroid insecticides. Although chemical control has long been practiced, no comprehensive analysis of Ae. aegypti susceptibility to insecticides has been conducted to date. The aim of this study was to determine the insecticide resistance status of Ae. aegypti in Bangladesh and investigate the role of detoxification enzymes and altered target site sensitivity as resistance mechanisms. METHODS: Eggs of Aedes mosquitoes were collected using ovitraps from five districts across Bangladesh and in eight neighborhoods of the capital city Dhaka, from August to November 2017. CDC bottle bioassays were conducted for permethrin, deltamethrin, malathion, and bendiocarb using 3- to 5-day-old F0-F2 non-blood-fed female mosquitoes. Biochemical assays were conducted to detect metabolic resistance mechanisms, and real-time PCR was performed to determine the frequencies of the knockdown resistance (kdr) mutations Gly1016, Cys1534, and Leu410. RESULTS: High levels of resistance to permethrin were detected in all Ae. aegypti populations, with mortality ranging from 0 to 14.8% at the diagnostic dose. Substantial resistance continued to be detected against higher (2×) doses of permethrin (5.1-44.4% mortality). Susceptibility to deltamethrin and malathion varied between populations while complete susceptibility to bendiocarb was observed in all populations. Significantly higher levels of esterase and oxidase activity were detected in most of the test populations as compared to the susceptible reference Rockefeller strain. A significant association was detected between permethrin resistance and the presence of Gly1016 and Cys1534 homozygotes. The frequency of kdr (knockdown resistance) alleles varied across the Dhaka Aedes populations. Leu410 was not detected in any of the tested populations. CONCLUSIONS: The detection of widespread pyrethroid resistance and multiple resistance mechanisms highlights the urgency for implementing alternate Ae. aegypti control strategies. In addition, implementing routine monitoring of insecticide resistance in Ae. aegypti in Bangladesh will lead to a greater understanding of susceptibility trends over space and time, thereby enabling the development of improved control strategies.

Mosquito Control Activities during Local Transmission of Zika Virus, Miami-Dade County, Florida, USA, 2016.

In 2016, four clusters of local mosquitoborne Zika virus transmission were identified in Miami-Dade County, Florida, USA, generating "red zones" (areas into which pregnant women were advised against traveling). The Miami-Dade County Mosquito Control Division initiated intensive control activities, including property inspections, community education, and handheld sprayer applications of larvicides and adulticides. For the first time, the Mosquito Control Division used a combination of areawide ultralow-volume adulticide and low-volume larvicide spraying to effectively control Aedes aegypti mosquitoes, the primary Zika virus vector within the county. The number of mosquitoes rapidly decreased, and Zika virus transmission was interrupted within the red zones immediately after the combination of adulticide and larvicide spraying.

Identification and Pilot Evaluation of Salivary Peptides from Anopheles albimanus as Biomarkers for Bite Exposure and Malaria Infection in Colombia.

Insect saliva induces significant antibody responses associated with the intensity of exposure to bites and the risk of disease in humans. Several salivary biomarkers have been characterized to determine exposure intensity to Old World Anopheles mosquito species. However, new tools are needed to quantify the intensity of human exposure to Anopheles bites and understand the risk of malaria in low-transmission areas in the Americas. To address this need, we conducted proteomic and bioinformatic analyses of immunogenic candidate proteins present in the saliva of uninfected Anopheles albimanus from two separate colonies-one originating from Central America (STECLA strain) and one originating from South America (Cartagena strain). A ~65 kDa band was identified by IgG antibodies in serum samples from healthy volunteers living in a malaria endemic area in Colombia, and a total of five peptides were designed from the sequences of two immunogenic candidate proteins that were shared by both strains. ELISA-based testing of human IgG antibody levels against the peptides revealed that the transferrin-derived peptides, TRANS-P1, TRANS-P2 and a salivary peroxidase peptide (PEROX-P3) were able to distinguish between malaria-infected and uninfected groups. Interestingly, IgG antibody levels against PEROX-P3 were significantly lower in people that have never experienced malaria, suggesting that it may be a good marker for mosquito bite exposure in naïve populations such as travelers and deployed military personnel. In addition, the strength of the differences in the IgG levels against the peptides varied according to location, suggesting that the peptides may able to detect differences in intensities of bite exposure according to the mosquito population density. Thus, the An. albimanus salivary peptides TRANS-P1, TRANS-P2, and PEROX-P3 are promising biomarkers that could be exploited in a quantitative immunoassay for determination of human-vector contact and calculation of disease risk.

Rapid Screening of Aedes aegypti Mosquitoes for Susceptibility to Insecticides as Part of Zika Emergency Response, Puerto Rico.

In response to the 2016 Zika outbreak, Aedes aegypti mosquitoes from 38 locations across Puerto Rico were screened using Centers for Disease Control and Prevention bottle bioassays for sensitivity to insecticides used for mosquito control. All populations were resistant to pyrethroids. Naled, an organophosphate, was the most effective insecticide, killing all mosquitoes tested.

Contrasting patterns of gene expression indicate differing pyrethroid resistance mechanisms across the range of the New World malaria vector Anopheles albimanus.

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.

Monitoring and Predicting Net Longevity by Measuring Surface Levels of Insecticide: Implementing a Faster, Cost Effective, Nondestructive, and Field-Ready Alternative to the World Health Organization Cone Test Bioassay.

An important component of malaria control programs is the ability to assess the effectiveness of the insecticide in insecticide-treated nets (ITNs) during normal usage. The standard technique to measure insecticidal activity is the World Health Organization (WHO) cone test, which in many circumstances, may be difficult to implement. We have evaluated an alternative technique, the colorimetric field test (CFT) on a group of 24-month-old Permanet® 2.0 (Vestergaard-Frandsen, Denmark) nets collected in Colombia. The CFT, which measures surface levels (SL) of deltamethrin is compared with standard high-performance liquid chromatography (HPLC) and the WHO cone test. Effective concentrations of deltamethrin for 80% mortality (EC80) were determined from the CFT and HPLC results. Distribution of insecticide SL after 24 months of use reveal that sampling of the midsection best represents the condition of the entire net. We conclude that the CFT is a practical alternative to the WHO cone test for assessing ITN efficacy.

Spatial variation of insecticide resistance in the dengue vector Aedes aegypti presents unique vector control challenges.

BACKGROUND: Dengue is a major public health problem in Mexico, where the use of chemical insecticides to control the principal dengue vector, Aedes aegypti, is widespread. Resistance to insecticides has been reported in multiple sites, and the frequency of kdr mutations associated with pyrethroid resistance has increased rapidly in recent years. In the present study, we characterized patterns of insecticide resistance in Ae. aegypti populations in five small towns surrounding the city of Merida, Mexico. METHODS: A cross-sectional, entomological survey was performed between June and August 2013 in 250 houses in each of the five towns. Indoor resting adult mosquitoes were collected in all houses and four ovitraps were placed in each study block. CDC bottle bioassays were conducted using F0-F2 individuals reared from the ovitraps and kdr allele (Ile1016 and Cys1534) frequencies were determined. RESULTS: High, but varying, levels of resistance to chorpyrifos-ethyl was detected in all study towns, complete susceptibility to bendiocarb in all except one town, and variations in resistance to deltamethrin between towns, ranging from 63-88% mortality. Significant associations were detected between deltamethrin resistance and the presence of both kdr alleles. Phenotypic resistance was highly predictive of the presence of both alleles, however, not all mosquitoes containing a mutant allele were phenotypically resistant. An analysis of genotypic differentiation (exact G test) between the five towns based on the adult female Ae. aegypti collected from inside houses showed highly significant differences (p < 0.0001) between genotypes for both loci. When this was further analyzed to look for fine scale differences at the block level within towns, genotypic differentiation was significant for both loci in San Lorenzo (Ile1016, p = 0.018 and Cys1534, p = 0.007) and for Ile1016 in Acanceh (p = 0.013) and Conkal (p = 0.031). CONCLUSIONS: The results from this study suggest that 3 years after switching chemical groups, deltamethrin resistance and a high frequency of kdr alleles persisted in Ae. aegypti populations. The spatial variation that was detected in both resistance phenotypes and genotypes has practical implications, both for vector control operations as well as insecticide resistance management strategies.

Novel mutations on the ace-1 gene of the malaria vector Anopheles albimanus provide evidence for balancing selection in an area of high insecticide resistance in Peru.

BACKGROUND: Resistance to multiple classes of insecticides has been detected in the malaria vector Anopheles albimanus in northwest Peru. Acetylcholinesterase (AChE) insensitivity has previously been associated with resistance to organophosphate (OP) and carbamate (CA) insecticides in arthropods. A single point mutation on the ace-1 gene (G119S) associated with resistance to OPs and CAs has been described previously in four anopheline species, but not in field-collected An. albimanus. The present study aimed to characterize the role of ace-1 in conferring resistance to both OPs and CAs in the An. albimanus population in Tumbes, Peru. METHODS: The frequency and intensity of resistance to OPs and CAs was quantified through bioassays of female An. albimanus collected between 2012 and 2014, and the presence of insensitive AChE was confirmed using biochemical assays. A portion of the ace-1 gene flanking codon 119 was amplified and sequenced from individuals used in the bioassays and biochemical assays, as well as from historical samples collected in 2008. Statistical analyses were conducted to determine: (1) associations between genotype and AChE insensitivity; and, (2) associations between genotype and resistance phenotype. RESULTS: After confirming high levels of resistance to fenitrothion, malathion, and bendiocarb through bioassays, two novel polymorphisms were identified at the first and second loci of codon 119, with all individuals from the 2012-2014 collections being heterozygous at the first base (G/T) and either heterozygous (G/C) or homozygous mutants (C/C) at the second base. Based on sequence data from historical samples, these mutations arose prior to 2008, but became fixed in the population between 2008 and 2012. Homozygotes at the second locus had significantly higher levels of AChE insensitivity than heterozygotes (p <0.05). Individuals phenotypically susceptible to OPs and CAs were more likely to be heterozygous at the second locus (p <0.01). Cloning identified four individuals each containing three distinct genotypes, suggesting that a duplication of the ace-1 gene may have occurred. CONCLUSIONS: The occurrence of heterozygotes at two loci and the presence of three genotypes in four individuals suggest that balancing selection could be maintaining OP and CA resistance in this population, while minimizing associated fitness costs.

The Influence of Diet on the Use of Near-Infrared Spectroscopy to Determine the Age of Female Aedes aegypti Mosquitoes.

Interventions targeting adult mosquitoes are used to combat transmission of vector-borne diseases, including dengue. Without available vaccines, targeting the primary vector, Aedes aegypti, is essential to prevent transmission. Older mosquitoes (≥ 7 days) are of greatest epidemiological significance due to the 7-day extrinsic incubation period of the virus. Age-grading of female mosquitoes is necessary to identify post-intervention changes in mosquito population age structure. We developed models using near-infrared spectroscopy (NIRS) to age-grade adult female Ae. aegypti. To determine if diet affects the ability of NIRS models to predict age, two identical larval groups were fed either fish food or infant cereal. Adult females were separated and fed sugar water ± blood, resulting in four experimental groups. Females were killed 1, 4, 7, 10, 13, or 16 days postemergence. The head/thorax of each mosquito was scanned using a near-infrared spectrometer. Scans from each group were analyzed, and multiple models were developed using partial least squares regression. The best model included all experimental groups, and positively predicted the age group (< or ≥ 7 days) of 90.2% mosquitoes. These results suggest both larval and adult diets can affect the ability of NIRS models to accurately assign age categories to female Ae. aegypti.

Evaluation of a rapid colorimetric field test to assess the effective life of long-lasting insecticide-treated mosquito nets in the Lao PDR.

BACKGROUND: Malaria morbidity and mortality have been significantly reduced through the proper use of insecticide-treated mosquito nets, but the extra protection afforded by the insecticide diminishes over time. The insecticide depletion rates vary according to location where wash frequency and wear are influenced by cultural habits as well as the availability of water. Monitoring of available insecticides on the net surface is essential for determining the effective life of the net. Therefore, a rapid and inexpensive colorimetric field test for cyanopyrethroids (Cyanopyrethroid Field Test or CFT) was used to measure surface levels of deltamethrin on insecticide-coated polyester nets (PowerNets™) in rural Lao PDR over a two-year period. METHODS: Net surface levels of deltamethrin were measured by wiping the net with filter paper and measuring the adsorbed deltamethrin using the CFT. A relationship between surface levels of deltamethrin and whole net levels was established by comparing results of the CFT with whole levels assayed by high-performance liquid chromatography (HPLC). An effective deltamethrin surface concentration (EC80) was determined by comparing mosquito mortality (WHO Cone Test) with CFT and HPLC results. Five positions (roof to bottom) on each of 23 matched nets were assayed for deltamethrin surface levels at 6, 12, and 24 months. Mosquito mortality assays (WHO Cone Tests) were performed on a subset of eleven 24-month old nets and compared with the proportion of failed nets as predicted by the CFT. RESULTS: At six months, the nets retained about 80% of the baseline (new net) levels of deltamethrin with no significant differences between net positions. At 12 months, ~15-40%, and at 24 months <10% of deltamethrin was retained on the nets, with significant differences appearing between positions. Results from the CFT show that 93% of the nets failed (deltamethrin surface levels