Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

Copy number variants underlie the major selective sweeps in insecticide resistance genes in Anopheles arabiensis from Tanzania.

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis, which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis.

Signatures of adaptation at key insecticide resistance loci in Anopheles gambiae in Southern Ghana revealed by reduced-coverage WGS.

Resistance to insecticides and adaptation to a diverse range of environments present challenges to Anopheles gambiae s.l. mosquito control efforts in sub-Saharan Africa. Whole-genome-sequencing is often employed for identifying the genomic basis underlying adaptation in Anopheles, but remains expensive for large-scale surveys. Reduced coverage whole-genome-sequencing can identify regions of the genome involved in adaptation at a lower cost, but is currently untested in Anopheles mosquitoes. Here, we use reduced coverage WGS to investigate population genetic structure and identify signatures of local adaptation in Anopheles mosquitoes across southern Ghana. In contrast to previous analyses, we find no structuring by ecoregion, with Anopheles coluzzii and Anopheles gambiae populations largely displaying the hallmarks of large, unstructured populations. However, we find signatures of selection at insecticide resistance loci that appear ubiquitous across ecoregions in An. coluzzii, and strongest in forest ecoregions in An. gambiae. Our study highlights resistance candidate genes in this region, and validates reduced coverage WGS, potentially to very low coverage levels, for population genomics and exploratory surveys for adaptation in Anopheles taxa.

Parallel evolution in mosquito vectors - a duplicated esterase locus is associated with resistance to pirimiphos-methyl in An. gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programmes. The organophosphate, pirimiphos-methyl, is a relatively new chemical in the vector control armoury but is now widely used in indoor residual spray campaigns. Whilst generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in Anopheles gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to organophosphates in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in An. gambiae, An. coluzzii and An. arabiensis. As in Cx. pipiens, copy number variation seems to play a role in the evolution of insecticide resistance at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in Anopheles gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programmes.

Kdr genotyping and the first report of V410L and V1016I kdr mutations in voltage-gated sodium channel gene in Aedes aegypti (Diptera: Culicidae) from Iran.

BACKGROUND: Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. METHODS: Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. RESULTS: Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. CONCLUSIONS: The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible.

Molecular surveillance of insecticide resistance in Phlebotomus argentipes targeted by indoor residual spraying for visceral leishmaniasis elimination in India.

Molecular surveillance of resistance is an increasingly important part of vector borne disease control programmes that utilise insecticides. The visceral leishmaniasis (VL) elimination programme in India uses indoor residual spraying (IRS) with the pyrethroid, alpha-cypermethrin to control Phlebotomus argentipes the vector of Leishmania donovani, the causative agent of VL. Prior long-term use of DDT may have selected for knockdown resistance (kdr) mutants (1014F and S) at the shared DDT and pyrethroid target site, which are common in India and can also cause pyrethroid cross-resistance. We monitored the frequency of these marker mutations over five years from 2017-2021 in sentinel sites in eight districts of north-eastern India covered by IRS. Frequencies varied markedly among the districts, though finer scale variation, among villages within districts, was limited. A pronounced and highly significant increase in resistance-associated genotypes occurred between 2017 and 2018, but with relative stability thereafter, and some reversion toward more susceptible genotypes in 2021. Analyses linked IRS with mutant frequencies suggesting an advantage to more resistant genotypes, especially when pyrethroid was under-sprayed in IRS. However, this advantage did not translate into sustained allele frequency changes over the study period, potentially because of a relatively greater net advantage under field conditions for a wild-type/mutant genotype than projected from laboratory studies and/or high costs of the most resistant genotype. Further work is required to improve calibration of each 1014 genotype with resistance, preferably using operationally relevant measures. The lack of change in resistance mechanism over the span of the study period, coupled with available bioassay data suggesting susceptibility, suggests that resistance has yet to emerge despite intensive IRS. Nevertheless, the advantage of resistance-associated genotypes with IRS and under spraying, suggest that measures to continue monitoring and improvement of spray quality are vital, and consideration of future alternatives to pyrethroids for IRS would be advisable.

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.

Advances in the genetic characterization of the malaria vector, Anopheles funestus, and implications for improved surveillance and control.

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

Age-dependence of susceptibility to single and repeated deltamethrin exposure in pyrethroid-resistant Aedes aegypti strains.

Monitoring insecticide resistance is crucial in disease-transmitting mosquitoes to allow assessment of viable candidate insecticides to use for control and to provide indication of changes in resistance. Insecticide resistance bioassays are typically performed on young female mosquitoes, yet disease is transmitted by older females, which may also have encountered insecticide multiple times during their adult life. If insecticide mortality rates increase with age directly, or indirectly via cumulative toxicity from repeated exposure, the strategy of testing young mosquitoes as the least susceptible cohort would be supported. We tested three hypotheses via examination of how age and cumulative exposure impact mortality rates to the pyrethroid deltamethrin in strains of Aedes aegypti from Jeddah, Saudi Arabia and the Cayman Islands, which show differences in resistance mechanisms. Females of different ages (5, 7, 10 and 14 days-old) were exposed using WHO tube assays to either a single dose of insecticide, or in a second experiment females (initially 5 days-old) were exposed daily over 10 days. Age only increased mortality in the Jeddah strain at 14 days-old and had no impact on the Cayman strain. This is consistent with greater impact linked to metabolic resistance in the Jeddah strain, though results from qPCR of four candidate genes, failed to provide evidence for a candidate underpinning an age-dependent change in resistance. With repeated exposure, mortality rates of surviving females decreased to very low levels, suggesting that surviving older cohorts of females may exhibit substantially lower susceptibility than young females in single exposure assays. Our results indicate that testing young females with a single insecticide exposure should capture minimum susceptibility for the majority of the population, but a small fraction of older females may prove particularly unresponsive to pyrethroid-based control measures.

Insecticide resistance management strategies for public health control of mosquitoes exhibiting polygenic resistance: A comparison of sequences, rotations, and mixtures.

Malaria control uses insecticides to kill Anopheles mosquitoes. Recent successes in malaria control are threatened by increasing levels of insecticide resistance (IR), requiring insecticide resistance management (IRM) strategies to mitigate this problem. Field trials of IRM strategies are usually prohibitively expensive with long timeframes, and mathematical modeling is often used to evaluate alternative options. Previous IRM models in the context of malaria control assumed IR to have a simple (monogenic) basis, whereas in natural populations, IR will often be a complex polygenic trait determined by multiple genetic variants. A quantitative genetics model was developed to model IR as a polygenic trait. The model allows insecticides to be deployed as sequences (continuous deployment until a defined withdrawal threshold, termed "insecticide lifespan", as indicated by resistance diagnosis in bioassays), rotations (periodic switching of insecticides), or full-dose mixtures (two insecticides in one formulation). These IRM strategies were compared based on their "strategy lifespan" (capped at 500 generations). Partial rank correlation and generalized linear modeling was used to identify and quantify parameters driving the evolution of resistance. Random forest models were used to identify parameters offering predictive value for decision-making. Deploying single insecticides as sequences or rotations usually made little overall difference to their "strategy lifespan", though rotations displayed lower mean and peak resistances. Deploying two insecticides in a full-dose mixture formulation was found to extend the "strategy lifespan" when compared to deploying each in sequence or rotation. This pattern was observed regardless of the level of cross resistance between the insecticides or the starting level of resistance. Statistical analysis highlighted the importance of insecticide coverage, cross resistance, heritability, and fitness costs for selecting an appropriate IRM strategy. Full-dose mixtures appear the most promising of the strategies evaluated, with the longest "strategy lifespans". These conclusions broadly corroborate previous results from monogenic models.

Association of 410L, 1016I and 1534C kdr mutations with pyrethroid resistance in Aedes aegypti from Ouagadougou, Burkina Faso, and development of a one-step multiplex PCR method for the simultaneous detection of 1534C and 1016I kdr mutations.

BACKGROUND: Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks, making dengue an increasingly important health concern for the country. Previous studies in Burkina Faso reported that resistance of Aedes aegypti to pyrethroid insecticides was associated with the F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides, likely supported by mutations in the voltage-gated sodium channel, here evidenced by genotyping the kdr SNPs V410L, V1016I and F1534C. We also describe a new multiplex PCR-based diagnostic of F1534C and V1016I kdr SNPs. METHODS: Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to permethrin (15 µg/ml) and deltamethrin (10 µg/ml) was tested using bottles and to malathion (5%) using WHO tube tests. All bioassays used 1-h exposure and mortality recorded 24 h post-exposure. Bioassay results were interpreted according to WHO thresholds for resistance diagnosis. The kdr mutations were screened using AS-PCR and TaqMan methods in exposed and non-exposed Aedes mosquitoes. RESULTS: Females from all health districts were resistant to permethrin and deltamethrin (< 20% mortality) but were fully susceptible to 5% malathion. The F1534C and V1016I kdr mutations were successfully detected using a newly developed multiplex PCR in perfect agreement with TaqMan method. The 1534C/1016I/410L haplotype was correlated with permethrin resistance but not with deltamethrin resistance; however, the test power was limited by a low frequency of dead individuals in deltamethrin exposure. CONCLUSIONS: Resistance to pyrethroid insecticides is associated with kdr mutant haplotypes, while the absence of substantial resistance to malathion suggests that it remains a viable option for dengue vector control in Ouagadougou.

Improving the efficiency of aerosolized insecticide testing against mosquitoes.

Developing robust and standardised approaches for testing mosquito populations against insecticides is vital for understanding the effectiveness of new active ingredients or formulations. Methods for testing mosquito susceptibility against contact insecticides or products, such as those delivered through public health programmes, are well-established and standardised. Nevertheless, approaches for testing volatile or aerosolized insecticides used in household products can be challenging to implement efficiently. We adapted WHO guidelines for household insecticides to develop a standardised and higher-throughput methodology for testing aerosolized products in a Peet Grady test chamber (PG-chamber) using caged mosquitoes and an efficient decontamination method. The new approach was validated using insecticide resistant and susceptible Aedes and Anopheles mosquito colonies. An added feature is the inclusion of cage-facing cameras to allow real-time quantification of knockdown following insecticide exposure. The wipe-based decontamination method was highly effective for removing pyrethroids' aerosolized oil-based residues from chamber surfaces, with < 2% mortality recorded for susceptible mosquitoes tested directly on the surfaces. There was no spatial heterogeneity for knockdown or mortality of caged mosquitoes within the PG chamber. The dual-cage approach we implement yields eight-times the throughput compared to a free-flight protocol, allows simultaneous testing of different mosquito strains and effectively discriminates susceptible and resistant mosquito colonies tested side-by-side.

Insecticide resistance status of Aedes aegypti in southern and northern Ghana.

BACKGROUND: Outbreaks of Aedes-borne arboviral diseases are becoming rampant in Africa. In Ghana, there is no organized arboviral control programme with interventions restricted to mitigate outbreaks. Insecticide application is a crucial part of outbreak responses and future preventative control measures. Thus, knowledge of the resistance status and underlying mechanisms of Aedes populations is required to ensure optimal insecticide choices. The present study assessed the insecticide resistance status of Aedes aegypti populations from southern Ghana (Accra, Tema and Ada Foah) and northern Ghana (Navrongo) respectively. METHODS: Phenotypic resistance was determined with WHO susceptibility tests using Ae. aegypti collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific PCR. Synergist assays were performed with piperonyl butoxide (PBO) to investigate the possible involvement of metabolic mechanisms in resistance phenotypes. RESULTS: Resistance to DDT was moderate to high across sites (11.3 to 75.8%) and, for the pyrethroids deltamethrin and permethrin, moderate resistance was detected (62.5 to 88.8%). The 1534C kdr and 1016I kdr alleles were common in all sites (0.65 to 1) and may be on a trajectory toward fixation. In addition, a third kdr mutant, V410L, was detected at lower frequencies (0.03 to 0.31). Pre-exposure to PBO significantly increased the susceptibility of Ae. aegypti to deltamethrin and permethrin (P < 0.001). This indicates that in addition to kdr mutants, metabolic enzymes (monooxygenases) may be involved in the resistance phenotypes observed in the Ae. aegypti populations in these sites. CONCLUSION: Insecticide resistance underpinned by multiple mechanisms in Ae. aegypti indicates the need for surveillance to assist in developing appropriate vector control strategies for arboviral disease control in Ghana.

Residual malaria transmission and the role of Anopheles arabiensis and Anopheles melas in central Senegal.

Understanding the behavior and ecology of local malaria vectors is essential for the effectiveness of the commonly used vector-targeted malaria control tools in areas of low malaria transmission. This study was conducted to determine species composition, biting behavior and infectivity of the major Anopheles vectors of Plasmodium falciparum in low transmission settings in central Senegal. Adult mosquitoes were collected using human landing catches during 2 consecutive nights and Pyrethrum Spray Catches in 30-40 randomly selected rooms, from July 2017 to December 2018 in 3 villages. Anopheline mosquitoes were morphologically identified using conventional keys; their reproductive status assessed by ovary dissections, and a sub-sample of Anopheles gambiae s.l. were identified to species level using polymerase chain reaction (PCR). Plasmodium sporozoite infections were detected using real-time quantitative PCR. During this study 3684 Anopheles were collected of which 97% were An. gambiae s.l., 0.6% were Anopheles funestus, and 2.4% were Anopheles pharoensis. Molecular identification of 1,877 An. gambiae s.l. revealed a predominance of Anopheles arabiensis (68.7%), followed by Anopheles melas (28.8%), and Anopheles coluzzii (2.1%). The overall human-biting rate of An. gambiae s.l. was highest in the inland site of Keur Martin with 4.92 bites per person per night, while it was similar in the deltaic site, Diofior (0.51) and the coastal site, Mbine Coly (0.67). Parity rates were similar in An. arabiensis (45%) and An. melas (42%). Sporozoite infections were detected in both An. arabiensis and An. melas with the respective infection rates of 1.39% (N = 8) and 0.41% (N = 1). Results suggest that low residual malaria in central Senegal is transmitted by An. arabiensis and An. melas. Consequently, both vectors will need to be targeted as part of malaria elimination efforts in this area of Senegal.

RNA-Seq-Pop: Exploiting the sequence in RNA sequencing-A Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae.

We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to-end analysis of RNA sequencing data sets. The workflow allows the user to perform quality control, perform differential expression analyses and call genomic variants. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure, and genome-wide selection scans, together with clear visualizations. RNA-Seq-Pop is applicable to any organism, and we demonstrate the utility of the workflow by investigating pyrethroid resistance in selected strains of the major malaria mosquito, Anopheles gambiae. The workflow provides additional modules specifically for An. gambiae, including estimating recent ancestry and determining the karyotype of common chromosomal inversions. The Busia laboratory colony used for selections was collected in Busia, Uganda, in November 2018. We performed a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-selected G28 offspring; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns consistent with that of laboratory colonization and selection, with the parental Busia strain exhibiting the highest nucleotide diversity, followed by the selected Busia offspring, and finally, Kisumu. Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S target-site mutation, upregulation of SAP genes, P450s and a cluster of carboxylesterases. During deltamethrin selections, the 2La chromosomal inversion rose in frequency (from 33% to 86%), supporting a previous link with pyrethroid resistance. RNA-Seq-Pop is hosted at: github.com/sanjaynagi/rna-seq-pop. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An. gambiae and other taxa.

Confirmation of the presence of Anopheles stephensi among internally displaced people's camps and host communities in Aden city, Yemen.

BACKGROUND: Declines in global malaria cases and deaths since the millennium are currently challenged by multiple factors including funding limitations, limits of, and resistance to vector control tools, and also recent spread of the invasive vector species, Anopheles stephensi-especially into novel urban settings where malaria rates are typically low. Coupled with general increases in urbanization and escalations in the number of conflicts creating rapid and unplanned population displacement into temporary shelter camps within host urban areas, particularly in the Middle East and sub-Saharan Africa, increased urban malaria is a major threat to control and elimination. METHODS: Entomological monitoring surveys (targeting Aedes aegypti) of water containers across urban areas hosting internally displaced people (IDP) communities in Aden city, Yemen, were performed by The MENTOR Initiative, a non-governmental organisation. As part of these surveys in 2021 23 larvae collected and raised to adults were morphologically identified as An. stephensi. Twelve of the samples were sent to Liverpool School of Tropical Medicine for independent morphological assessment and genetic analysis by sequencing the ribosomal ITS2 region and the mitochondrial COI gene. RESULTS: All twelve samples were confirmed morphologically and by sequence comparison of the single ITS2 and COI haplotype detected to the NCBI BLAST database as An. stephensi. Phylogenetic analysis with comparable COI sequences indicated close relationship to haplotypes found in Djibouti and Ethiopia. CONCLUSION: The study results confirm the presence of An. stephensi in Yemen. Confirmation of the species in multiple urban communities hosting thousands of IDPs living in temporary shelters with widescale dependency on open water containers is of particular concern due to the vulnerability of the population and abundance of favourable breeding sites for the vector. Proactive monitoring and targeted integrated vector management are required to limit impacts in this area of typically low malaria transmission, and to prevent further the spread of An. stephensi within the region.

Characterizing pyrethroid resistance and mechanisms in Anopheles gambiae (s.s.) and Anopheles arabiensis from 11 districts in Uganda.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n â€‹= â€‹3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n â€‹= â€‹4); and where IRS had been sustained since 2014 ('IRS active', n â€‹= â€‹4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P â€‹< â€‹0.001; deltamethrin: 24.6 vs 83.7%, P â€‹< â€‹0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P â€‹< â€‹0.001; deltamethrin: 25.6 vs 88.9%, P â€‹< â€‹0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control.

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae s.l.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of the most widespread tools currently used to control malaria. The genetic underpinnings of resistance are still only partially understood, with much of the variance in resistance phenotype left unexplained. We performed a multi-country large scale genome-wide association study of resistance to two insecticides widely used in malaria control: deltamethrin and pirimiphos-methyl. Using a bioassay methodology designed to maximise the phenotypic difference between resistant and susceptible samples, we sequenced 969 phenotyped female An. gambiae and An. coluzzii from ten locations across four countries in West Africa (Benin, Côte d'Ivoire, Ghana and Togo), identifying single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) segregating in the populations. The patterns of resistance association were highly multiallelic and variable between populations, with different genomic regions contributing to resistance, as well as different mutations within a given region. While the strongest and most consistent association with deltamethrin resistance came from the region around Cyp6aa1 , this resistance was based on a combination of several independent CNVs in An. coluzzii , and on a non-CNV bearing haplotype in An. gambiae . Further signals involved a range of cytochrome P450, mitochondrial, and immunity genes. Similarly, for pirimiphos-methyl, while the strongest signal came from the region of Ace1 , more widespread signals included cytochrome P450s, glutathione S-transferases, and a subunit of the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes were associated with resistance to both insecticide classes, suggesting possible cross-resistance mechanisms. These locally-varying, multigenic and multiallelic patterns highlight the challenges involved in genomic monitoring and surveillance of resistance, and form the basis for improvement of methods used to detect and predict resistance. Based on simulations of resistance variants, we recommend that yet larger scale studies, exceeding 500 phenotyped samples per population, are required to better identify associated genomic regions.

Asymptomatic Plasmodium infection among primary schoolchildren and Anopheles-mediated malaria transmission: A cross-sectional study in Ouidah; south-western Benin.

Understanding the contribution of asymptomatic Plasmodium carriers in malaria transmission might be helpful to design and implement new control measures. The present study explored the prevalence of asymptomatic and symptomatic Plasmodium infections (asexual and sexual stages) and the contribution of asymptomatic P. falciparum carriers to Anopheles-mediated malaria transmission in Ouidah (Benin). Thick and thin blood smears were examined from finger-prick blood specimens using light microscopy, and the density of both asexual and sexual stages of Plasmodium species was calculated. Infectivity of gametocyte-infected blood samples to Anopheles gambiae was assessed through direct membrane feeding assays. The prevalence of asymptomatic Plasmodium infections was 28.73% (289/1006). All the asymptomatic gametocyte-carriers (19/19), with gametocytaemia ranging from 10 - 1200 gametocytes/µL of blood, were infectious to An. gambiae mosquitoes. The mean oocyst prevalences varied significantly (χ 2  = 16.42, df = 7, p = 0.02) among laboratory mosquito strains (6.9 - 39.4%) and near-field mosquitoes (4.9 - 27.2%). Likewise, significant variation (χ 2  = 56.85, df = 7, p = 6.39 × 10-10) was observed in oocyst intensity. Our findings indicate that asymptomatic Plasmodium carriers could significantly contribute to malaria transmission. Overall, this study highlights the importance of diagnosing and treating asymptomatic and symptomatic infection carriers during malaria control programmes.