Species composition and insecticide resistance in malaria vectors in Ellibou, southern Côte d'Ivoire and first finding of Anopheles arabiensis in Côte d'Ivoire.

BACKGROUND: Knowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d'Ivoire. METHODS: A 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR). RESULTS: Overall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2. CONCLUSION: This is the first documented presence of An. arabiensis in Côte d'Ivoire. Its detection - together with a recent finding further north of the country - confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

Fine-scale spatial distribution of deltamethrin resistance and population structure of Anopheles funestus and Anopheles arabiensis populations in Southern Mozambique.

BACKGROUND: Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages. METHODS: Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. RESULTS: Phenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy-Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (- 0.00457 to 0.04213), Nm values were high (9.4-71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations. CONCLUSION: Despite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

Anopheles rufipes implicated in malaria transmission both indoors and outdoors alongside Anopheles funestus and Anopheles arabiensis in rural south-east Zambia.

BACKGROUND: The primary malaria vector-control interventions, indoor residual spraying and long-lasting insecticidal nets, are effective against indoor biting and resting mosquito species. Consequently, outdoor biting and resting malaria vectors might elude the primary interventions and sustain malaria transmission. Varied vector biting and resting behaviour calls for robust entomological surveillance. This study investigated the bionomics of malaria vectors in rural south-east Zambia, focusing on species composition, their resting and host-seeking behaviour and sporozoite infection rates. METHODS: The study was conducted in Nyimba District, Zambia. Randomly selected households served as sentinel houses for monthly collection of mosquitoes indoors using CDC-light traps (CDC-LTs) and pyrethrum spray catches (PSC), and outdoors using only CDC-LTs for 12 months. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further identified using molecular techniques. Plasmodium falciparum sporozoite infection was determined using sandwich enzyme-linked immunosorbent assays. RESULTS: From 304 indoor and 257 outdoor light trap-nights and 420 resting collection, 1409 female Anopheles species mosquitoes were collected and identified morphologically; An. funestus (n = 613; 43.5%), An. gambiae sensu lato (s.l.)(n = 293; 20.8%), Anopheles pretoriensis (n = 282; 20.0%), Anopheles maculipalpis (n = 130; 9.2%), Anopheles rufipes (n = 55; 3.9%), Anopheles coustani s.l. (n = 33; 2.3%), and Anopheles squamosus (n = 3, 0.2%). Anopheles funestus sensu stricto (s.s.) (n = 144; 91.1%) and Anopheles arabiensis (n = 77; 77.0%) were the dominant species within the An. funestus group and An. gambiae complex, respectively. Overall, outdoor CDC-LTs captured more Anopheles mosquitoes (mean = 2.25, 95% CI 1.22-3,28) than indoor CDC-LTs (mean = 2.13, 95% CI 1.54-2.73). Fewer resting mosquitoes were collected with PSC (mean = 0.44, 95% CI 0.24-0.63). Sporozoite infectivity rates for An. funestus, An. arabiensis and An. rufipes were 2.5%, 0.57% and 9.1%, respectively. Indoor entomological inoculation rates (EIRs) for An. funestus s.s, An. arabiensis and An. rufipes were estimated at 4.44, 1.15 and 1.20 infectious bites/person/year respectively. Outdoor EIRs for An. funestus s.s. and An. rufipes at 7.19 and 4.31 infectious bites/person/year, respectively. CONCLUSION: The findings of this study suggest that An. rufipes may play an important role in malaria transmission alongside An. funestus s.s. and An. arabiensis in the study location.

Rosette formation by Plasmodium vivax gametocytes favors the infection in Anopheles aquasalis.

Plasmodium vivax is a public health problem and the most common type of malaria outside sub-Saharan Africa. The capacity of cytoadhesion, rosetting, and liver latent phase development could impact treatment and disease control. Although the ability to P. vivax gametocyte develop rosetting is known, it is not yet clear which role it plays during the infection and transmission process to the mosquito. Here, we used ex vivo approaches for evaluate the rosetting P. vivax gametocytes capacity and we have investigated the effect of this adhesive phenotype on the infection process in the vector Anopheles aquasalis mosquito. Rosette assays were performed in 107 isolates, and we have observed an elevated frequency of cytoadhesive phenomena (77,6%). The isolates with more than 10% of rosettes have presented a higher infection rate in Anopheles aquasalis (p=0.0252). Moreover, we found a positive correlation between the frequency of parasites in rosetting with the infection rate (p=0.0017) and intensity (p=0.0387) in the mosquito. The disruption of P. vivax rosette formation through mechanical rupture assay confirmed the previously findings, since the paired comparison showed that isolates with disrupted rosettes have a lower infection rate (p<0.0001) and intensity (p=0.0003) compared to the control group (no disruption). Herein we have demonstrated for the first time a potential effect of the rosette phenomenon on the infection process in the mosquito vector An. aquasalis, favoring its capacity and intensity of infection, thus allowing the perpetuation of the parasite cycle life.

The interplay between malaria vectors and human activity accounts for high residual malaria transmission in a Burkina Faso village with universal ITN coverage.

BACKGROUND: Mosquito and human behaviour interaction is a key determinant of the maximum level of protection against malaria that can be provided by insecticide-treated nets (ITNs). Nevertheless, scant literature focuses on this interaction, overlooking a fundamental factor for efficient malaria control. This study aims to estimate malaria transmission risk in a Burkina Faso village by integrating vector biting rhythms with some key information about human habits. METHODS: Indoor/outdoor human landing catches were conducted for 16 h (16:00-08:00) during 8 nights (September 2020) in Goden village. A survey about net usage and sleeping patterns was submitted to half the households (October-December 2020). A subsample of collected specimens of Anopheles gambiae sensu lato was molecularly processed for species identification, Plasmodium detection from heads-thoraxes and L1014F pyrethroid-resistance allele genotyping. Hourly mosquito abundance was statistically assessed by GLM/GAM, and the entomological inoculation rate (EIR) was corrected for the actual ITN usage retrieved from the questionnaire. RESULTS: Malaria transmission was mainly driven by Anopheles coluzzii (68.7%) followed by A. arabiensis (26.2%). The overall sporozoite rate was 2% with L1014F estimated frequency of 0.68 (N = 1070 out of 15,201 A. gambiae s.l. collected). No major shift in mosquito biting rhythms in response to ITN or differences between indoor and outdoor catches were detected. Impressive high biting pressure (mean 30.3 mosquitoes/person/hour) was exerted from 20:00 to 06:00 with a peak at 4:00. Human survey revealed that nearly all inhabitants were awake before 20:00 and after 7:00 and at least 8.7% had no access to bednets. Adjusting for anthropological data, the EIR dropped from 6.7 to 1.2 infective bites/person/16 h. In a scenario of full net coverage and accounting only for the human sleeping patterns, the daily malaria transmission risk not targetable by ITNs was 0.69 infective bites. CONCLUSIONS: The high mosquito densities and interplay between human/vector activities means that an estimated 10% of residual malaria transmission cannot be prevented by ITNs in the village. Locally tailored studies, like the current one, are essential to explore the heterogeneity of human exposure to infective bites and, consequently, to instruct the adoption of new vector control tools strengthening individual and community protection.

Trends of insecticide resistance monitoring in mainland Tanzania, 2004-2020.

BACKGROUND: Insecticide resistance is a serious threat to the continued effectiveness of insecticide-based malaria vector control measures, such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). This paper describes trends and dynamics of insecticide resistance and its underlying mechanisms from annual resistance monitoring surveys on Anopheles gambiae sensu lato (s.l.) populations conducted across mainland Tanzania from 2004 to 2020. METHODS: The World Health Organization (WHO) standard protocols were used to assess susceptibility of the wild female An. gambiae s.l. mosquitoes to insecticides, with mosquitoes exposed to diagnostic concentrations of permethrin, deltamethrin, lambdacyhalothrin, bendiocarb, and pirimiphos-methyl. WHO test papers at 5× and 10× the diagnostic concentrations were used to assess the intensity of resistance to pyrethroids; synergist tests using piperonyl butoxide (PBO) were carried out in sites where mosquitoes were found to be resistant to pyrethroids. To estimate insecticide resistance trends from 2004 to 2020, percentage mortalities from each site and time point were aggregated and regression analysis of mortality versus the Julian dates of bioassays was performed. RESULTS: Percentage of sites with pyrethroid resistance increased from 0% in 2004 to more than 80% in the 2020, suggesting resistance has been spreading geographically. Results indicate a strong negative association (p = 0.0001) between pyrethroids susceptibility status and survey year. The regression model shows that by 2020 over 40% of An. gambiae mosquitoes survived exposure to pyrethroids at their respective diagnostic doses. A decreasing trend of An. gambiae susceptibility to bendiocarb was observed over time, but this was not statistically significant (p = 0.8413). Anopheles gambiae exhibited high level of susceptibility to the pirimiphos-methyl in sampled sites. CONCLUSIONS: Anopheles gambiae Tanzania's major malaria vector, is now resistant to pyrethroids across the country with resistance increasing in prevalence and intensity and has been spreading geographically. This calls for urgent action for efficient malaria vector control tools to sustain the gains obtained in malaria control. Strengthening insecticide resistance monitoring is important for its management through evidence generation for effective malaria vector control decision.

The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites.

Malaria is caused by the unicellular parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, Saglin can be detected in high amounts in the midgut of mosquitoes after blood ingestion, possibly indicating a previously unknown host-pathogen interaction between Saglin and midgut stages of Plasmodium. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.

Resistance to Pyrethroids in the Malaria Vector Anopheles albimanus in Two Important Villages in the Soconusco Region of Chiapas, Mexico, 2022.

Chiapas State comprises the largest malaria foci from Mexico, and 57% of the autochthonous cases in 2021, all with Plasmodium vivax infections, were reported in this State. Southern Chiapas is at constant risk of cases imported due to migratory human flow. Since chemical control of vector mosquitoes is the main entomological action implemented for the prevention and control of vector-borne diseases, this work aimed to investigate the susceptibility of Anopheles albimanus to insecticides. To this end, mosquitoes were collected in cattle in two villages in southern Chiapas in July-August 2022. Two methods were used to evaluate the susceptibility: the WHO tube bioassay and the CDC bottle bioassay. For the latter, diagnostic concentrations were calculated. The enzymatic resistance mechanisms were also analyzed. CDC diagnostic concentrations were obtained; 0.7 µg/mL deltamethrin, 12 µg/mL permethrin, 14.4 µg/mL malathion, and 2 µg/mL chlorpyrifos. Mosquitoes from Cosalapa and La Victoria were susceptible to organophosphates and to bendiocarb, but resistant to pyrethroids, with mortalities between 89% and 70% (WHO), and 88% and 78% (CDC), for deltamethrin and permethrin, respectively. High esterase levels are suggested as the resistance mechanism involved in the metabolism of pyrethroids in mosquitoes from both villages. Mosquitoes from La Victoria might also involve cytochrome P450. Therefore, organophosphates and carbamates are suggested to currently control An. albimanus. Its use might reduce the frequency of resistance genes to pyrethroids and vector abundance and may impede the transmission of malaria parasites.

Mapping current and future thermal limits to suitability for malaria transmission by the invasive mosquito Anopheles stephensi.

BACKGROUND: Anopheles stephensi is a malaria-transmitting mosquito that has recently expanded from its primary range in Asia and the Middle East, to locations in Africa. This species is a competent vector of both Plasmodium falciparum and Plasmodium vivax malaria. Perhaps most alarming, the characteristics of An. stephensi, such as container breeding and anthropophily, make it particularly adept at exploiting built environments in areas with no prior history of malaria risk. METHODS: In this paper, global maps of thermal transmission suitability and people at risk (PAR) for malaria transmission by An. stephensi were created, under current and future climate. Temperature-dependent transmission suitability thresholds derived from recently published species-specific thermal curves were used to threshold gridded, monthly mean temperatures under current and future climatic conditions. These temperature driven transmission models were coupled with gridded population data for 2020 and 2050, under climate-matched scenarios for future outcomes, to compare with baseline predictions for 2020 populations. RESULTS: Using the Global Burden of Disease regions approach revealed that heterogenous regional increases and decreases in risk did not mask the overall pattern of massive increases of PAR for malaria transmission suitability with An. stephensi presence. General patterns of poleward expansion for thermal suitability were seen for both P. falciparum and P. vivax transmission potential. CONCLUSIONS: Understanding the potential suitability for An. stephensi transmission in a changing climate provides a key tool for planning, given an ongoing invasion and expansion of the vector. Anticipating the potential impact of onward expansion to transmission suitable areas, and the size of population at risk under future climate scenarios, and where they occur, can serve as a large-scale call for attention, planning, and monitoring.

Insights into the elimination of vivax malaria in China.

BACKGROUND: Malaria is caused by multiple parasitic species of the genus Plasmodium. Plasmodium vivax is the most geographically widespread and poses challenges in elimination due to its unique biological and epidemiological characteristics. The aim of study was to highlight the practices and experience targeting vivax malaria control and elimination in China. MAIN BODY: P. vivax malaria was historically endemic in more than 70% of counties in China, with reported vivax malaria cases as high as 26 million a year. After around 70 years of effort, China was certified as malaria-free in June of 2021. The key insights into China's vivax malaria control and elimination were offered, including radical cure strategies, comprehensive but adaptive strategies targeting species of Plasmodium and Anopheles, mass drug administration, and case-/focus-centred surveillance and response systems. CONCLUSION: The complete global eradication of P. vivax and eventually malaria will be more difficult, and China's practices and experience could be a valuable reference in this campaign.

Accounting for complex intracluster correlations in longitudinal cluster randomized trials: a case study in malaria vector control.

BACKGROUND: The effectiveness of malaria vector control interventions is often evaluated using cluster randomized trials (CRT) with outcomes assessed using repeated cross-sectional surveys. A key requirement for appropriate design and analysis of longitudinal CRTs is accounting for the intra-cluster correlation coefficient (ICC). In addition to exchangeable correlation (constant ICC over time), correlation structures proposed for longitudinal CRT are block exchangeable (allows a different within- and between-period ICC) and exponential decay (allows between-period ICC to decay exponentially). More flexible correlation structures are available in statistical software packages and, although not formally proposed for longitudinal CRTs, may offer some advantages. Our objectives were to empirically explore the impact of these correlation structures on treatment effect inferences, identify gaps in the methodological literature, and make practical recommendations. METHODS: We obtained data from a parallel-arm CRT conducted in Tanzania to compare four different types of insecticide-treated bed-nets. Malaria prevalence was assessed in cross-sectional surveys of 45 households in each of 84 villages at baseline, 12-, 18- and 24-months post-randomization. We re-analyzed the data using mixed-effects logistic regression according to a prespecified analysis plan but under five different correlation structures as well as a robust variance estimator under exchangeable correlation and compared the estimated correlations and treatment effects. A proof-of-concept simulation was conducted to explore general conclusions. RESULTS: The estimated correlation structures varied substantially across different models. The unstructured model was the best-fitting model based on information criteria. Although point estimates and confidence intervals for the treatment effect were similar, allowing for more flexible correlation structures led to different conclusions based on statistical significance. Use of robust variance estimators generally led to wider confidence intervals. Simulation results showed that under-specification can lead to coverage probabilities much lower than nominal levels, but over-specification is more likely to maintain nominal coverage. CONCLUSION: More flexible correlation structures should not be ruled out in longitudinal CRTs. This may be particularly important in malaria trials where outcomes may fluctuate over time. In the absence of robust methods for selecting the best-fitting correlation structure, researchers should examine sensitivity of results to different assumptions about the ICC and consider robust variance estimators.

Feeding rates of malaria vectors from a prototype attractive sugar bait station in Western Province, Zambia: results of an entomological validation study.

BACKGROUND: Attractive targeted sugar bait (ATSB) stations are a promising new approach to malaria vector control that could compliment current tools by exploiting the natural sugar feeding behaviors of mosquitoes. Recent proof of concept work with a prototype ATSB® Sarabi Bait Station (Westham Co., Hod-Hasharon, Israel) has demonstrated high feeding rates and significant reductions in vector density, human biting rate, and overall entomological inoculation rate for Anopheles gambiae sensu lato (s.l.) in the tropical savannah of western Mali. The study reported here was conducted in the more temperate, rainier region of Western Province, Zambia and was designed to confirm the primary vector species in region and to estimate corresponding rates of feeding from prototype attractive sugar bait (ASB) Sarabi Bait Stations. METHODS: The product evaluated was the Sarabi v1.1.1 ASB station, which did not include insecticide but did include 0.8% uranine as a dye allowing for the detection, using UV fluorescence light microscopy, of mosquitoes that have acquired a sugar meal from the ASB. A two-phase, crossover study design was conducted in 10 village-based clusters in Western Province, Zambia. One study arm initially received 2 ASB stations per eligible structure while the other initially received 3. Primary mosquito sampling occurred via indoor and outdoor CDC Miniature UV Light Trap collection from March 01 through April 09, 2021 (Phase 1) and from April 19 to May 28, 2021 (Phase 2). RESULTS: The dominant vector in the study area is Anopheles funestus s.l., which was the most abundant species group collected (31% of all Anophelines; 45,038/144,5550), had the highest sporozoite rate (3.16%; 66 positives out of 2,090 tested), and accounted for 94.3% (66/70) of all sporozoite positive specimens. Of those An. funestus specimens further identified to species, 97.2% (2,090/2,150) were An. funestus sensu stricto (s.s.). Anopheles gambiae s.l. (96.8% of which were Anopheles arabiensis) is a likely secondary vector and Anopheles squamosus may play a minor role in transmission. Overall, 21.6% (9,218/42,587) of An. funestus specimens and 10.4% (201/1,940) of An. gambiae specimens collected were positive for uranine, translating into an estimated daily feeding rate of 8.9% [7.7-9.9%] for An. funestus (inter-cluster range of 5.5% to 12.7%) and 3.9% [3.3-4.7%] for An. gambiae (inter-cluster range of 1.0-5.2%). Feeding rates were no different among mosquitoes collected indoors or outdoors, or among mosquitoes from clusters with 2 or 3 ASBs per eligible structure. Similarly, there were no correlations observed between feeding rates and the average number of ASB stations per hectare or with weekly rainfall amounts. CONCLUSIONS: Anopheles funestus and An. gambiae vector populations in Western Province, Zambia readily fed from the prototype Sarabi v1.1.1 ASB sugar bait station. Observed feeding rates are in line with those thought to be required for ATSB stations to achieve reductions in malaria transmission when used in combination with conventional control methods (IRS or LLIN). These results supported the decision to implement a large-scale, epidemiological cluster randomized controlled trial of ATSB in Zambia, deploying 2 ATSB stations per eligible structure.

Enrichment of phenotype among biological forms of Anopheles stephensi Liston through establishment of isofemale lines.

BACKGROUND: Vector management programs rely on knowledge of the biology and genetic make-up of mosquitoes. Anopheles stephensi is a major invasive urban malaria vector, distributed throughout the Indian subcontinent and Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs and varying vectorial competence, An. stephensi is a perfect species for developing isofemale lines, which can be tested for insecticide susceptibility and vectorial competence of various biological forms. METHODS: We describe key steps involved in establishment and validation of isofemale lines. Isofemale colonies were further used for the characterization of insecticide susceptibility and differential vector competence. The results were statistically evaluated through descriptive and inferential statistics using Vassar Stat and Prism GraphPad software packages. RESULTS: Through a meticulous selection process, we overcame an initial inbreeding depression and found no significant morphometric differences in wings and egg size between the parental and respective isofemale lines in later generations. IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all four major classes. We observed a significant change in vectorial competence between the respective isofemale and parental lines. CONCLUSIONS: Isofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses characterization of fitness traits among two lines through a transgenerational study. Furthermore, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into differential susceptibility status of the parental and isofemale lines to different insecticides belonging to the same class. Corroborating an earlier hypothesis, we demonstrate the high vector competence of the type form relative to the intermediate form using homozygous lines. Using these lines, it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave the way for developing better vector management strategies.

Standing genetic variation and chromosome differences drove rapid ecotype formation in a major malaria mosquito.

Species distributed across heterogeneous environments often evolve locally adapted ecotypes, but understanding of the genetic mechanisms involved in their formation and maintenance in the face of gene flow is incomplete. In Burkina Faso, the major African malaria mosquito Anopheles funestus comprises two strictly sympatric and morphologically indistinguishable yet karyotypically differentiated forms reported to differ in ecology and behavior. However, knowledge of the genetic basis and environmental determinants of An. funestus diversification was impeded by lack of modern genomic resources. Here, we applied deep whole-genome sequencing and analysis to test the hypothesis that these two forms are ecotypes differentially adapted to breeding in natural swamps versus irrigated rice fields. We demonstrate genome-wide differentiation despite extensive microsympatry, synchronicity, and ongoing hybridization. Demographic inference supports a split only ~1,300 y ago, closely following the massive expansion of domesticated African rice cultivation ~1,850 y ago. Regions of highest divergence, concentrated in chromosomal inversions, were under selection during lineage splitting, consistent with local adaptation. The origin of nearly all variations implicated in adaptation, including chromosomal inversions, substantially predates the ecotype split, suggesting that rapid adaptation was fueled mainly by standing genetic variation. Sharp inversion frequency differences likely facilitated adaptive divergence between ecotypes by suppressing recombination between opposing chromosomal orientations of the two ecotypes, while permitting free recombination within the structurally monomorphic rice ecotype. Our results align with growing evidence from diverse taxa that rapid ecological diversification can arise from evolutionarily old structural genetic variants that modify genetic recombination.

Seasonal assessment on the effects of time of night, temperature and humidity on the biting profile of Anopheles farauti in north Queensland, Australia using a population naive to malaria vector control pressures.

BACKGROUND: Anopheles farauti is one of the major vectors of malaria in the Southwest Pacific region and is responsible for past outbreaks in Australia. With an adaptable biting profile conducive to behavioural resistance to indoor residual spraying (IRS) and insecticide-treated nets (ITNs), its all-night biting behaviour can switch to biting mostly in the early evening. With limited insight into the biting profile of An. farauti populations in areas that have not encountered IRS or ITNs, the aim of this study was to develop insights on the biting behaviour of a malaria control naive population of An. farauti. METHODS: Biting profiles of An. farauti were conducted at Cowley Beach Training Area, in north Queensland, Australia. Initially, encephalitis virus surveillance (EVS) traps were used to document the 24-h biting profile of An. farauti and then human landing collections (HLC) were used to follow the 18.00-06.00 h biting profile. The human landing catches (HLC) were performed at both the end of the wet (April) and dry (October) seasons. RESULTS: Data exploration using a Random Forest Model shows that time of night is the most important variable for predicting An. farauti biting activity. Temperature was found to be the next important predictor, followed by humidity, trip, collector, and season. The significant effect of time of night and peak in time of night biting, between 19.00 and 20.00 h was also observed in a generalized linear model. The main effect of temperature was significant and non-linear and appears to have a positive effect on biting activity. The effect of humidity is also significant but its relationship with biting activity is more complex. This population's biting profile is similar to populations found in other parts of its range prior to insecticide intervention. A tight timing for the onset of biting was identified with more variation with the end of biting, which is likely underpinned by an endogenous circadian clock rather than any light intensity. CONCLUSION: This study sees the first record of a relationship between biting activity and the decreasing temperature during the night for the malaria vector, Anopheles farauti.

Cattle feeding tendency of Anopheles mosquitoes and their infection rates in Aradum village, North Wollo, Ethiopia: an implication for animal-based malaria control strategies.

BACKGROUND: Surveillance of indoor and outdoor resting malaria vector populations is crucial to monitor possible changes in vector resting and feeding behaviours. This study was conducted to assess the resting behaviour, blood meal sources and circumsporozoite (CSP) rates of Anopheles mosquito in Aradum village, Northern Ethiopia. METHODS: Mosquito collection was conducted from September 2019 to February 2020 using clay pots (indoor and outdoor), pit shelter and pyrethrum spray catches (PSC). The species of Anopheles gambiae complex and Anopheles funestus group were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was done to determine CSP and blood meal sources of malaria vectors. RESULTS: A total of 775 female Anopheles mosquitoes were collected using the clay pot, PSC and pit shelter. Seven Anopheles mosquito species were identified morphologically, of which Anopheles demeilloni (593; 76.5%) was the dominant species followed by An. funestus group (73; 9.4%). Seventy-three An. funestus group screened by PCR, 91.8% (67/73) were identified as Anopheles leesoni and only 2.7% (2/73) were found to be Anopheles parensis. The molecular speciation of 71 An. gambiae complex confirmed 91.5% (65/71) of Anopheles arabiensis. The majority of Anopheles mosquitoes were collected from outdoor pit shelter (42.2%) followed by outdoor clay pots. The majority of the blood meal of An. demeilloni (57.5%; 161/280), An. funestus sensu lato 10 (43.5%) and An. gambiae (33.3%; 14/42) originated from bovine. None of the 364 Anopheles mosquitoes tested for Plasmodium falciparum and Plasmodium vivax sporozoite infections were positive. CONCLUSION: Since the Anopheles mosquitoes in the area prefer to bite cattle, it may be best to target them with an animal-based intervention. Clay pots could be an alternative tool for outdoor monitoring of malaria vectors in areas where pit shelter construction is not possible.

Human landing catches provide a useful measure of protective efficacy for the evaluation of volatile pyrethroid spatial repellents.

BACKGROUND: The human landing catch (HLC) method, in which human volunteers collect mosquitoes that land on them before they can bite, is used to quantify human exposure to mosquito vectors of disease. Comparing HLCs in the presence and absence of interventions such as repellents is often used to measure protective efficacy (PE). Some repellents have multiple actions, including feeding inhibition, whereby mosquitoes may be unable to bite even if they land on a host. A comparison was made between the PE of the volatile pyrethroid spatial repellent (VPSR) transfluthrin determined using a landing method (HLC) and a biting method (allowing the mosquitoes that landed to blood-feed) to evaluate whether HLC is a suitable method for the estimation of the personal PE of a VPSR. METHODS: A fully balanced, two-arm crossover design study was conducted using a 6 × 6 × 2-m netted cage within a semi-field system. Hessian strips (4 m × 0.1 m) treated with a 5-, 10-, 15-, or 20-g dose of transfluthrin were evaluated against a paired negative control for three strains of laboratory-reared Anopheles and Aedes aegypti mosquitoes. Six replicates were performed per dose using either the landing or the biting method. The number of recaptured mosquitoes was analysed by negative binomial regression, and the PEs calculated using the two methods were compared by Bland-Altman plots. RESULTS: For Anopheles, fewer mosquitoes blood-fed in the biting arm than landed in the landing arm (incidence rate ratio = 0.87, 95% confidence interval 0.81-0.93, P < 0.001). For Ae. aegypti, biting was overestimated by around 37% with the landing method (incidence rate ratio = 0.63, 95% confidence interval 0.57-0.70, P = 0.001). However, the PEs calculated for each method were in close agreement when tested by the Bland Altman plot. CONCLUSIONS: The HLC method led to underestimation of mosquito feeding inhibition as a mode of action of transfluthrin, and there were species- and dose-dependent differences in the relationship between landing and biting. However, the estimated PEs were similar between the two methods. The results of this study indicate that HLC can be used as a proxy for personal PE for the evaluation of a VPSR, especially when the difficulties associated with enumerating blood-fed mosquitoes in a field setting are taken into consideration.

Combined effect of physico-chemical and microbial quality of breeding habitat water on oviposition of malarial vector Anopheles subpictus.

Mosquitoes prefer diverse water bodies for egg laying and larval survival. Present study was performed with an objective to characterize physico-chemical properties and microbial profiling of breeding habitat water bodies of Anopheles subpictus mosquitoes. A field survey was accomplished to check the presence of An. subpictus larvae to record per dip larval density in various breeding habitats throughout the year. Physico-chemical and bacteriological properties in relation to mosquito oviposition were assessed. Dissolved oxygen content, pH and alkalinity were found to have major impacts and ponderosity on the prevalence of An. subpictus larvae. Larval density showed significant positive correlation with dissolved oxygen content of water and significant negative correlation with pH and alkalinity of habitat water. Comparatively higher population (cfu/mL) of Bacillus spp. competent with starch hydrolyzing and nitrate reducing properties were recorded all the breeding habitat water bodies of An. subpictus. Higher amplitude of anopheline larvae was portrayed during monsoon and post-monsoon season in clear water with an inclining trend to high dissolved oxygen content and neutral pH. B. cereus, B. megaterium, B. subtilis and B. tequilensis prevalent in all habitat water bodies were marked as oviposition attractants of gravid An. subpictus mosquitoes. Microbial population played key roles in the modulation of physico-chemical parameters of habitat water with a view to enhance its acceptability by gravid mosquitoes in relation to their oviposition. Better understanding of the interactions along with the control of oviposition attractant bacterial strains from mosquito breeding habitats might contribute to the vector management programme.

Distribution of Anopheles gambiae thioester-containing protein 1 alleles along malaria transmission gradients in The Gambia.

BACKGROUND: Thioester-containing protein 1 (TEP1) is a highly polymorphic gene playing an important role in mosquito immunity to parasite development and associated with Anopheles gambiae vectorial competence. Allelic variations in TEP1 could render mosquito either susceptible or resistant to parasite infection. Despite reports of TEP1 genetic variations in An. gambiae, the correlation between TEP1 allelic variants and transmission patterns in malaria endemic settings remains unclear. METHODS: TEP1 allelic variants were characterized by PCR from archived genomic DNA of > 1000 An. gambiae mosquitoes collected at 3 time points between 2009 and 2019 from eastern Gambia, where malaria transmission remains moderately high, and western regions with low transmission. RESULTS: Eight common TEP1 allelic variants were identified at varying frequencies in An. gambiae from both transmission settings. These comprised the wild type TEP1, homozygous susceptible genotype, TEP1s; homozygous resistance genotypes: TEP1rA and TEP1rB, and the heterozygous resistance genotypes: TEP1srA, TEP1srB, TEP1rArB and TEP1srArB. There was no significant disproportionate distribution of the TEP1 alleles by transmission setting and the temporal distribution of alleles was also consistent across the transmission settings. TEP1s was the most common in all vector species in both settings (allele frequencies: East = 21.4-68.4%. West = 23.5-67.2%). In Anopheles arabiensis, the frequency of wild type TEP1 and susceptible TEP1s was significantly higher in low transmission setting than in high transmission setting (TEP1: Z = - 4.831, P < 0.0001; TEP1s: Z = - 2.073, P = 0.038). CONCLUSIONS: The distribution of TEP1 allele variants does not distinctly correlate with malaria endemicity pattern in The Gambia. Further studies are needed to understand the link between genetic variations in vector population and transmission pattern in the study settings. Future studies on the implication for targeting TEP1 gene for vector control strategy such as gene drive systems in this settings is also recommended.

Laboratory evaluation of the efficacy of deltamethrin-laced attractive toxic sugar bait formulation on Anopheles stephensi.

BACKGROUND: Attractive toxic sugar bait (ATSB) is a promising "attract and kill"-based approach for mosquito control. It is a combination of flower nectar/fruit juice to attract the mosquitoes, sugar solution to stimulate feeding, and a toxin to kill them. Selecting an effective attractant and optimizing concentration of toxicant is significant in the formulation of ATSB. METHODS: Current study formulated an ATSB using fruit juice, sugar and deltamethrin, a synthetic pyrethroid. It was evaluated against two laboratory strains of Anopheles stephensi. Initial studies evaluated comparative attractiveness of nine different fruit juices to An. stephensi adults. Nine ASBs were prepared by adding fermented juices of plum, guava, sweet lemon, orange, mango, pineapple, muskmelon, papaya, and watermelon with 10% sucrose solution (w/v) in 1:1 ratio. Cage bioassays were conducted to assess relative attraction potential of ASBs based on the number of mosquito landings on each and the most effective ASB was identified. Ten ATSBs were prepared by adding the identified ASB with different deltamethrin concentrations (0.015625-8.0 mg/10 mL) in 1:9 ratio. Each ATSB was assessed for the toxic potential against both the strains of An. stephensi. The data was statistically analysed using PASW (SPSS) software 19.0 program. RESULTS: The cage bioassays with nine ASBs revealed higher efficacy (p < 0.05) of Guava juice-ASB > Plum juice-ASB > Mango juice-ASB in comparison to rest of the six ASB's. The bioassay with these three ASB's ascertained the highest attractancy potential of guava juice-ASB against both the strains of An. stephensi. The ATSB formulations resulted in 5.1-97.9% mortality in Sonepat (NIMR strain) with calculated LC30, LC50, and LC90 values of 0.17 mg deltamethrin/10 mL, 0.61 mg deltamethrin/10 mL, and 13.84 mg deltamethrin/10 mL ATSB, respectively. Whereas, 6.12-86.12% mortality was recorded in the GVD-Delhi (AND strain) with calculated LC30, LC50, and LC90 values of 0.25 mg deltamethrin/10 mL, 0.73 mg deltamethrin/10 mL and 10.22 mg deltamethrin/10 mL ATSB, respectively. CONCLUSION: The ATSB formulated with guava juice-ASB and deltamethrin (0.0015625-0.8%) in 9:1 ratio showed promising results against two laboratory strains of An. stephensi. Field assessment of these formulations is being conducted to estimate their feasibility for use in mosquito control.