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The Arizona State University (ASU) Tempe campus is inhabited by some 55,000 enrolled students as well as several mosquito species that can transmit West Nile virus, dengue, Zika, chikungunya, and yellow fever. The time of host-seeking of these vectors has not been quantified on the ASU Tempe campus, but this information is important to inform ground and/or truck-mount fogging operations targeting mosquitoes to prevent or control disease outbreaks. We quantified the time of host-seeking of the predominant mosquito vector species at the ASU Tempe campus during the post-monsoon season in 2021, using collection bottle rotators with encephalitis vector survey traps that were baited with CO2, at 3 h intervals during a full day. Culex quinquefasciatus, Aedes aegypti, and Culex tarsalis were the most abundant species captured. Pre-midnight host-seeking (18:00-00:00) accounted for 52% of all captures, whereas post-midnight host-seeking (00:00-06:00) accounted for 35% of all captures. Peak activity times were between 21:00 and 00:00 for Cx. quinquefasciatus and Cx. tarsalis, and between 15:00 and 18:00 for Ae. aegypti. Data can be used to inform local mosquito surveillance and control programs., (Copyright © 2024 by The American Mosquito Control Association, Inc.)

Background: The control and prevention of mosquito-borne diseases is mostly achieved with insecticides. However, their use has led to the rapid development and spread of insecticide resistance worldwide. Health experts have called for intensified efforts to find new approaches to reduce mosquito populations and human-mosquito contact. A promising new tool is the use of electrical fields (EFs), whereby mosquitoes are repelled by charged particles in their flight path. Such particles move between two or more conductors, and the use of uninsulated copper or aluminum plates as conductors has been proven to be effective at repelling mosquitoes. Here, for the first time, we assess if EFs generated using a single row of insulated conductor wires (ICWs) can also successfully repel mosquitoes, and whether mosquitoes are equally repelled at the same EF strength when the electrodes are a) orientated differently (horizontal vs. vertical placement), and b) spaced more apart., Methodology/principal Findings: Over a period of 23 hours, the number of host-seeking female Aedes aegypti mosquitoes that were successfully repelled by EFs, using ICWs, at EF strengths ranging from 0 kV/cm (control) to 9.15 kV/cm were quantified. Mosquitoes were released inside a 220×220×180 cm room and lured into a BG-Pro trap that was equipped with a BG-counter and baited with CO2 using dry ice. Mosquitoes had to pass through an EF window, that contained a single row of ICWs with alternating polarity, to reach the bait. The baseline interaction between EF strength and repellency was assessed first, after which the impact of different ICW orientations and ICW distances on repellency were determined. Over 50% of mosquitoes were repelled at EF strengths of ≥ 3.66 kV/cm. A linear regression model showed that a vertical ICW orientation (vertical vs. horizontal) had a small but insignificant increased impact on mosquito repellency (p = 0.059), and increasing ICW distance (while maintaining the same EF strength) significantly reduced repellency (p = 0.01)., Conclusions/significance: ICWs can be used to generate EFs that partially repel host-seeking mosquitoes, which will reduce human-mosquito contact. While future studies need to assess if (i) increased repellency can be achieved, and (ii) a repellency of 50-60% is sufficient to impact disease transmission, it is encouraging that EF repellency using ICWs is higher compared to that of some spatial repellent technologies currently in development. This technology can be used in the housing improvement toolkit (i.e. preventing mosquito entry through eaves, windows, and doors). Moreover, the use of cheap, over-the-counter ICWs will mean that the technology is more accessible worldwide, and easier to manufacture and implement locally., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors declare the following potential conflicts of interest: KPP is an inventor in US Patent No. 11,672,244B2 and/or in other patent applications related to this work. NBJ and KPP are inventors in US Patent No. 63/519.181., (Copyright: © 2024 Jobe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

West Nile virus (WNV) is the leading mosquito-borne disease causing-pathogen in the United States. Concerningly, there are no prophylactics or drug treatments for WNV and public health programs rely heavily on vector control efforts to lessen disease incidence. Insecticides can be effective in reducing vector numbers if implemented strategically, but can diminish in efficacy and promote insecticide resistance otherwise. Vector control programs which employ mass-fogging applications of insecticides, often conduct these methods during the late-night hours, when diel temperatures are coldest, and without a-priori knowledge on daily mosquito activity patterns. This study's aims were to 1) quantify the effect of temperature on the toxicity of two conventional insecticides used in fogging applications (malathion and deltamethrin) to Culex tarsalis, an important WNV vector, and 2) quantify the time of host-seeking of Cx. tarsalis and other local mosquito species in Maricopa County, Arizona. The temperature-toxicity relationship of insecticides was assessed using the WHO tube bioassay, and adult Cx. tarsalis, collected as larvae, were exposed to three different insecticide doses at three temperature regimes (15, 25, and 35°C; 80% RH). Time of host-seeking was assessed using collection bottle rotators with encephalitis vector survey traps baited with dry ice, first at 3h intervals during a full day, followed by 1h intervals during the night-time. Malathion became less toxic at cooler temperatures at all doses, while deltamethrin was less toxic at cooler temperatures at the low dose. Regarding time of host-seeking, Cx. tarsalis, Aedes vexans, and Culex quinquefasciatus were the most abundant vectors captured. During the 3-hour interval surveillance over a full day, Cx. tarsalis were most-active during post-midnight biting (00:00-06:00), accounting for 69.0% of all Cx. tarsalis, while pre-midnight biting (18:00-24:00) accounted for 30.0% of Cx. tarsalis. During the 1-hour interval surveillance overnight, Cx. tarsalis were most-active during pre-midnight hours (18:00-24:00), accounting for 50.2% of Cx. tarsalis captures, while post-midnight biting (00:00-06:00) accounted for 49.8% of Cx. tarsalis. Our results suggest that programs employing large-scale applications of insecticidal fogging should consider temperature-toxicity relationships coupled with time of host-seeking data to maximize the efficacy of vector control interventions in reducing mosquito-borne disease burden., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Arizona is home to many mosquito species, some of which are known vectors of infectious diseases that harm both humans and animals. Here, we provide an overview of the 56 mosquito species that have been identified in the State to date, but also discuss their known feeding preference and the diseases they can (potentially) transmit to humans and animals. This list is unlikely to be complete for several reasons: (i) Arizona's mosquitoes are not systematically surveyed in many areas, (ii) surveillance efforts often target specific species of interest, and (iii) doubts have been raised by one or more scientists about the accuracy of some collection records, which has been noted in this article. There needs to be an integrated and multifaceted surveillance approach that involves entomologists and epidemiologists, but also social scientists, wildlife ecologists, ornithologists, representatives from the agricultural department, and irrigation and drainage districts. This will allow public health officials to (i) monitor changes in current mosquito species diversity and abundance, (ii) monitor the introduction of new or invasive species, (iii) identify locations or specific populations that are more at risk for mosquito-borne diseases, and (iv) effectively guide vector control.

Background: The Magude Project assessed the feasibility of eliminating malaria in Magude district, a low transmission setting in southern Mozambique, using a package of interventions, including long-lasting insecticidal nets (LLINs). As the efficacy of LLINs depends in part on their physical integrity, this metric was quantified for Olyset® Nets post mass-distribution, in addition to net use, care and handling practices and other risk factors associated with net physical integrity., Methods: Nets were collected during a cross-sectional net evaluation, nine months after the Magude project commenced, which was 2 years after the nets were distributed by the National Malaria Control Programme (NMCP). The physical integrity of the nets was assessed by counting and sizing the holes at different positions on each net. A structured questionnaire was administered to assess how the selected net was used and treated (care, wash and repair). Net bio-efficacy was assessed following the standard World Health Organization (WHO) cone bioassay procedures., Results: Out of the 170 Olyset® Nets included in the analysis, 63.5% had been used the night before. The main reason for not using a net was the notion that there were no mosquitoes present. The average number of people using each net was 1.79. Two thirds of the nets had only been washed once or twice since distribution. Most nets (80.9%) were holed and 18% were torn, but none of the risk factors were significantly associated with net integrity, except for presence of mice in the household. Less than half of the participants noticed holes in holed nets, and of those only 38.6% attempted to repair those. None of the six nets that were tested for bio-efficacy passed the WHO threshold of 80% mosquito mortality., Conclusion: Overall the majority of Olyset® Nets were in serviceable condition two years post-distribution, but their insecticidal effect may have been lost. This study-together with previous evidence on suboptimal access to and use of LLINs in Magude district-highlights that LLINs as an intervention could have been optimized during the Magude project to achieve maximum intervention impact., (© 2024. The Author(s).)

Background: Different anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni)., Methods: Female anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination., Results: Out of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison., Conclusion: The reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species., (© 2024. The Author(s).)

Chemical-based interventions are mostly used to control insects that are harmful to human health and agriculture or that simply cause a nuisance. An overreliance on these insecticides however raises concerns for the environment, human health, and the development of resistance, not only in the target species. As such, there is a critical need for the development of novel nonchemical technologies to control insects. Electrocution traps using UV light as an attractant are one classical nonchemical approach to insect control but lack the specificity necessary to target only pest insects and to avoid harmless or beneficial species. Here we review the fundamental physics behind electric fields (EFs) and place them in context with electromagnetic fields more broadly. We then focus on how novel uses of strong EFs, some of which are being piloted in the field and laboratory, have the potential to repel, capture, or kill (electrocute) insects without the negative side effects of other classical approaches. As EF-insect science remains in its infancy, we provide recommendations for future areas of research in EF-insect science., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Public health insecticides play a crucial role in malaria control and elimination programmes. Many other arthropods, including mechanical and biological vectors of infectious diseases, have similar indoor feeding or resting behaviours, or both, as malaria mosquitoes, and could be exposed to the same insecticides. In this Personal View, we show that little is known about the insecticide susceptibility status and the extent of exposure to malaria interventions of other arthropod species. We highlight that there is an urgent need to better understand the selection pressure for insecticide resistance in those vectors, to ensure current and future active ingredients remain effective in targeting a broad range of arthropod species, allowing us to prevent and control future outbreaks of infectious diseases other than malaria., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We compared the effectiveness of 4 different carbon dioxide (CO2) sources (sugar-fermented BG-CO2, sugar-fermented Fleischmann yeast, dry ice, and compressed gas cylinders) in attracting different mosquito species in 2 separate 4 × 4 Latin square trials. The CO2 generated by dry ice and the gas cylinders collected more Culex quinquefasciatus than the sugar-fermented BG-CO2 and Fleischmann yeasts during the 1st trial (16-h surveillance periods), but there was no significant difference in Aedes aegypti numbers. There were no significant differences between the different CO2 sources in collecting Cx. quinquefasciatus and Ae. aegypti mosquitoes in the 2nd trial (24-h surveillance periods). Catches for Culiseta inornata and Cx. tarsalis were too low in both experiments for formal statistical analysis. Data can be used to inform local mosquito surveillance programs, but the selection of a CO2 source will also depend on financial and logistical constraints., (Copyright © 2023 by The American Mosquito Control Association, Inc.)

The Magude Project assessed the feasibly of eliminating malaria in a low transmission setting in southern Mozambique using a package of interventions. This study measured the ownership, access and use of long-lasting insecticide treated nets (LLINs) and inequalities in these indicators across household wealth, size and population subgroups, to understand the protection that LLINs provided during the project. Data were obtained from various household surveys. At least 31% of the nets distributed during the 2014 and 2017 campaigns were lost during the first year post-distribution. Most nets (77.1%) present in the district were Olyset Nets. LLIN access never exceeded 76.3% and use varied seasonally between 40% and 76.4%. LLIN access limited LLIN use during the project, especially during the high transmission season. LLIN ownership, access and use were lower in harder-to-reach localities, in poorer and larger households. Children and women below 30 had poorer access to LLINs than the overall population. Net use was lowest among school-aged children and young adults, especially among young males, and highest in children under 5, pregnant women, in older adults and in households that received indoor residual spraying (IRS). This study revealed that LLIN mass-distribution campaigns alone are not sufficient to achieve the high level of net protection needed during elimination programs and that reviewing the LLIN allocation scheme, top-up distributions and/or community engagement campaigns is needed, also to reduce inequalities in populations' access to LLINs., Competing Interests: The authors have read the journal’s policy and have the following competing interests: Abt Associates Inc. provided support in the form of salaries for author NC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE’s policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2023 Fernández Montoya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 F ST values were low for both anophelines (- 0.00457 to 0.04213), N m 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., (© 2023. The Author(s).)

Progress in reducing both malaria cases and deaths has stalled with regression seen in many geographies. While significant attention is given to the contributing challenges of drug and insecticide resistance, 'residual' malaria is often diminished to transmission resulting from outdoor-biting or zoophagic/opportunistic mosquito vectors. These specific vector bionomic traits are only part of the problem, as residual transmission may be driven by (a combination of) (1) sub-optimal intervention coverage, quality, acceptance, and/or usage, (2) drug resistance, (3) insecticide resistance, (4) refractory, resistant and adaptive vector and human behaviours that lower intervention effectiveness, (5) lack of, limited access to, and/or willingness to use healthcare systems, (6) diagnostic sensitivity along with the parallel issue of hrp2/3 mutations, (7) (inter)national policy, (8) the research and development pipeline, and (9) external factors such as natural disasters and conflict zones. Towards combating the minimization of this extensive and multipronged issue among the scientific community, funding agencies, and public health officials responsible for guiding or developing malaria programmes, an alternative way of describing this transmission is proposed by focusing in on the causative 'gaps in protection'. Defining and wording it as such zeros in on the drivers that result in the observed remaining (or increasing) transmission, allowing the malaria community to focus on solutions by identifying the actual causes. Outlining, defining and quantifying the gaps in protection for a given system is of utmost importance to understand what needs to be done, differentiating what can be done versus what cannot be tackled at that moment, along with delineating the technical and financial capacity required., (© 2023. The Author(s).)

Indoor residual spraying (IRS) has been and remains an important malaria control intervention in southern Mozambique, South Africa and Eswatini. A better understanding of the effectiveness of IRS campaigns is critical to guide future elimination efforts. We analyze the three IRS campaigns conducted during a malaria elimination demonstration project in southern Mozambique, the "Magude project", and propose a new method to calculate the efficacy of IRS campaigns adjusting for IRS coverage, pace of house spraying and IRS residual efficacy on different wall types. Anopheles funestus sensu lato (s.l.) and An. gambiae s.l. were susceptible to pirimiphos-methyl and DDT. Anopheles funestus s.l. was resistant to pyrethroids, with 24h post-exposure mortality being lower for An. funestus sensu stricto (s.s.) than for An. parensis (collected indoors). The percentage of structures sprayed was above 90% and percentage of people covered above 86% in all three IRS campaigns. The percentage of households sprayed was above 83% in 2015 and 2016, but not assessed in 2017. Mosquito mortality 24h post-exposure stayed above 80% for 196 days after the 2016 IRS campaign and 222 days after the 2017 campaign and was 1.5 months longer on mud walls than on cement walls. This was extended by up to two months when 120h post-exposure mortality was considered. The district-level realized IRS efficacy was 113 days after the 2016 campaign. While the coverage of IRS campaigns in Magude were high, IRS protection did not remain optimal for the entire high malaria transmissions season. The use of a longer-lasting IRS product could have further supported the interruption of malaria transmission in the district. To better estimate the protection afforded by IRS campaigns, National Malaria Control Programs and partners are encouraged to adjust the calculation of IRS efficacy for IRS coverage, pace of house spraying during the campaign and IRS efficacy on different wall types combined with wall type distribution in the sprayed area., Competing Interests: The authors have declared that no competing interests exist. Abt Associates Inc. provided support in the form of salaries for author NC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE’s policies on sharing data and materials.

Characterizing persistent malaria transmission that occurs after the combined deployment of indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) is critical to guide malaria control and elimination efforts. This requires a detailed understanding of both human and vector behaviors at the same temporal and spatial scale. Cross-sectional human behavior evaluations and mosquito collections were performed in parallel in Magude district, Mozambique. Net use and the exact time when participant moved into each of five environments (outdoor, indoor before bed, indoor in bed, indoor after getting up, and outdoor after getting up) were recorded for individuals from three different age groups and both sexes during a dry and a rainy season. Malaria mosquitoes were collected with CDC light traps in combination with collection bottle rotators. The percentage of residual exposure to host-seeking vectors that occurred in each environment was calculated for five local malaria vectors with different biting behaviors, and the actual (at observed levels of LLIN use) and potential (i.e. if all residents had used an LLIN) personal protection conferred by LLINs was estimated. Anopheles arabiensis was responsible for more than 74% of residents' residual exposure to host-seeking vectors during the Magude project. The other four vector species (An. funestus s.s., An. parensis, An. squamosus and An. merus) were responsible for less than 10% each. The personal protection conferred by LLINs prevented only 39.2% of the exposure to host-seeking vectors that survived the implementation of both IRS and LLINs, and it differed significantly across seasons, vector species and age groups. At the observed levels of bednet use, 12.5% of all residual exposure to host-seeking vectors occurred outdoor during the evening, 21.9% indoor before going to bed, almost two thirds (64%) while people were in bed, 1.4% indoors after getting up and 0.2% outdoor after leaving the house. Almost a third of the residual exposure to host-seeking vectors (32.4%) occurred during the low transmission season. The residual bites of An. funestus s.s. and An. parensis outdoors and indoor before bedtime, of An. arabiensis indoors when people are in bed, and of An. squamosus both indoors and outdoors, are likely to have sustained malaria transmission throughout the Magude project. By increasing LLIN use, an additional 24.1% of exposure to the remaining hosts-seeking vectors could have been prevented. Since An. arabiensis, the most abundant vector, feeds primarily while people are in bed, increasing net use and net feeding inhibition (through e.g. community awareness activities and the selection of more effective LLINs) could significantly reduce the exposure to remaining host-seeking mosquitoes. Nonetheless, supplementary interventions aiming to reduce human-vector contact outdoors and/or indoors before people go to bed (e.g. through larval source management, window and eave screening, eave tubes, and spatial repellents) will be needed to reduce residual exposure to the outdoor and early biting An. funestus s.s. and An. parensis., Competing Interests: The authors have declared that no competing interests exist. Abt Associates Inc. provided support in the form of salaries for author NC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS One’s policies on sharing data and materials.

The "Magude project" aimed but failed to interrupt local malaria transmission in Magude district, southern Mozambique, by using a comprehensive package of interventions, including indoor residual spraying (IRS), pyrethroid-only long-lasting insecticide treated nets (LLINs) and mass-drug administration (MDA). Here we present detailed information on the vector species that sustained malaria transmission, their association with malaria incidence and behaviors, and their amenability to the implemented control interventions. Mosquitoes were collected monthly between May 2015 and October 2017 in six sentinel sites in Magude district, using CDC light traps both indoors and outdoors. Anopheles arabiensis was the main vector during the project, while An. funestus s.s., An. merus, An. parensis and An. squamosus likely played a secondary role. The latter two species have never previously been found positive for Plasmodium falciparum in southern Mozambique. The intervention package successfully reduced vector sporozoite rates in all species throughout the project. IRS was effective in controlling An. funestus s.s. and An. parensis, which virtually disappeared after its first implementation, but less effective at controlling An. arabiensis. Despite suboptimal use, LLINs likely provided significant protection against An. arabiensis and An. merus that sought their host largely indoors when people where in bed. Adding IRS on top of LLINs and MDA likely added value to the control of malaria vectors during the Magude project. Future malaria elimination attempts in the area could benefit from i) increasing the use of LLINs, ii) using longer-lasting IRS products to counteract the increase in vector densities observed towards the end of the high transmission season, and iii) a higher coverage with MDA to reduce the likelihood of human infection. However, additional interventions targeting vectors that survive IRS and LLINs by biting outdoors or indoors before people go to bed, will be likely needed to achieve local malaria elimination., Competing Interests: The authors have declared that no competing interests exist.

Background: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics., Methods: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps., Results: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled., Conclusion: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary malaria vector suggesting that IRS can reduce malaria transmission, but the presence of other vector species both indoors and outdoors suggests that alternative vector control interventions that target these gaps in protection may increase the impact of vector control in southern Mozambique., (© 2022. The Author(s).)

Monitoring local mosquito populations for insecticide resistance is critical for effective vector-borne disease control. However, widely used phenotypic assays, which are designed to monitor the emergence and spread of insecticide resistance (technical resistance), do not translate well to the efficacy of vector control products to suppress mosquito numbers in the field (practical resistance). This is because standard testing conditions such as environmental conditions, exposure dose, and type of substrate differ dramatically from those experienced by mosquitoes under field conditions. In addition, field mosquitoes have considerably different physiological characteristics such as age and blood-feeding status. Beyond this, indirect impacts of insecticide resistance and/or exposure on mosquito longevity, pathogen development, host-seeking behavior, and blood-feeding success impact disease transmission. Given the limited number of active ingredients currently available and the observed discordance between resistance and disease transmission, we conclude that additional testing guidelines are needed to determine practical resistance-the efficacy of vector control tools under relevant local conditions- in order to obtain programmatic impact., Competing Interests: AN, NJ, KP, SH No competing interests declared, (© 2021, Namias et al.)

Long-lasting insecticidal nets, or LLINs, have significantly reduced malaria morbidity and mortality over the past two decades. The net provides a physical barrier that decreases human-mosquito contact and the impregnated insecticide kills susceptible mosquito vectors upon contact and may repel them. However, the future of LLINs is threatened as resistance to pyrethroids is now widespread, the chemical arsenal for LLINs is very limited, time from discovery of next-generation insecticides to market is long, and persistent transmission is frequently caused by vector populations avoiding contact with LLINs. Here we ask the question whether, given these challenges, insecticides should be incorporated in nets at all. We argue that developing long-lasting nets without insecticide(s) can still reduce vector populations and provide both personal and community protection, if combined with other approaches or technologies. Taking the insecticide out of the equation (i) allows for a faster response to the current pyrethroid resistance crisis, (ii) avoids an LLIN-treadmill aimed at replacing failing bed nets due to insecticide resistance, and (iii) permits the utilization of our current and future insecticidal arsenal for other vector control tools to target persistent malaria transmission.

Malaria prevalence has significantly reduced since 2000, largely due to the scale-up of vector control interventions, mainly indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs). Given their success, these tools remain the frontline interventions in the fight against malaria. Their effectiveness relies on three key ingredients: the intervention, the mosquito vector and the end-user. Regarding the intervention, factors such as the insecticide active ingredient(s) used and the durability and/or bio-efficacy of the tool over time are critical. For the vectors, these factors include biting and resting behaviours and the susceptibility to insecticides. Finally, the end-users need to accept and properly use the intervention. Whilst human attitude and behaviour towards LLINs are well-documented both during and after distribution, only initial coverage is monitored for IRS and in a few geographic settings the residual efficacy of the used product. Here, the historical evidence on end-users modifying their wall surfaces post-spraying is presented, a behaviour that has the potential to reduce actual IRS coverage, effectiveness and impact, as fewer people are truly protected. Therefore, clear guidelines on how to monitor IRS acceptability and/or coverage, both before, during and after spraying, are urgently needed as part of the Monitoring and Evaluation of malaria programmes.

Background: Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for P. falciparum resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance. Anopheles funestus were collected in southern Mozambique using CDC light traps and manual collections. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of five mosquitoes. These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr1, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS., Results: Among the 846 samples screened for P. falciparum, 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for k13 and pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/pfdhps mutations were near fixation and about half of the isolates contained the pfmdr184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing., Conclusions: Pooled deep sequencing of P. falciparum isolates extracted from mosquitoes is a promising, efficient and cost-effective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogeneous geographical range.

Background: It is anticipated that malaria elimination efforts in Africa will be hampered by increasing resistance to the limited arsenal of insecticides approved for use in public health. However, insecticide susceptibility status of vector populations evaluated under standard insectary test conditions can give a false picture of the threat, as the thermal environment in which the insect and insecticide interact plays a significant role in insecticide toxicity., Methods: The effect of temperature on the expression of the standard WHO insecticide resistance phenotype was examined using Anopheles arabiensis and Anopheles funestus strains: a susceptible strain and the derived resistant strain, selected in the laboratory for resistance to DDT or pyrethroids. The susceptibility of mosquitoes to the pyrethroid deltamethrin or the carbamate bendiocarb was assessed at 18, 25 or 30 °C. The ability of the pyrethroid synergist piperonyl-butoxide (PBO) to restore pyrethroid susceptibility was also assessed at these temperatures., Results: Temperature impacted the toxicity of deltamethrin and bendiocarb. Although the resistant An. funestus strain was uniformly resistant to deltamethrin across temperatures, increasing temperature increased the resistance of the susceptible An. arabiensis strain. Against susceptible An. funestus and resistant An. arabiensis females, deltamethrin exposure at temperatures both lower and higher than standard insectary conditions increased mortality. PBO exposure completely restored deltamethrin susceptibility at all temperatures. Bendiocarb displayed a consistently positive temperature coefficient against both susceptible and resistant An. funestus strains, with survival increasing as temperature increased., Conclusions: Environmental temperature has a marked effect on the efficacy of insecticides used in public health against important African malaria vectors. Caution must be exercised when drawing conclusions about a chemical's efficacy from laboratory assays performed at only one temperature, as phenotypic resistance can vary significantly even over a temperature range that could be experienced by mosquitoes in the field during a single day. Similarly, it might be inappropriate to assume equal efficacy of a control tool over a geographic area where local conditions vary drastically. Additional studies into the effects of temperature on the efficacy of insecticide-based interventions under field conditions are warranted.

Since 2000, the world has made significant progress in reducing malaria morbidity and mortality, and several countries in Africa, South America and South-East Asia are working hard to eliminate the disease. These elimination efforts continue to rely heavily on antimalarial drugs and insecticide-based interventions, which remain the cornerstones of malaria treatment and prevention. However, resistance has emerged against nearly every antimalarial drug and insecticide that is available. In this review we discuss the evolutionary consequences of the way we currently implement antimalarial interventions, which is leading to resistance and may ultimately lead to control failure, but also how evolutionary principles can be applied to extend the lifespan of current and novel interventions. A greater understanding of the general evolutionary principles that are at the core of emerging resistance is urgently needed if we are to develop improved resistance management strategies with the ultimate goal to achieve a malaria-free world.

Temperature is a key environmental driver of Anopheles mosquito population dynamics; understanding its central role is important for these malaria vectors. Mosquito population responses to temperature fluctuations, though important across the life history, are poorly understood at a population level. We used stage-structured, temperature-dependent delay-differential equations to conduct a detailed exploration of the impacts of diurnal and annual temperature fluctuations on mosquito population dynamics. The model allows exploration of temperature-driven temporal changes in adult age structure, giving insights into the population's capacity to vector malaria parasites. Because of temperature-dependent shifts in age structure, the abundance of potentially infectious mosquitoes varies temporally, and does not necessarily mirror the dynamics of the total adult population. In addition to conducting the first comprehensive theoretical exploration of fluctuating temperatures on mosquito population dynamics, we analysed observed temperatures at four locations in Africa covering a range of environmental conditions. We found both temperature and precipitation are needed to explain the observed malaria season in these locations, enhancing our understanding of the drivers of malaria seasonality and how temporal disease risk may shift in response to temperature changes. This approach, tracking both mosquito abundance and age structure, may be a powerful tool for understanding current and future malaria risk.

The effectiveness of conventional malaria vector control is being threatened by the spread of insecticide resistance. One promising alternative to chemicals is the use of naturally-occurring insect-killing fungi. Numerous laboratory studies have shown that isolates of fungal pathogens such as Beauveria bassiana can infect and kill adult mosquitoes, including those resistant to chemical insecticides.Unlike chemical insecticides, fungi may take up to a week or more to kill mosquitoes following exposure. This slow kill speed can still reduce malaria transmission because the malaria parasite itself takes at least eight days to complete its development within the mosquito. However, both fungal virulence and parasite development rate are strongly temperature-dependent, so it is possible that biopesticide efficacy could vary across different transmission environments.We examined the virulence of a candidate fungal isolate against two key malaria vectors at temperatures from 10-34 °C. Regardless of temperature, the fungus killed more than 90% of exposed mosquitoes within the predicted duration of the malarial extrinsic incubation period, a result that was robust to realistic diurnal temperature variation.We then incorporated temperature sensitivities of a suite of mosquito, parasite and fungus life-history traits that are important determinants of malaria transmission into a stage-structured malaria transmission model. The model predicted that, at achievable daily fungal infection rates, fungal biopesticides have the potential to deliver substantial reductions in the density of malaria-infectious mosquitoes across all temperatures representative of malaria transmission environments. Synthesis and applications . Our study combines empirical data and theoretical modelling to prospectively evaluate the potential of fungal biopesticides to control adult malaria vectors. Our results suggest that Beauveria bassiana could be a potent tool for malaria control and support further development of fungal biopesticides to manage infectious disease vectors.

Background: Chemical insecticides are crucial to malaria control and elimination programmes. The frontline vector control interventions depend mainly on pyrethroids; all long-lasting insecticidal nets (LLINs) and more than 80% of indoor residual spraying (IRS) campaigns use chemicals from this class. This extensive use of pyrethroids imposes a strong selection pressure for resistance in mosquito populations, and so continuous resistance monitoring and evaluation are important. As pyrethroids have also been used for many years in the Manhiça District, an area in southern Mozambique with perennial malaria transmission, an assessment of their efficacy against the local malaria vectors was conducted., Methods: Female offspring of wild-caught Anopheles funestus s.s. females were exposed to deltamethrin, lambda-cyhalothrin and permethrin using the World Health Organization (WHO) insecticide-resistance monitoring protocols. The 3-min WHO cone bioassay was used to evaluate the effectiveness of the bed nets distributed or available for purchase in the area (Olyset, permethrin LLIN; PermaNet 2.0, deltamethrin LLIN) against An. funestus. Mosquitoes were also exposed to PermaNet 2.0 for up to 8 h in time-exposure assays., Results: Resistance to pyrethroids in An. funestus s.s. was extremely high, much higher than reported in 2002 and 2009. No exposure killed more than 25.8% of the mosquitoes tested (average mortality, deltamethrin: 6.4%; lambda-cyhalothrin: 5.1%; permethrin: 19.1%). There was no significant difference in the mortality generated by 3-min exposure to any net (Olyset: 9.3% mortality, PermaNet 2.0: 6.0%, untreated: 2.0%; p = 0.2). Six hours of exposure were required to kill 50% of the An. funestus s.s. on PermaNet 2.0., Conclusions: Anopheles funestus s.s. in Manhiça is extremely resistant to pyrethroids, and this area is clearly a pyrethroid-resistance hotspot. This could severely undermine vector control in this district if no appropriate countermeasures are undertaken. The National Malaria Control Programme (NMCP) of Mozambique is currently improving its resistance monitoring programme, to design and scale up new management strategies. These actions are urgently needed, as the goal of the NMCP and its partners is to reach elimination in southern Mozambique by 2020.

Extrinsic environmental factors influence the distribution and population dynamics of many organisms, including insects that are of concern for human health and agriculture. This is particularly true for vector-borne infectious diseases like malaria, which is a major source of morbidity and mortality in humans. Understanding the mechanistic links between environment and population processes for these diseases is key to predicting the consequences of climate change on transmission and for developing effective interventions. An important measure of the intensity of disease transmission is the reproductive number R0. However, understanding the mechanisms linking R0 and temperature, an environmental factor driving disease risk, can be challenging because the data available for parameterization are often poor. To address this, we show how a Bayesian approach can help identify critical uncertainties in components of R0 and how this uncertainty is propagated into the estimate of R0. Most notably, we find that different parameters dominate the uncertainty at different temperature regimes: bite rate from 15 degrees C to 25 degrees C; fecundity across all temperatures, but especially approximately 25-32 degrees C; mortality from 20 degrees C to 30 degrees C; parasite development rate at degrees 15-16 degrees C and again at approximately 33-35 degrees C. Focusing empirical studies on these parameters and corresponding temperature ranges would be the most efficient way to improve estimates of R0. While we focus on malaria, our methods apply to improving process-based models more generally, including epidemiological, physiological niche, and species distribution models.

Background: Insecticides are critical components of malaria control programmes. In a variety of insect species, temperature plays a fundamental role in determining the outcome of insecticide exposure. However, surprisingly little is known about how temperature affects the efficacy of chemical interventions against malaria vectors., Methods: Anopheles stephensi, with no recent history of insecticide exposure, were exposed to the organophosphate malathion or the pyrethroid permethrin at 12, 18, 22, or 26°C, using the WHO tube resistance-monitoring assay. To evaluate the effect of pre-exposure temperature on susceptibility, adult mosquitoes were kept at 18 or 26°C until just before exposure, and then moved to the opposite temperature. Twenty-four hours after exposure, mosquitoes exposed at <26°C were moved to 26°C and recovery was observed. Susceptibility was assessed in terms of survival 24 hours after exposure; data were analysed as generalized linear models using a binomial error distribution and logit link function., Results: Lowering the exposure temperature from the laboratory standard 26°C can strongly reduce the susceptibility of female An. stephensi to the WHO resistance-discriminating concentration of malathion (χ2(df=3) = 29.0, p < 0.001). While the susceptibility of these mosquitoes to the resistance-discriminating concentration of permethrin was not as strongly temperature-dependent, recovery was observed in mosquitoes moved from 12, 18 or 22°C to 26°C 24 hours after exposure. For permethrin especially, the thermal history of the mosquito was important in determining the ultimate outcome of insecticide exposure for survival (permethrin: pre-exposure temperature: F1,29 = 14.2, p < 0.001; exposure temp: F1,29 = 1.1, p = 0.3; concentration: F1,29 = 85.2, p < 0.001; exposure temp x conc: F1,29 = 5.8, p = 0.02). The effect of acclimation temperature on malathion susceptibility depended on the exposure temperature (exposure temp: F1,79 = 98.4, p < 0.001; pre-exposure temp: F1,79 = 0.03, p = 0.9; pre-exp temp x exp temp F1,79 = 6.0, p = 0.02)., Conclusions: A single population of An. stephensi could be classified by WHO criteria as susceptible or resistant to a given chemical, depending on the temperature at which the mosquitoes were exposed. Investigating the performance of vector control tools under different temperature conditions will augment the ability to better understand the epidemiological significance of insecticide resistance and select the most effective products for a given environment.

The parasites that cause malaria depend on Anopheles mosquitoes for transmission; because of this, mosquito population dynamics are a key determinant of malaria risk. Development and survival rates of both the Anopheles mosquitoes and the Plasmodium parasites that cause malaria depend on temperature, making this a potential driver of mosquito population dynamics and malaria transmission. We developed a temperature-dependent, stage-structured delayed differential equation model to better understand how climate determines risk. Including the full mosquito life cycle in the model reveals that the mosquito population abundance is more sensitive to temperature than previously thought because it is strongly influenced by the dynamics of the juvenile mosquito stages whose vital rates are also temperature-dependent. Additionally, the model predicts a peak in abundance of mosquitoes old enough to vector malaria at more accurate temperatures than previous models. Our results point to the importance of incorporating detailed vector biology into models for predicting the risk for vector borne diseases.

Ectotherms are considered to be particularly vulnerable to climate warming. Descriptions of habitat temperatures and predicted changes in climate usually consider mean monthly, seasonal or annual conditions. Ectotherms, however, do not simply experience mean conditions, but are exposed to daily fluctuations in habitat temperatures. Here, we highlight how temperature fluctuation can generate 'realized' thermal reaction (fitness) norms that differ from the 'fundamental' norms derived under standard constant temperatures. Using a mosquito as a model organism, we find that temperature fluctuation reduces rate processes such as development under warm conditions, increases processes under cool conditions, and reduces both the optimum and the critical maximum temperature. Generalizing these effects for a range of terrestrial insects reveals that prevailing daily fluctuations in temperature should alter the sensitivity of species to climate warming by reducing 'thermal safety margins'. Such effects of daily temperature dynamics have generally been ignored in the climate change literature., (© 2013 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Background: Environmental temperature is an important driver of malaria transmission dynamics. Both the parasite and vector are sensitive to mean ambient temperatures and daily temperature variation. To understand transmission ecology, therefore, it is important to determine the range of microclimatic temperatures experienced by malaria vectors in the field., Methods: A pilot study was conducted in the Indian city of Chennai to determine the temperature variation in urban microclimates and characterize the thermal ecology of the local transmission setting. Temperatures were measured in a range of probable indoor and outdoor resting habitats of Anopheles stephensi in two urban slum malaria sites. Mean temperatures and daily temperature fluctuations in local transmission sites were compared with standard temperature measures from the local weather station. The biological implications of the different temperatures were explored using temperature-dependent parasite development models to provide estimates of the extrinsic incubation period (EIP) of Plasmodium vivax and Plasmodium falciparum., Results: Mean daily temperatures within the urban transmission sites were generally warmer than those recorded at the local weather station. The main reason was that night-time temperatures were higher (and hence diurnal temperature ranges smaller) in the urban settings. Mean temperatures and temperature variation also differed between specific resting sites within the transmission environments. Most differences were of the order of 1-3°C but were sufficient to lead to important variation in predicted EIPs and hence, variation in estimates of transmission intensity., Conclusions: Standard estimates of environmental temperature derived from local weather stations do not necessarily provide realistic measures of temperatures within actual transmission environments. Even the small differences in mean temperatures or diurnal temperature ranges reported in this study can lead to large variations in key mosquito and/or parasite life history traits that determine transmission intensity. Greater effort should be directed at quantifying adult mosquito resting behaviour and determining the temperatures actually experienced by mosquitoes and parasites in local transmission environments. In the absence of such highly resolved data, the approach used in the current study provides a framework for improved thermal characterization of transmission settings.

The ecology of mosquito vectors and malaria parasites affect the incidence, seasonal transmission and geographical range of malaria. Most malaria models to date assume constant or linear responses of mosquito and parasite life-history traits to temperature, predicting optimal transmission at 31 °C. These models are at odds with field observations of transmission dating back nearly a century. We build a model with more realistic ecological assumptions about the thermal physiology of insects. Our model, which includes empirically derived nonlinear thermal responses, predicts optimal malaria transmission at 25 °C (6 °C lower than previous models). Moreover, the model predicts that transmission decreases dramatically at temperatures > 28 °C, altering predictions about how climate change will affect malaria. A large data set on malaria transmission risk in Africa validates both the 25 °C optimum and the decline above 28 °C. Using these more accurate nonlinear thermal-response models will aid in understanding the effects of current and future temperature regimes on disease transmission., (© 2012 Blackwell Publishing Ltd/CNRS.)

A mosquito needs to bite at least twice for malaria transmission to occur: once to acquire parasites and, after these parasites complete their development in their mosquito host, once to transmit the parasites to the next vertebrate host. Here we investigate the relationship between temperature, parasite development, and biting frequency in a mosquito-rodent malaria model system. We show that the pre-bloodmeal period (the time lag between mosquito emergence and first bloodmeal) increases at lower temperatures. In addition, parasite development time and feeding exhibit different thermal sensitivities such that mosquitoes might not be ready to feed at the point at which the parasite is ready to be transmitted. Exploring these effects using a simple theoretical model of human malaria shows that delays in infection and transmission can reduce the vectorial capacity of malaria mosquitoes by 20 to over 60%, depending on temperature. These delays have important implications for disease epidemiology and control, and should be considered in future transmission models.

Temperature is an important determinant of malaria transmission. Recent work has shown that mosquito and parasite biology are influenced not only by average temperature, but also by the extent of the daily temperature variation. Here we examine how parasite development within the mosquito (Extrinsic Incubation Period) is expected to vary over time and space depending on the diurnal temperature range and baseline mean temperature in Kenya and across Africa. Our results show that under cool conditions, the typical approach of using mean monthly temperatures alone to characterize the transmission environment will underestimate parasite development. In contrast, under warmer conditions, the use of mean temperatures will overestimate development. Qualitatively similar patterns hold using both outdoor and indoor temperatures. These findings have important implications for defining malaria risk. Furthermore, understanding the influence of daily temperature dynamics could provide new insights into ectotherm ecology both now and in response to future climate change.

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.

Over the last 20 years, ecological immunology has provided much insight into how environmental factors shape host immunity and host-parasite interactions. Currently, the application of this thinking to the study of mosquito immunology has been limited. Mechanistic investigations are nearly always conducted under one set of conditions, yet vectors and parasites associate in a variable world. We highlight how environmental temperature shapes cellular and humoral immune responses (melanization, phagocytosis and transcription of immune genes) in the malaria vector, Anopheles stephensi. Nitric oxide synthase expression peaked at 30°C, cecropin expression showed no main effect of temperature and humoral melanization, and phagocytosis and defensin expression peaked around 18°C. Further, immune responses did not simply scale with temperature, but showed complex interactions between temperature, time and nature of immune challenge. Thus, immune patterns observed under one set of conditions provide little basis for predicting patterns under even marginally different conditions. These quantitative and qualitative effects of temperature have largely been overlooked in vector biology but have significant implications for extrapolating natural/transgenic resistance mechanisms from laboratory to field and for the efficacy of various vector control tools.

The development rate of parasites and pathogens within vectors typically increases with temperature. Accordingly, transmission intensity is generally assumed to be higher under warmer conditions. However, development is only one component of parasite/pathogen life history and there has been little research exploring the temperature sensitivity of other traits that contribute to transmission intensity. Here, using a rodent malaria, we show that vector competence (the maximum proportion of infectious mosquitoes, which implicitly includes parasite survival across the incubation period) tails off at higher temperatures, even though parasite development rate increases. We also show that the standard measure of the parasite incubation period (i.e. time until the first mosquitoes within a cohort become infectious following an infected blood-meal) is incomplete because parasite development follows a cumulative distribution, which itself varies with temperature. Including these effects in a simple model dramatically alters estimates of transmission intensity and reduces the optimum temperature for transmission. These results highlight the need to understand the interactive effects of environmental temperature on multiple host-disease life-history traits and challenge the assumptions of many current disease models that ignore this complexity.

The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasmodium chabaudi, co-infection with drug-sensitive parasites can prevent the transmission of initially rare resistant parasites to mosquitoes. Removal of drug-sensitive parasites following chemotherapy enabled resistant parasites to transmit to mosquitoes as successfully as sensitive parasites in the absence of treatment. We also show that the genetic composition of gametocyte populations in host venous blood accurately reflects the genetic composition of gametocytes taken up by mosquitoes. Our data demonstrate that, at least for this mouse model, aggressive chemotherapy leads to very effective transmission of highly resistant parasites that are present in an infection, the very parasites which undermine the long term efficacy of front-line drugs.

Background: The majority of the mosquito and parasite life-history traits that combine to determine malaria transmission intensity are temperature sensitive. In most cases, the process-based models used to estimate malaria risk and inform control and prevention strategies utilize measures of mean outdoor temperature. Evidence suggests, however, that certain malaria vectors can spend large parts of their adult life resting indoors., Presentation of Hypothesis: If significant proportions of mosquitoes are resting indoors and indoor conditions differ markedly from ambient conditions, simple use of outdoor temperatures will not provide reliable estimates of malaria transmission intensity. To date, few studies have quantified the differential effects of indoor vs outdoor temperatures explicitly, reflecting a lack of proper understanding of mosquito resting behaviour and associated microclimate., Testing the Hypothesis: Published records from 8 village sites in East Africa revealed temperatures to be warmer indoors than outdoors and to generally show less daily variation. Exploring the effects of these temperatures on malaria parasite development rate suggested indoor-resting mosquitoes could transmit malaria between 0.3 and 22.5 days earlier than outdoor-resting mosquitoes. These differences translate to increases in transmission risk ranging from 5 to approaching 3,000%, relative to predictions based on outdoor temperatures. The pattern appears robust for low- and highland areas, with differences increasing with altitude., Implications of the Hypothesis: Differences in indoor vs outdoor environments lead to large differences in the limits and the intensity of malaria transmission. This finding highlights a need to better understand mosquito resting behaviour and the associated microclimate, and to broaden assessments of transmission ecology and risk to consider the potentially important role of endophily.

Virulence (speed of kill) of a fungal entomopathogen against a particular host insect depends on biological properties of the specific isolate-host combination, together with factors such as fungal dose. How these intrinsic and extrinsic factors affect the actual pattern and extent of fungal growth invivo is poorly understood. In this study we exposed adult house flies (Muscadomestica L.) to surfaces treated with high and low doses of Beauveriabassiana (isolates BbGHA and Bb5344), Metarhiziumanisopliae (strain MaF52) and M.anisopliae var. acridum (isolate Ma189) and used quantitative real-time PCR with species-specific primers to examine the relationship between fungal growth kinetics and virulence. At the highest dose, all fungal isolates killed flies significantly faster than controls, with BbGHA, Bb5344 and MaF52 roughly equivalent in virulence (median survival time (±SE)=5.0±0.10, 5.0±0.08 and 5.0±0.12days, respectively) and Ma189 killing more slowly (MST=8.0±0.20days). At the lower dose, effective virulence was reduced and only flies exposed to isolates BbGHA and Bb5344 died significantly faster than controls (MST=12±1.36, 15±0.64, 18±0.86 and 21.0±0.0days for BbGHA, Bb5344, MaF52 and Ma189, respectively). Real-time PCR assays revealed that flies exposed to surfaces treated with the high dose of spores had greater spore pickup than flies exposed to the low dose for each isolate. After pickup, a general pattern emerged for all isolates in which there was a significant reduction of recovered fungal DNA 48h after exposure followed by a brief recovery phase, a stable period of little net change in fungal sequence counts, and then a dramatic increase in sequence counts of up to three orders of magnitude around the time of host death. However, while the patterns of growth were similar, there were quantitative differences such that higher final sequence counts were recovered in insects infected with the most lethal isolates and with the higher dose. These results suggest that variation in virulence between isolates, species and doses is determined more by quantitative rather than qualitative differences in fungal growth kinetics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Most studies on the ability of insect populations to transmit pathogens consider only constant temperatures and do not account for realistic daily temperature fluctuations that can impact vector-pathogen interactions. Here, we show that diurnal temperature range (DTR) affects two important parameters underlying dengue virus (DENV) transmission by Aedes aegypti. In two independent experiments using different DENV serotypes, mosquitoes were less susceptible to virus infection and died faster under larger DTR around the same mean temperature. Large DTR (20 °C) decreased the probability of midgut infection, but not duration of the virus extrinsic incubation period (EIP), compared with moderate DTR (10 °C) or constant temperature. A thermodynamic model predicted that at mean temperatures <18 °C, DENV transmission increases as DTR increases, whereas at mean temperatures >18 °C, larger DTR reduces DENV transmission. The negative impact of DTR on Ae. aegypti survival indicates that large temperature fluctuations will reduce the probability of vector survival through EIP and expectation of infectious life. Seasonal variation in the amplitude of daily temperature fluctuations helps to explain seasonal forcing of DENV transmission at locations where average temperature does not vary seasonally and mosquito abundance is not associated with dengue incidence. Mosquitoes lived longer and were more likely to become infected under moderate temperature fluctuations, which is typical of the high DENV transmission season than under large temperature fluctuations, which is typical of the low DENV transmission season. Our findings reveal the importance of considering short-term temperature variations when studying DENV transmission dynamics.

Background: As the ecology of mosquito larvae can be complex there is need to develop a rational framework for undertaking larval ecological studies. Local environmental characteristics, such as altitude, climate and land use, can significantly impact on phenology and population dynamics of mosquito larvae, and indirectly affect the dynamics of mosquito-borne diseases. The aim of this study was to assess the feasibility of implementing an integrated approach to larval source management under the distinct ecological settings., Methods: The study was conducted in two highland villages and one village, at a lower altitude, in the Lake Victoria basin, where malaria is endemic and transmitted by the same Anopheles mosquito species. In each village the stability of mosquito larval habitats was classified as either temporary or permanent. The productivity of these habitat types was quantified by carrying out weekly larval sampling using a standard dipping method for a period of two years. During sampling the physical characteristic of the larval habitat, including the vegetation cover were noted. Ambient temperature, rainfall and relative humidity were recorded on a 21 × Micro-datalogger in each study site., Results: Anopheles gambiae sensu lato larvae were found in all study sites. Anopheles arabiensis was more abundant (93%) in Nyalenda (Lake Victoria basin) and Fort Ternan (highland area; 71%). In Lunyerere (highland area), An. gambiae sensu stricto comprised 93% of the total An. gambiae s.l. larvae. Larvae of An. gambiae s.l. mosquitoes were present in both temporary and permanent habitats with monthly variations dependent on rainfall intensity and location. Anopheles larvae were more likely to be found in man-made as opposed to natural habitats. Grassy habitats were preferred and were, therefore, more productive of Anopheles larvae compared to other habitat types. Weekly rainfall intensity led to an increase or decrease in mosquito larval abundance depending on the location., Conclusion: The majority of mosquito breeding habitats were man made in all sites. Both temporary and permanent habitats were suitable for An. gambiae breeding. In Fort Ternan temporary sites were favoured for mosquito breeding above permanent sites. Significant differences in larval abundance were found depending on weekly rainfall intensity. Larval source management programmes should target permanent and temporary habitats equally and work closely with land and home owners as a majority of the breeding habitats are man made.

Malaria transmission is strongly influenced by environmental temperature, but the biological drivers remain poorly quantified. Most studies analyzing malaria-temperature relations, including those investigating malaria risk and the possible impacts of climate change, are based solely on mean temperatures and extrapolate from functions determined under unrealistic laboratory conditions. Here, we present empirical evidence to show that, in addition to mean temperatures, daily fluctuations in temperature affect parasite infection, the rate of parasite development, and the essential elements of mosquito biology that combine to determine malaria transmission intensity. In general, we find that, compared with rates at equivalent constant mean temperatures, temperature fluctuation around low mean temperatures acts to speed up rate processes, whereas fluctuation around high mean temperatures acts to slow processes down. At the extremes (conditions representative of the fringes of malaria transmission, where range expansions or contractions will occur), fluctuation makes transmission possible at lower mean temperatures than currently predicted and can potentially block transmission at higher mean temperatures. If we are to optimize control efforts and develop appropriate adaptation or mitigation strategies for future climates, we need to incorporate into predictive models the effects of daily temperature variation and how that variation is altered by climate change.

Background: The relationship between mosquito development and temperature is one of the keys to understanding the current and future dynamics and distribution of vector-borne diseases such as malaria. Many process-based models use mean air temperature to estimate larval development times, and hence adult vector densities and/or malaria risk., Methods: Water temperatures in three different-sized water pools, as well as the adjacent air temperature in lowland and highland sites in western Kenya were monitored. Both air and water temperatures were fed into a widely-applied temperature-dependent development model for Anopheles gambiae immatures, and subsequently their impact on predicted vector abundance was assessed., Results: Mean water temperature in typical mosquito breeding sites was 4-6 degrees C higher than the mean temperature of the adjacent air, resulting in larval development rates, and hence population growth rates, that are much higher than predicted based on air temperature. On the other hand, due to the non-linearities in the relationship between temperature and larval development rate, together with a marginal buffering in the increase in water temperature compared with air temperature, the relative increases in larval development rates predicted due to climate change are substantially less., Conclusions: Existing models will tend to underestimate mosquito population growth under current conditions, and may overestimate relative increases in population growth under future climate change. These results highlight the need for better integration of biological and environmental information at the scale relevant to mosquito biology.

The incubation period for malaria parasites within the mosquito is exquisitely temperature-sensitive, so that temperature is a major determinant of malaria risk. Epidemiological models are increasingly used to guide allocation of disease control resources and to assess the likely impact of climate change on global malaria burdens. Temperature-based malaria transmission is generally incorporated into these models using mean monthly temperatures, yet temperatures fluctuate throughout the diurnal cycle. Here we use a thermodynamic malaria development model to demonstrate that temperature fluctuation can substantially alter the incubation period of the parasite, and hence malaria transmission rates. We find that, in general, temperature fluctuation reduces the impact of increases in mean temperature. Diurnal temperature fluctuation around means >21 degrees C slows parasite development compared with constant temperatures, whereas fluctuation around <21 degrees C speeds development. Consequently, models which ignore diurnal variation overestimate malaria risk in warmer environments and underestimate risk in cooler environments. To illustrate the implications further, we explore the influence of diurnal temperature fluctuation on malaria transmission at a site in the Kenyan Highlands. Based on local meteorological data, we find that the annual epidemics of malaria at this site cannot be explained without invoking the influence of diurnal temperature fluctuation. Moreover, while temperature fluctuation reduces the relative influence of a subtle warming trend apparent over the last 20 years, it nonetheless makes the effects biologically more significant. Such effects of short-term temperature fluctuations have not previously been considered but are central to understanding current malaria transmission and the consequences of climate change.

The present paper reports the occurrence of competition between larvae of the malaria mosquito sibling species Anopheles arabiensis and An. gambiae under ambient conditions in western Kenya. Larvae of both species were reared at the same density and under the same food conditions outdoors in single-species and mixed-species populations (species ratio 1:1) in transparent cups that floated in small and large semi-natural pools, which experienced different diurnal variations in water temperature. In a second experiment, both species were reared at similar densities and under the same food conditions in trays in either single-species or mixed-species populations at different proportions (species ratio 1:1, 1:3 or 3:1). Competition affected the development rate of both species in an opposite way: the development time of larvae of An. arabiensis increased whereas the development time of larvae of An. gambiae decreased in the presence of its sibling species. In small pools larvae developing in mixed-species populations experienced a higher mortality than larvae reared in single-species populations, whereas no such effect was observed in the large pools. In both species the time to pupation was longer and emerging females were larger in the small pools. Larval mortality of An. arabiensis was lower in the small pools compared to the large pools, whereas An. gambiae showed the opposite trend. Overall An. arabiensis showed reduced development rates, higher mortality rates and emerged with a larger body size compared to An. gambiae. The implication of these competitive interactions between larvae of An. arabiensis and An. gambiae under semi-filed conditions needs to be considered in the design and implementation of programmes that aim to reduce malaria transmission as competition may alter the species composition in the field.

Water temperature is a critical regulator in the growth and development of malaria mosquito immatures, as they are poikilothermic. Measuring or estimating the diurnal temperature ranges to which these immatures are exposed is of the utmost importance, as these immatures will develop into adults that can transmit malaria. Recent attempts to predict the daily water temperature dynamics in mosquito breeding sites in Kenya have been successful. However, the developed model may be too complex, as the sophisticated equipment that was used for detailed meteorological observations is not widely distributed in Africa, making it difficult to predict the daily water temperature dynamics on a local scale. Therefore, we compared two energy budget models with earlier made observations of the daily water temperature dynamics in a small, shallow and clear water pool (diameter 0.96 m, depth 0.32 m) in Kenya. This paper describes (1) a complex 1-Dimensional model, and (2) a simplified second model, and (3) shows that both models mimic the water temperature dynamics in the water pool accurately. The latter model has the advantage that it only needs common weather data (air temperature, air humidity, wind speed and cloud cover) to estimate the diurnal temperature dynamics in breeding sites of African malaria mosquitoes.

Water temperature is an important determinant in many aquatic biological processes, including the growth and development of malaria mosquito (Anopheles arabiensis and A. gambiae) immatures. Water turbidity affects water temperature, as suspended particles in a water column absorb and scatter sunlight and hence determine the extinction of solar radiation. To get a better understanding of the relationship between water turbidity and water temperature, a series of semi-natural larval habitats (diameter 0.32 m, water depth 0.16 m) with increasing water turbidity was created. Here we show that at midday (1300 hours) the upper water layer (thickness of 10 mm) of the water pool with the highest turbidity was on average 2.8 degrees C warmer than the same layer of the clearest water pool. Suspended soil particles increase the water temperature and furthermore change the temperature dynamics of small water collections during daytime, exposing malaria mosquito larvae, which live in the top water layer, longer to higher temperatures.

For aquatic biological processes, diurnal and annual cycles of water temperature are very important to plants as well as to animals and microbes living in the water. An existing one-dimensional model has been extended to simulate the temperature profile within a small water body. A year-round outdoor experiment has been conducted to estimate the model input parameters and to verify the model. Both model simulations and measurements show a strong temperature stratification in the water during daytime. Throughout the night, however, a well-mixed layer starting at the water surface develops. Because the water body is relatively small, it appears that the sediment heat flux has a strong effect on the behaviour of the water temperature throughout the seasons. In spring, the water temperature remains relatively low due to the cold surrounding soil, while in autumn the opposite occurs due to the relatively warm soil. It appears that, in small water bodies, the total amount of incoming long wave radiation is sensitive to the sky view factor. In our experiments, the intensity of precipitation also appears to have a small effect on the stratification of the water temperature.