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Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Malaria is among the world's deadliest diseases, predominantly affecting Sub-Saharan Africa and killing over half a million people annually. Controlling the principal vector, the mosquito Anopheles gambiae, as well as other anophelines, is among the most effective methods to control disease spread. Here, we develop a genetic population suppression system termed Ifegenia (inherited female elimination by genetically encoded nucleases to interrupt alleles) in this deadly vector. In this bicomponent CRISPR-based approach, we disrupt a female-essential gene, femaleless (fle), demonstrating complete genetic sexing via heritable daughter gynecide. Moreover, we demonstrate that Ifegenia males remain reproductively viable and can load both fle mutations and CRISPR machinery to induce fle mutations in subsequent generations, resulting in sustained population suppression. Through modeling, we demonstrate that iterative releases of nonbiting Ifegenia males can act as an effective, confinable, controllable, and safe population suppression and elimination system.

Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when ca. 40 adult females are sampled biweekly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with ca. 120 larvae sampled biweekly over the same period. The methods are also effective at detecting intervention-induced increases in adult mortality and decreases in population size. As the cost of genome sequencing declines, CKMR holds great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

Mosquitoes are important vectors for pathogens that infect humans and other vertebrate animals. Some aspects of adult mosquito behavior and mosquito ecology play an important role in determining the capacity of vector populations to transmit pathogens. Here, we re-examine factors affecting the transmission of pathogens by mosquitoes using a new approach. Unlike most previous models, this framework considers the behavioral states and state transitions of adult mosquitoes through a sequence of activity bouts. We developed a new framework for individual-based simulation models called MBITES (Mosquito Bout-based and Individual-based Transmission Ecology Simulator). In MBITES, it is possible to build models that simulate the behavior and ecology of adult mosquitoes in exquisite detail on complex resource landscapes generated by spatial point processes. We also developed an ordinary differential equation model which is the Kolmogorov forward equations for models developed in MBITES under a specific set of simplifying assumptions. While mosquito infection and pathogen development are one possible part of a mosquito's state, that is not our main focus. Using extensive simulation using some models developed in MBITES, we show that vectorial capacity can be understood as an emergent property of simple behavioral algorithms interacting with complex resource landscapes, and that relative density or sparsity of resources and the need to search can have profound consequences for mosquito populations' capacity to transmit pathogens.

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.

Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.

As Africa-wide malaria prevalence declines, an understanding of human movement patterns is essential to inform how best to target interventions. We fitted movement models to trip data from surveys conducted at 3-5 sites throughout each of Mali, Burkina Faso, Zambia and Tanzania. Two models were compared in terms of their ability to predict the observed movement patterns - a gravity model, in which movement rates between pairs of locations increase with population size and decrease with distance, and a radiation model, in which travelers are cumulatively "absorbed" as they move outwards from their origin of travel. The gravity model provided a better fit to the data overall and for travel to large populations, while the radiation model provided a better fit for nearby populations. One strength of the data set was that trips could be categorized according to traveler group - namely, women traveling with children in all survey countries and youth workers in Mali. For gravity models fitted to data specific to these groups, youth workers were found to have a higher travel frequency to large population centers, and women traveling with children a lower frequency. These models may help predict the spatial transmission of malaria parasites and inform strategies to control their spread.

BackgroundResidual malaria transmission has been reported in many areas even with adequate indoor vector control coverage, such as long-lasting insecticidal nets (LLINs). The increased insecticide resistance in Anopheles mosquitoes has resulted in reduced efficacy of the widely used indoor tools and has been linked with an increase in outdoor malaria transmission. There are considerations of incorporating outdoor interventions into integrated vector management (IVM) to achieve malaria elimination; however, more information on the combination of tools for effective control is needed to determine their utilization.MethodsA spatial individual-based model was modified to simulate the environment and malaria transmission activities in a hypothetical, isolated African village setting. LLINs and outdoor attractive toxic sugar bait (ATSB) stations were used as examples of indoor and outdoor interventions, respectively. Different interventions and lengths of efficacy periods were tested. Simulations continued for 420 days, and each simulation scenario was repeated 50 times. Mosquito populations, entomologic inoculation rates (EIRs), probabilities of local mosquito extinction, and proportion of time when the annual EIR was reduced below one were compared between different intervention types and efficacy periods.ResultsIn the village setting with clustered houses, the combinational intervention of 50% LLINs plus outdoor ATSBs significantly reduced mosquito population and EIR in short term, increased the probability of local mosquito extinction, and increased the time when annual EIR is less than one per person compared to 50% LLINs alone; outdoor ATSBs alone significantly reduced mosquito population in short term, increased the probability of mosquito extinction, and increased the time when annual EIR is less than one compared to 50% LLINs alone, but there was no significant difference in EIR in short term between 50% LLINs and outdoor ATSBs. In the village setting with dispersed houses, the combinational intervention of 50% LLINs plus outdoor ATSBs significantly reduced mosquito population in short term, increased the probability of mosquito extinction, and increased the time when annual EIR is less than one per person compared to 50% LLINs alone; outdoor ATSBs alone significantly reduced mosquito population in short term, but there were no significant difference in the probability of mosquito extinction and the time when annual EIR is less than one between 50% LLIN and outdoor ATSBs; and there was no significant difference in EIR between all three interventions. A minimum of 2 months of efficacy period is needed to bring out the best possible effect of the vector control tools, and to achieve long-term mosquito reduction, a minimum of 3 months of efficacy period is needed.ConclusionsThe results highlight the value of incorporating outdoor vector control into IVM as a supplement to traditional indoor practices for malaria elimination in Africa, especially in village settings of clustered houses where LLINs alone is far from sufficient.

BackgroundAs malaria prevalence declines in many parts of the world due to widescale control efforts and as drug-resistant parasites begin to emerge, a quantitative understanding of human movement is becoming increasingly relevant to malaria control. However, despite its importance, significant knowledge gaps remain regarding human movement, particularly in sub-Saharan Africa.MethodsA quantitative survey of human movement patterns was conducted in four countries in sub-Saharan Africa: Mali, Burkina Faso, Zambia, and Tanzania, with three to five survey locations chosen in each country. Questions were included on demographic and trip details, malaria risk behaviour, children accompanying travellers, and mobile phone usage to enable phone signal data to be better correlated with movement. A total of 4352 individuals were interviewed and 6411 trips recorded.ResultsA cluster analysis of trips highlighted two distinct traveller groups of relevance to malaria transmission: women travelling with children (in all four countries) and youth workers (in Mali). Women travelling with children were more likely to travel to areas of relatively high malaria prevalence in Mali (OR = 4.46, 95% CI = 3.42-5.83), Burkina Faso (OR = 1.58, 95% CI = 1.23-1.58), Zambia (OR = 1.50, 95% CI = 1.20-1.89), and Tanzania (OR = 2.28, 95% CI = 1.71-3.05) compared to other travellers. They were also more likely to own bed nets in Burkina Faso (OR = 1.77, 95% CI = 1.25-2.53) and Zambia (OR = 1.74, 95% CI = 1.34 2.27), and less likely to own a mobile phone in Mali (OR = 0.50, 95% CI = 0.39-0.65), Burkina Faso (OR = 0.39, 95% CI = 0.30-0.52), and Zambia (OR = 0.60, 95% CI = 0.47-0.76). Malian youth workers were more likely to travel to areas of relatively high malaria prevalence (OR = 23, 95% CI = 17-31) and for longer durations (mean of 70 days cf 21 days, p < 0.001) compared to other travellers.ConclusionsWomen travelling with children were a remarkably consistent traveller group across all four countries surveyed. They are expected to contribute greatly towards spatial malaria transmission because the children they travel with tend to have high parasite prevalence. Youth workers were a significant traveller group in Mali and are expected to contribute greatly to spatial malaria transmission because their movements correlate with seasonal rains and hence peak mosquito densities. Interventions aimed at interrupting spatial transmission of parasites should consider these traveller groups.

As the world's fastest spreading vector-borne disease, dengue was estimated to infect more than 390 million people in 2010, a 30-fold increase in the past half century. Although considered to be a non-endemic country, mainland China had 55,114 reported dengue cases from 2005 to 2014, of which 47,056 occurred in 2014. Furthermore, 94% of the indigenous cases in this time period were reported in Guangdong Province, 83% of which were in Guangzhou City. In order to determine the possible determinants of the unprecedented outbreak in 2014, a population-based deterministic model was developed to describe dengue transmission dynamics in Guangzhou. Regional sensitivity analysis (RSA) was adopted to calibrate the model and entomological surveillance data was used to validate the mosquito submodel. Different scenarios were created to investigate the roles of the timing of an imported case, climate, vertical transmission from mosquitoes to their offspring, and intervention. The results suggested that an early imported case was the most important factor in determining the 2014 outbreak characteristics. Precipitation and temperature can also change the transmission dynamics. Extraordinary high precipitation in May and August, 2014 appears to have increased vector abundance. Considering the relatively small number of cases in 2013, the effect of vertical transmission was less important. The earlier and more frequent intervention in 2014 also appeared to be effective. If the intervention in 2014 was the same as that in 2013, the outbreak size may have been over an order of magnitude higher than the observed number of new cases in 2014.The early date of the first imported and locally transmitted case was largely responsible for the outbreak in 2014, but it was influenced by intervention, climate and vertical transmission. Early detection and response to imported cases in the spring and early summer is crucial to avoid large outbreaks in the future.

BackgroundThe development of insecticide resistance and the increased outdoor-biting behaviour of malaria vectors reduce the efficiency of indoor vector control methods. Attractive toxic sugar baits (ATSBs), a method targeting the sugar-feeding behaviours of vectors both indoors and outdoors, is a promising supplement to indoor tools. The number and configuration of these ATSB stations needed for malaria control in a community needs to be determined.MethodsA hypothetical village, typical of those in sub-Saharan Africa, 600 × 600 m, consisting of houses, humans and essential resource requirements of Anopheles gambiae (sugar sources, outdoor resting sites, larval habitats) was simulated in a spatial individual-based model. Resource-rich and resource-poor environments were simulated separately. Eight types of configurations and different densities of ATSB stations were tested. Anopheles gambiae population size, human biting rate (HBR) and entomological inoculation rates (EIR) were compared between different ATSB configurations and densities. Each simulated scenario was run 50 times.ResultsCompared to the outcomes not altered by ATSB treatment in the control scenario, in resource-rich and resource-poor environments, respectively, the optimum ATSB treatment reduced female abundance by 98.22 and 91.80 %, reduced HBR by 99.52 and 98.15 %, and reduced EIR by 99.99 and 100 %. In resource-rich environments, n × n grid design, stations at sugar sources, resting sites, larval habitats, and random locations worked better in reducing vector population and HBRs than other configurations (P < 0.0001). However, there was no significant difference of EIR reductions between all ATSB configurations (P > 0.05). In resource-poor environments, there was no significant difference of female abundances, HBRs and EIRs between all ATSB configurations (P > 0.05). The optimum number of ATSB stations was about 25 for resource-rich environments and nine for resource-poor environments.ConclusionsATSB treatment reduced An. gambiae population substantially and reduced EIR to near zero regardless of environmental resource availability. In resource-rich environments, dispersive configurations worked better in reducing vector population, and stations at or around houses worked better in preventing biting and parasite transmission. In resource-poor environments, all configurations worked similarly. Optimum numbers of bait stations should be adjusted according to seasonality when resource availability changes.

BackgroundAgent-based modelling (ABM) has been used to simulate mosquito life cycles and to evaluate vector control applications. However, most models lack sugar-feeding and resting behaviours or are based on mathematical equations lacking individual level randomness and spatial components of mosquito life. Here, a spatial individual-based model (IBM) incorporating sugar-feeding and resting behaviours of the malaria vector Anopheles gambiae was developed to estimate the impact of environmental sugar sources and resting sites on survival and biting behaviour.MethodsA spatial IBM containing An. gambiae mosquitoes and humans, as well as the village environment of houses, sugar sources, resting sites and larval habitat sites was developed. Anopheles gambiae behaviour rules were attributed at each step of the IBM: resting, host seeking, sugar feeding and breeding. Each step represented one second of time, and each simulation was set to run for 60 days and repeated 50 times. Scenarios of different densities and spatial distributions of sugar sources and outdoor resting sites were simulated and compared.ResultsWhen the number of natural sugar sources was increased from 0 to 100 while the number of resting sites was held constant, mean daily survival rate increased from 2.5% to 85.1% for males and from 2.5% to 94.5% for females, mean human biting rate increased from 0 to 0.94 bites per human per day, and mean daily abundance increased from 1 to 477 for males and from 1 to 1,428 for females. When the number of outdoor resting sites was increased from 0 to 50 while the number of sugar sources was held constant, mean daily survival rate increased from 77.3% to 84.3% for males and from 86.7% to 93.9% for females, mean human biting rate increased from 0 to 0.52 bites per human per day, and mean daily abundance increased from 62 to 349 for males and from 257 to 1120 for females. All increases were significant (P < 0.01). Survival was greater when sugar sources were randomly distributed in the whole village compared to clustering around outdoor resting sites or houses.ConclusionsIncreases in densities of sugar sources or outdoor resting sites significantly increase the survival and human biting rates of An. gambiae mosquitoes. Survival of An. gambiae is more supported by random distribution of sugar sources than clustering of sugar sources around resting sites or houses. Density and spatial distribution of natural sugar sources and outdoor resting sites modulate vector populations and human biting rates, and thus malaria parasite transmission.

BackgroundThe use of genetically modified mosquitoes (GMMs) for the control of malaria and other mosquito-borne diseases has been proposed in malaria-endemic countries, such as Nigeria, which has the largest burden in Africa. Scientists are major stakeholders whose opinions and perceptions can adversely affect the success of the trials of GMMs if they are not involved early. Unfortunately, information on the awareness of Nigerians scientists and their overall perception of the GMMs is practically non-existent in the literature. Therefore, this study aimed at understanding how receptive Nigerian scientists are to a potential release of GMMs for the control of malaria.MethodsThe sample consisted of 164 scientists selected from academic and research institutions in Nigeria. Data were collected from participants using a semi-structured, self-administered questionnaire. Questions were asked about the cause and prevention of malaria, genetic modification and biotechnology. Specific questions on perception and acceptable conditions for the potential release of GM mosquitoes in Nigeria were also covered.ResultsAll participants cited mosquitoes as one of several causes of malaria and used various methods for household control of mosquitoes. The main concerns expressed by the scientists were that GMMs can spread in an uncontrolled way beyond their release sites (89%) and will mate with other mosquito species to produce hybrids with unknown consequences (94.5%). Most participants (92.7%) agreed that it was important that before approving the release of GMMs in Nigeria, there had to be evidence of contingency measures available to remove the GMMs should a hazard become evident during the course of the release. In general, a majority (83.5%) of scientists who participated in this study were sceptical about a potential release in Nigeria, while 16.5% of the participants were in support.ConclusionsAlthough a majority of the participants are sceptical about GMMs generally, most encourage the use of genetic modification techniques to make mosquitoes incapable of spreading diseases provided that there are contingency measures to remove GMMs if a hazard becomes evident during the course of the release.

Insects act as vectors for diseases of plants, animals, and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.

Gene drive systems provide novel opportunities for insect population suppression by driving genes that confer a fitness cost into pest or disease vector populations; however regulatory issues arise when genes are capable of spreading across international borders. Gene drive systems displaying threshold properties provide a solution since they can be confined to local populations and eliminated through dilution with wild-types. We propose a novel, threshold-dependent gene drive system, Medusa, capable of inducing a local and reversible population crash. Medusa consists of four components--two on the X chromosome, and two on the Y chromosome. A maternally-expressed, X-linked toxin and a zygotically-expressed, Y-linked antidote results in suppression of the female population and selection for the presence of the transgene-bearing Y because only male offspring of Medusa-bearing females are protected from the effects of the toxin. At the same time, the combination of a zygotically-expressed, Y-linked toxin and a zygotically-expressed, X-linked antidote selects for the transgene-bearing X in the presence of the transgene-bearing Y. Together these chromosomes create a balanced lethal system that spreads while selecting against females when present above a certain threshold frequency. Simple population dynamic models show that an all-male release of Medusa males, carried out over six generations, is expected to induce a population crash within 12 generations for modest release sizes on the order of the wild population size. Re-invasion of non-transgenic insects into a suppressed population can result in a population rebound; however this can be prevented through regular releases of modest numbers of Medusa males. Finally, we outline how Medusa could be engineered with currently available molecular tools.

BackgroundCurrent vector control strategies focus largely on indoor measures, such as long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS); however mosquitoes frequently feed on sugar sources outdoors, inviting the possibility of novel control strategies. Attractive toxic sugar baits (ATSB), either sprayed on vegetation or provided in outdoor bait stations, have been shown to significantly reduce mosquito densities in these settings.MethodsSimple models of mosquito sugar-feeding behaviour were fitted to data from an ATSB field trial in Mali and used to estimate sugar-feeding rates and the potential of ATSB to control mosquito populations. The model and fitted parameters were then incorporated into a larger integrated vector management (IVM) model to assess the potential contribution of ATSB to future IVM programmes.ResultsIn the Mali experimental setting, the model suggests that about half of female mosquitoes fed on ATSB solution per day, dying within several hours of ingesting the toxin. Using a model incorporating the number of gonotrophic cycles completed by female mosquitoes, a higher sugar-feeding rate was estimated for younger mosquitoes than for older mosquitoes. Extending this model to incorporate other vector control interventions suggests that an IVM programme based on both ATSB and LLINs may substantially reduce mosquito density and survival rates in this setting, thereby substantially reducing parasite transmission. This is predicted to exceed the impact of LLINs in combination with IRS provided ATSB feeding rates are 50% or more of Mali experimental levels. In addition, ATSB is predicted to be particularly effective against Anopheles arabiensis, which is relatively exophilic and therefore less affected by IRS and LLINs.ConclusionsThese results suggest that high coverage with a combination of LLINs and ATSB could result in substantial reductions in malaria transmission in this setting. Further field studies of ATSB in other settings are needed to assess the potential of ATSB as a component in future IVM malaria control strategies.