Journal of Medical Entomology

Mosquitoes such as Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are vectors of many pathogens that greatly affect humankind. The maintenance of these mosquitoes in laboratory permit different studies that can help understanding their biology, as well as the vector-pathogen interaction. In addition to sugar meals, the blood feeding is essential for maintenance of the reproductive cycle in several vectors. The main blood sources for many mosquito colonies are direct feeding on live animal or the use of human/animal blood through artificial feeders. However, this latter process has some disadvantages, as artificial feeders can be very laborious for assembly and decontamination. Based on these observations, a simplified technique for feeding and artificial infection was developed with cotton-pads soaked (CS) and blood or blood and viral supernatant to simulate an artificial infection. The efficiency of the CS technique was investigated through the number of mosquitoes fed/infected, when compared to their respective control group. The CS technique, with blood at room temperature, promoted a feeding rate of 61.4% for Ae. albopictus, 70.8% for Cx. quinquefasciatus and 17% for Ae. aegypti. The control group (Hemotec-feeding) presented 47.9%, 16.5% and 59.1% of feeding success, respectively. To improve the CS technique for Ae. aegypti mosquitoes, the procedure was then performed with blood at 38{degrees}C, which was possible to observe a feeding rate of 47.3%, in comparison to 53.2% for the control group (Hemotec). When using the CS technique for artificial infection with Mayaro virus, more than 80% of infection was observed for Ae. aegypti and 100% for Ae. albopictus. In the traditional infection technique (glass feeder), the infection rate was 90% (Ae. aegypti) and 96.6% (Ae. albopictus). For Cx. quinquefasciatus, the infection was positive only with the CS technique, resulting in 1 (5%) mosquito infected with Mayaro virus. Our results suggest that this simplified technique of low-cost feeding and easy assembly, offers good results for feeding (maintenance of colonies) and artificial infection of different species of mosquitoes.

Dengue, Zika, and chikungunya, all transmitted by Aedes aegypti mosquitoes, present a significant and growing global health challenge. Contact-based insecticide such as pyrethroids, organophosphates, and organochlorines, are a core component of strategies to control Ae. aegypti. However, reliance on these chemical insecticides alone presents long-term challenges, particularly around increasing insecticide resistance. There are relatively few novel tools available that target adult mosquitoes through alternative mechanisms. Innovative, complementary approaches that can enhance the effectiveness and sustainability of existing mosquito control programs are urgently needed. Newly explored vector management tools are attractive toxic sugar baits (ATSBs), targeting the sugar-feeding behavior of both male and female mosquitoes, and allow for an ingested insecticide. Dried Attractive Bait Stations (DABS), a novel variant of ATSBs, have shown promising results for indoor Ae. aegypti control. We conducted cage and experimental house trials of DABS on laboratory-reared Ae. aegypti mosquitoes in Ponce, Puerto Rico. In cage trials, both male and female Ae. Aegypti that were exposed to DABS for 48 hours experienced near complete mortality, while control groups showed universal survival. All tested concentrations (1%, 2%, and 4% boric acid compounds) significantly reduced survival compared to controls (p < 0.001), with no significant differences between concentrations (p = 0.50). Water availability reduced DABS efficacy, lowering mortality by 47% in females (p < 0.001) and 32% in males (p < 0.001). In experimental house trials, DABS reduced mosquito survival by [~]75% (p < 0.001), with deployment of four DABS devices per room suggesting the greatest reduction in mosquito survival (p = 0.06-0.10). Optimal performance was observed at placing DABS 0.75 m above the floor (p < 0.001) and results suggest that medium-sized DABS may be more effective than larger (p = 0.09). Natural light increased mosquito survival (p = 0.028), but its interaction with DABS treatment was not significant (p = 0.322) and room-to-room variability was minimal. A notable limitation was the interference of ants during household trials, which led to the removal of mosquito carcasses and potential data loss, highlighting the need to further investigate alternative sugar sources and DABS interaction with non-target organisms. Despite these challenges, the results support DABS as a promising, scalable Aedes aegypti control tool. Future research should confirm effectiveness under varied field conditions, and assess the community acceptability for broader deployment.

We report the collection of three Aedes aegypti adult female mosquitoes in a Rockville, Maryland backyard in late July, 2023, followed by the emergence of 15 adults collected as larvae in a residential backyard in Baltimore, Maryland in mid-September, 2023. Aedes aegypti is a species primarily associated with tropical and subtropical regions, known for its significance as a vector of arboviruses, including Yellow Fever, Dengue, Zika, and Chikungunya among others. In the continental United States, Ae. aegypti populations are mostly found in the southeast, and in several isolated locations such as southern California and Arizona. This finding raises questions about the potential establishment and survival of Ae. aegypti in temperate climates, emphasizing the critical importance of robust vector surveillance programs in preventing potential outbreaks of vector-borne diseases in regions not traditionally considered endemic for this species.

Aedes aegypti mosquitoes are the primary vectors for dengue, yellow fever, chikungunya and zika virus transmission, posing significant public health risks. In Kenya, these viruses drive disease outbreaks especially, dengue and chikungunya with coastal Kenya being the most affected. Low-level circulation of the Zika virus has been reported in parts of the Kenyan coast, with confirmed cases reported in Lamu County between August to September 2024. Except for yellow fever, there are no approved vaccines or therapeutics, hence vector control remains the most effective means of protection. However, prolonged exposure to insecticides can lead to resistance, threatening these interventions. Therefore, monitoring resistance in mosquito populations is critical to allow for appropriate interventions using effective chemical classes to prevent disease outbreaks. This study aimed to establish the levels of resistance to pyrethroids, and associated markers, among Ae. aegypti populations in sections of Lamu County where there had been a recent localized outbreak of Zika. Mosquito eggs were collected from Mkomani, Kashmir, and Kandahar villages in Lamu County, reared and tested for susceptibility to three pyrethroid insecticides (0.75% permethrin, 0.05% Alpha-cypermethrin and 0.05% deltamethrin) using WHO tube assays. Genotyping of knockdown resistance (kdr) mutations L982W, S989P, A1007G, V1016G/I, and F1534C was done using Sanger sequencing. Association between resistant phenotypes and genotypes were inferred. The results varied between the three pyrethroids with high resistance to permethrin observed (6-15% mortality), for deltamethrin mortality ranged between 53-57%, while for alphacypermethrin 88%-99% mortality was observed. Two mutation types and six genotypes were identified at F1534. No other kdr mutations were detected. The CC genotype was significantly associated with 0.75% permethrin resistance in Ae. aegypti populations (OR = 2.87, 95% CI: 1.34-6.17, P = 0.0036). The current data show that Ae. aegypti from Kandahar, Kashmir and Mkomani villages in Lamu County have developed very high resistance to permethrin, and varying resistance to other pyrethroids, thus threatening pyrethroid-based control strategies in this region, highlighting the need for alternative strategies to control the vector for arboviruses.

Aedes aegypti is the primary vector species for dengue, Zika, and chikungunya viruses, and is adapted to thrive in urban environments. Their proximity to human populations and the adverse health outcomes associated with arboviral infections make mosquito control essential for reducing the risk of outbreaks and disease spread. Source reduction and larvicide applications are central components of mosquito control because they reduce adult mosquito emergence by targeting immature life stages. Therefore, our objective was to identify urban aquatic habitats used by Ae. aegypti and assess temporal variation in their contribution to immature mosquito production. To distinguish habitat use from habitat productivity, we considered larval presence as an indicator of oviposition or early-stage survival and pupal presence as evidence that a habitat supported development through most of the immature life cycle. Between July 2018 and October 2019, 2,482 inspections in Miami-Dade County, Florida, identified 2,756 aquatic habitats from which 19,466 Ae. aegypti larvae and 3,648 pupae were collected. Unique temporal trends were observed for both larval and pupal presence and abundance at county and habitat resolutions. County-level trends showed the highest larval and pupal production during Summer and the lowest pupal production during Winter. However, temporal patterns differed across habitats, supporting a context-dependent interpretation of habitat conduciveness, in which the same habitat type may support different levels of immature mosquito production depending on local environmental conditions. Our results show that habitats with frequent larval occurrence did not consistently have high pupal presence, indicating that larval presence does not necessarily reflect habitat productivity. Control strategies that prioritize habitats repeatedly associated with pupal production by season may improve year-round mosquito control, outbreak preparedness, and resource allocation.

Understanding the susceptibility status of mosquitoes to insecticides is critical for effective decision making regarding the use or rotation of insecticides in control programs. In this study, we demonstrated the use of amino acid profiling for the detection of deltamethrin-resistant Aedes albopictus (L.). Mosquitoes collected in the field were first tested with WHO adulticide bioassay kits, and the amino acid profiles of the resistant mosquitoes were compared with the susceptible strain of Ae. albopictus. Samples were lyophilized and derived by silylation and then analyzed by gas chromatography-mass spectrometry (GC-MS). Using standardized, known concentrations of amino acids, we quantified the amino acids in both resistant and susceptible strains. An independent t-test was performed to compare the concentrations of each amino acid between strains. Logistic regression was then performed to assess the relationship between amino acid concentrations and susceptibility status of the mosquitoes. Our results showed that the amino acids in resistant mosquitoes differed significantly from those in susceptible mosquitoes, with the exception of serine. Further regression analysis showed that seven amino acids significantly predicted susceptibility, suggesting that they are suitable as biological indicators for rapid assessment of resistance status in field mosquitoes. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/604257v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@7c3c91org.highwire.dtl.DTLVardef@6c7ad1org.highwire.dtl.DTLVardef@1f36900org.highwire.dtl.DTLVardef@172b13d_HPS_FORMAT_FIGEXP M_FIG C_FIG Identification of deltamethrin-resistant mosquitoes based on differences in the amino acid profile: Deltamethrin-susceptible and -resistant mosquito strains were lyophilised and converted into esters by silylation, which were then analysed using a gas chromatography-mass spectrometer (GC-MS). The difference between susceptible and resistant could be classified by developing a classification model with logistic regression.

BackgroundUrbanization and climate change exacerbate mosquito-borne disease transmission by expanding mosquito habitats in densely populated areas. Traditional mosquito control methods, such as eliminating breeding sites or using insecticides, face challenges in urban settings due to practicality and resistance issues. This study introduces Human-Controlled Breeding Sites (HCBS), a novel, low-cost, and environmentally friendly mosquito control method tailored for urban and suburban households. HCBS provides controlled oviposition sites to attract gravid female mosquitoes, disrupting their reproductive cycle without chemical deterrents. MethodsThe HCBS method involves deploying devices designed to mimic natural breeding sites, encouraging mosquitoes to lay eggs in a controlled environment. After 10 days, eggs, larvae, and pupae are collected and destroyed. The HCBS device, fabricated using 3D-printed ABS material in five colors (white, black, red, green, blue), was tested in a semi-controlled laboratory in La Molina, Peru. The experiment evaluated oviposition preferences of Aedes aegypti by counting larvae and pupae across five 10-day cycles, with device colors randomized to minimize bias. ResultsHCBS devices successfully attracted gravid Aedes aegypti for oviposition, with green devices showing the highest initial preference (2-17 larvae/pupae per cycle), though larvae were later observed in all colors, indicating color is not essential. The method achieved 100% effectiveness in disrupting the mosquito life cycle by eliminating eggs, larvae, and pupae within the 10-day timeframe. The low-cost design and use of household materials enhance its scalability and community engagement potential. ConclusionsHCBS offers a sustainable alternative to conventional mosquito control by targeting early life stages, reducing populations with minimal environmental impact. Its adaptability to household settings and cost-effectiveness make it suitable for urban and suburban areas. Further research is needed to optimize device design, assess long-term efficacy, and integrate HCBS into disaster risk management systems for broader public health applications, particularly in regions like Peru with documented insecticide resistance.

Vector-borne diseases remain a significant global health concern, with the invasive mosquito Aedes albopictus playing a key role in the transmission of arboviruses including dengue, chikungunya, and Zika viruses. As this species expands in novel territories, effective vector control strategies are increasingly critical. Densoviruses (DVs) have emerged as potential biological control agents, either through direct pathogenic effects on mosquito populations or via paratransgenesis. However, the influence of combined environmental factors, such as temperature and densovirus infection on mosquito life-history traits remains largely unexplored. In this study, we investigated the effects of different temperatures (28{degrees}C, 31{degrees}C, and 34{degrees}C) and exposure to the densovirus AalDV2 on the survival of Ae. albopictus and on several mosquito life-history traits including its development time, size and symmetry at adult stage. Larvae were individually reared under controlled conditions and exposed to AalDV2 or a control treatment. Temperature had a strong nonlinear effect on survival, with dramatic mortality increases at 34{degrees}C. Unexpectedly, AalDV2-infected larvae showed significantly higher survival than controls at this extreme temperature, suggesting a protective effect under thermal stress. Across all temperatures, viral infection delayed pupation in a sex-dependent manner, with females experiencing greater delays and reduced adult wing size. Quantitative PCR revealed high infection rates (>96%) across all conditions with a viral load increasing with higher temperature in a sex-dependent manner. Our findings reveal a paradoxical outcome: while AalDV2 imposes fitness costs through delayed development and reduced body size, it confers an unexpected survival advantage at the extreme temperature. This represents the first documentation of temperature-dependent protective effects by an entomopathogenic virus in mosquitoes, challenging conventional assumptions about pathogen impacts. These results have critical implications for vector biocontrol strategies in a warming climate, as densovirus deployment could inadvertently enhance mosquito resilience in heat-stressed regions. Further research is needed to elucidate the underlying mechanisms and assess the impact on mosquito population dynamics.

Locally acquired arboviral infections are an increasing public health concern in the United States. Aedes aegypti and Culex quinquefasciatus, vectors of dengue virus and West Nile virus, respectively, are established in regions where local transmission has been reported. Seasonal variation in mosquito abundance affects vector density, human-vector contact, and arbovirus transmission risk. The aim of this study is to investigate seasonal variations in Ae. aegypti and Cx. quinquefasciatus population dynamics in Miami-Dade County, Florida; Maricopa County, Arizona; and Clark County, Nevada. Monthly relative abundance, average mosquitoes collected per trap-night, normalized abundance ratios, and seasonal-trend decomposition were used to evaluate species- and location-specific seasonal patterns. Mosquito population dynamics differed by species and location. In Miami-Dade County, the two species showed seasonal turnover, with Cx. quinquefasciatus peaking during winter and spring and Ae. aegypti peaking during summer. In Maricopa and Clark counties, both species peaked primarily between August and September. Species dominance also differed by site, with Ae. aegypti more abundant than Cx. quinquefasciatus in Maricopa County and Cx. quinquefasciatus more abundant than Ae. aegypti in Clark and Miami-Dade counties. Seasonal decomposition showed that Ae. aegypti peaked earlier in Miami-Dade County than in Maricopa and Clark counties, whereas Cx. quinquefasciatus showed a spring peak in Miami-Dade County and bimodal seasonal patterns in Maricopa and Clark counties. These results suggest that mosquito population dynamics are species- and location-specific and support the use of local surveillance data to guide the timing of mosquito control and arbovirus preparedness.

Aedes transmitted arboviral human cases are increasing worldwide and spreading to new areas of the United States of America (USA). These diseases continue to re-emerge likely due to changes in vector ecology, urbanization, human migration, and larger range of climatic suitability. Recent shifts in landscape and weather variables are predicted to impact the habitat patterns of urban mosquitoes such as Aedes aegypti and Aedes albopictus. Miami (FL) is in the tropical zone and an established hotspot for arboviruses, while Charleston (SC) is in the humid subtropical zone and newly vulnerable. Although these coastal cities have distinct climates, both have hot summers. To understand mosquito infestation in both cities and potentiate our surveillance effort, we performed egg collections in the warmest season. We applied remote sensing with land-use cover and weather variation to identify mosquito infestation patterns. Our study found predominant occurrence of Ae. aegypti and, to a lesser extent, Ae. albopictus in both cities. We detected statistically significant positive and negative associations between entomological indicators and most weather variables in combined data from both cities. For all entomological indices, weekly wind speed and relative humidity were significantly positively associated, while precipitation and maximum temperature were significantly negatively associated. Aedes egg abundance was significantly positively associated with open land in Charleston but was negatively associated with vegetation cover in combined data. There is a clear need for further observational studies to determine the impact of climate change on Ae. aegypti and Ae. albopictus infestation in the Southeastern region of the USA.

Pyrethroid resistance in Aedes aegypti has become widespread after almost two decades of frequent applications to reduce the transmission of mosquito-borne diseases. Because few insecticide classes are available for public health use, insecticide resistance management (IRM) is proposed as a strategy to retain their use. A key hypothesis of IRM assumes that negative fitness is associated with resistance, and when insecticides are removed from use, susceptibility is restored. In Tapachula, Mexico, pyrethroids (PYRs) were used exclusively by dengue control programs for 15 years, thereby contributing to selection for high PYR resistance in mosquitoes and failure in dengue control. In 2013, PYRs were replaced by organophosphates--insecticides from a class with a different mode of action. To test the hypothesis that PYR resistance is reversed in the absence of PYRs, we monitored Ae. aegyptis PYR resistance from 2016 to 2021 in Tapachula. We observed significant declining rates in the lethal concentration 50 (LC50), for permethrin and deltamethrin. For each month following the discontinuation of PYR use by vector control programs, we observed increases in the odds of mosquitoes dying by 1.5% and 8.4% for permethrin and deltamethrin, respectively. Also, knockdown-resistance mutations (kdr) in the voltage-gated sodium channel explained the variation in the permethrin LC50s, whereas variation in the deltamethrin LC50s was only explained by time. This trend was rapidly offset by application of a mixture of neonicotinoid and PYRs by vector control programs. Our results suggest that IRM strategies can be used to reverse PYR resistance in Ae. aegypti; however, long-term commitment by operational and community programs will be required for success. Author summaryThe mosquito Aedes aegypti is the principal urban vector of the viruses that cause three globally significant diseases: dengue fever, chikungunya, and Zika fever, for which vaccines and effective treatments are currently absent. The only way to control dengue, chikungunya, and Zika fever outbreaks is to diminish vector populations. During epidemics, the most frequent way of targeting adult mosquitoes is outdoor spatial spraying of insecticides. Control of Ae. aegypti is difficult because limited insecticide classes are available for public health, leading to operational practices that overuse single molecules for long periods of time, imposing great selection pressure on mosquito populations for insecticide resistance. Insecticide resistance management (IRM) is proposed as a strategy to prevent resistance and avoid depleting the susceptibility resource in mosquito populations. IRM strategies assume that alternation of insecticides with different toxicological modes of action will prevent the selection of resistance. Unfortunately, very few field evaluations have reported IRM schemes to control Ae. aegypti. In our study, the exclusive use of pyrethroids in vector control programs in Mexico from 1999 to 2013, led in the selection of knockdown resistance (kdr) to pyrethroid insecticides. To address this issue, vector control programs temporarily phased out pyrethroids from 2013 to 2019, substituting them with an insecticide class with a different mode of action: organophosphates (OPs). During six years, we monitored pyrethroid resistance in 24 mosquito populations from Tapachula, Mexico. We show that discontinuing pyrethroids for six years led in pyrethroid resistance reversal in Ae. aegypti in the field. However, high levels of pyrethroid resistance continue to jeopardize operational application, necessitating longer periods of pyrethroid cessation and novel IRM strategies to achieve lower resistance thresholds.

We report the first records of Aedes euedes Howard, Dyar, and Knab, and Coquillettidia perturbans (Walker) from Canadas Yukon Territory, and the first record of Ae. decticus Howard, Dyar, and Knab from British Columbia. We also report range extensions in northern BC for the western treehole mosquito, Aedes sierrensis (Ludlow), the common house mosquito, Culex pipiens L., and the cool weather mosquito Culiseta incidens (Thomson).

BackgroundResistance to insecticides is spreading among populations of Aedes aegypti, the primary vector of important human arboviruses. The escalating insecticide resistance poses a significant threat to dengue vector control, with an expanding number of countries affected by the disease. To gain a deeper insight into the evolution of insecticide resistance, it is essential to have longitudinal surveillance results, which are currently lacking, particularly from African Ae. aegypti populations. Here we report on three-years of surveillance of Ae. aegypti susceptibility to insecticide resistance phenotypes and associated kdr mutations in Burkina Faso, a country with regular dengue outbreaks. MethodsAe. aegypti susceptibility to insecticides and the V410L, V1016I, and F1534C kdr target site mutations linked to pyrethroid insecticide resistance were monitored in Ouagadougou from 2016 to 2018. Larvae were collected from artificial containers at two sites and reared to adulthood in an insectary. Bioassays were conducted on female adults, alongside with a laboratory-susceptible strain, following standard WHO protocols. Allele-specific PCR genotyping assays were utilized to identify the V410L, V1016I, and F1534C kdr pyrethroid target site mutations. ResultsBioassays revealed a high level of resistance to permethrin and deltamethrin that progressively increased over the three-year period in both localities. The 1534C mutation was nearly fixed throughout the three years at each locality, and while the closely related 410L and 1016I mutations did not vary between localities, their frequency notably increased from 2016 to 2018. Interestingly, Ae. aegypti populations in both areas remained susceptible to bendiocarb, fenitrothion, and malathion. Modeling the mortality data further confirmed the escalating resistance trend over the years and emphasized the significant role played by the three kdr mutations in conferring resistance to pyrethroids. ConclusionMortality rates indicate that Ae. aegypti populations from Ouagadougou are becoming increasingly resistant to pyrethroid insecticides, likely due to an increase in the frequencies of the 410L and 1016I kdr mutations. Organophosphate insecticides are likely to be better alternative options for control.

Precision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. The deployment of pgSIT shows the greatest promise and does not have a near peer competitor in the fight to eradicate malaria. Here we explore the cost effectiveness of using this approach in Africa using mathematical modeling and economical analysis. Overall, we find that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits. SummaryPrecision-guided sterile insect technique (pgSIT) is an extremely promising vector control intervention that can reduce and potentially eliminate the unacceptable malaria burden, particularly in sub-Saharan Africa. pgSIT is a safe, innovative, and highly targeted approach to mosquito control that combines the principles of the sterile insect technique (SIT) with advanced state-of-the-art technologies of genetic engineering (Akbari et al. 2023; M. Li et al. 2021; Kandul, Liu, and Akbari 2021; Kandul et al. 2019, 2022). The technique involves inundative releases of genetically sterile male mosquitoes into the environment to mate with their wild counterparts, sterilizing them in the process. After multiple releases, this method can suppress, and even eradicate pest populations without the use of chemical pesticides or other less specific agents. The use of pgSIT has the potential to be a lasting, safe, cost-effective, sustainable, and environmentally friendly method to suppress a target species, acting as a chemical-free, species-specific insecticide. Before this work is considered for field application, however, we need to have a robust, data-driven modeling framework that will accurately predict the outcome of pgSIT release scenarios for malaria control, and we need an assessment of the costs and health benefits of implementing this technology in a region in Africa. This cost assessment evaluates the economic feasibility of both capacity building and establishing the infrastructure for a pgSIT facility in The Gambia to control the deadly malaria mosquito vector Anopheles gambiae. We focused on the Upper River region (URR) for three key reasons: (1) this area is known to have the highest per capita malaria rates in The Gambia (2) this region has more comprehensive historical and current data on malaria incidence and prevalence, malaria-associated healthcare and prevention costs, and human demographic data and (3) its location could feasibly demonstrate that a single pgSIT production facility can support phased suppression on a regional scale and then upon local extinction be repurposed to support vector suppression on a country or continent-wide scale. The pgSIT treatment of the URR ([~]2069 km2) is predicted to prevent approximately 230 deaths and about 48,000 sick days per year. This estimate is based on a model for localized extinction of A. gambiae, reaching full epidemiological impact by the third year. There are multiple ways to calculate the value of this intervention monetarily, from the value of statistical life (VSL) to quality adjusted life years (QALY), which ranges from 367 million to 880 million USD saved in the first ten years of the facility being active. Other metrics such as willingness-to-pay (WTP), estimates the willingness of locals to contribute to malaria prevention financially, and estimates based on gross domestic product (GDP) growth predict this model to save either 53 million or 551 million USD, respectively, in the first ten years of intervention. This model assumes localized extinction of A. gambiae by the second year of intervention with repeated releases to maintain extinction despite seasonal mosquito migration from beyond the treatment area. Localized extinction, however, is expected to have a year-to-year suppression effect making it easier to suppress mosquito populations with reduced sterile male releases in subsequent years. It is, therefore, likely an underestimate of the costs and benefits of pgSIT sterile male releases. In later years, the release of sterile males from this facility could be redirected to new areas to expand the suppression region. Additionally, this facility would have a significant off-season where the facility is not producing sterile male A. gambiae. This off-season could, therefore, produce sterile males to suppress A. gambiae populations in other regions with seasonal malaria during the off-season in The Gambiae. It could even be used to mass produce pgSIT sterile males for other mosquito species, such as the dengue vector, Aedes aegypti, which have eggs that can be stored for many months and consequently can be stockpiled to aid in the suppression of dengue outbreaks, which are also common in the area. Initially, however, the off-season can be used to build local capacity for genetically engineered (GE) mosquitos, which would consist of training staff, optimizing procedures, and troubleshooting any issues that arose during the higher production phases. The costs for this approach will vary as there are some unknowns and variability in the expected efficiencies of the facility, equipment, and procedures. In particular, mosquito survival rates and fecundity may vary more widely at scale and selection of the rearing equipment and protocols, and the mosquito sorting technology will dictate production levels and procedures. This variability is factored into the cost, and, therefore, a range of technologies and costs are considered. The expected start-up costs range from 6 to 11.5 million USD, which includes all necessary development, field trials, equipment, facility construction, staffing, and other establishment costs. The major upfront costs vary by facility size and capital equipment, much of which is dependent on mosquito rearing efficiencies. The annual estimated operational costs range from approximately 315,000 USD, depending on equipment selection and the size of the facility. Annually, this intervention costs less than 1 USD per person to suppress mosquitoes in the URR and prevent malaria transmission, which is about 6% of a malaria prevention WTP based on current interventions or 0.3-11% when using the VSL or QALY metrics, with most variation from the VSL calculation method. This intervention costs 15 to 124 USD (2022) to save one life-year and prevents malaria infections at 13 to 113 USD per case prevented, making this method competitive with many current interventions. WTP demonstrates that this facility could be made locally sustainable long term by local funding. Overall, the estimates of the capital and operational costs associated with the production of pgSIT sterile males, and the construction and management of the production facility indicate the cost savings associated with the annual decrease in morbidity and mortality (value of life) resulting from the use of pgSIT are significantly higher than the implementation costs. These estimates suggest that pgSIT represents a cost-effective and promising approach to A. gambiae control in The Gambia, with the potential to deliver significant economic and social benefits.

Carausius morosus, the Indian stick insect, is a slender twig-like insect endemic to India. Though widely introduced through captivity around the world and commonly used in laboratories or kept as a household pet, standardized animal husbandry laboratory protocols are lacking. Here we report detailed laboratory culture conditions for C. morosus. We maintain stocks at 23 {degrees}C, 70% relative humidity, and a 12:12 hour light-dark photoperiod. This culture has been successfully sustained under these conditions for over two years, with standardized protocols in place for dietary and cage setup conditions. We also report methods for egg and hatchling care to support ongoing experiments with C. morosus. These standardized methods improve reproducibility and accessibility, enabling the broader use of C. morosus as a laboratory model system for developmental, behavioral, and physiological studies. SummaryThis paper outlines detailed protocols for maintaining a Carausius morosus laboratory colony, including key procedures for animal husbandry, egg and hatchling care, and an overview of the species lifespan and biological characteristics.

Several endocrine signals are known to mediate mosquito egg development including insulin-like peptide, 20-hydroxyecdysone, and juvenile hormone. The objective of this study was to determine the effects of prostaglandin E2 (PGE2) as an additional mediator of oogenesis in the mosquitoes, Aedes albopictus and Anopheles gambiae. The injection of aspirin (an inhibitor of cyclooxygenase) shortly after blood-feeding significantly inhibited egg development at choriogenesis in a dose-dependent manner in Ae. albopictus. Moreover, oral administration of aspirin to An. albopictus and An. gambiae also inhibited egg production. The aspirin treatment suppressed expression of the genes (Yellow-g and Yellow-g2) associated with exochorion darkening and led to the production of a malformed egg shell in Ae. albopictus. These inhibitory effects of aspirin on egg development were rescued by the addition of PGE2, confirming the specificity of aspirin in inhibiting prostaglandin production. To validate these results, we identified a putative PGE2 receptor (Aa-PGE2R) in Ae. albopictus. Aa-PGE2R expression was highly inducible in adult ovary after blood-feeding. RNA interference of Aa-PGE2R expression resulted in the significant suppression of choriogenesis similar to aspirin treatment, where the addition of PGE2 to Aa-PGE2R-silenced females failed to rescue egg production. Together, these results suggest that PG synthesis and signaling are required for egg development across diverse mosquito taxa. Author SummaryProgstaglandins (PGs) play crucial roles in mediating various physiological processes in insects. Aspirin (ASP) inhibits PG biosynthesis and has been used as an anti-inflammatory drug. ASP injection or feeding to mosquitoes of Aedes albopictus or Anopheles gambiae significantly inhibits egg production at chorion formation. This led to significant reduction in fecundity and egg hatchability. PG signal is interrupted by RNA interference (RNAi) of PGE2 receptor. The RNAi treatment also gave a similar damage to females in egg production as seen in ASP treatment. Thus, PG signal is required for egg production of these mosquitoes. Data Availability StatementAll relevant data are within the manuscript and its Supporting Information file.

West Nile virus (WNV) transmission risk is typically estimated from pooled whole-mosquito infection data, which may overestimate the proportion of mosquitoes capable of transmission. To assess natural viral dissemination in field-collected Culex tarsalis, we tested infection rates in tissues of 1,793 individual mosquitoes collected from Northern Colorado in August 2023. Abdomens were screened for WNV RNA, and corresponding thorax and head tissues from positive mosquitoes were tested. Fifteen mosquitoes had detectable abdominal infections, but WNV RNA was detected in only 53% (8/15) of both the thorax and head tissues, while another 27% (4/15) had WNV RNA detected in either the thorax or the head alone. Logistic regression suggests an inconsistent relationship between abdominal viral ribonucleic acid (RNA) load and virus dissemination, whereas receiver operating characteristic analysis identifies a threshold of [~]59,000 RNA copies in the abdomen predictive of dissemination (AUC 0.80, 95% CI: 0.545, 1). These results suggest whole-body RNA detection may overestimate transmission potential from field-captured mosquitoes, and that incorporating infection data could refine surveillance-based risk indices for WNV.

The susceptibility to six pyrethroid insecticides (permethrin, deltamethrin, alpha-cypermethrin, cypermethrin, lambda-cyhalothrin and bifenthrin), each at the recommended concentration, was evaluated for the two stable fly species Stomoxys calcitrans (Linnaeus) and Stomoxys indicus Picard, through tarsal contact using a World Health Organization (WHO) cone bioassay procedure. The field populations of stable flies were collected from three study sites (Songkhla, Phattalung and Satun provinces) in Thailand. The stable flies were exposed to insecticide-treated filter paper for 30 min and their knockdown counts at 30 min and 60 min and mortality counts at 12 hr and 24 hr were recorded. The S. calcitrans and S. indicus in Songkhla and Phattalung populations were moderately susceptible to pyrethroids for 24-hr mortality. Nonetheless, the Satun population of S. indicus was completely susceptible to permethrin with 100% mortality and the lowest susceptible to deltamethrin and bifenthrin. The results indicate a generally low susceptibility of stable flies to pyrethroids in the southern provinces of Thailand.

Mating is a vital behavior for mosquito reproduction, yet it remains poorly understood under captive conditions. We examined the copulation dynamics of two key malaria vectors, Anopheles funestus, and Anopheles arabiensis, in controlled laboratory settings in Tanzania. We observed how variations in mosquito age and artificial lighting influence mating success for these two mosquito species within cages under controlled conditions. We conducted observations in 24-hour cycles, monitoring copulation events and insemination in females. We used generalized linear mixed models (GLMMs) for statistical analyses to assess how environmental conditions influence mating behavior. We found that Anopheles arabiensis exhibited rapid copulation, with 32.4% of individuals mating by Day 3 post-emergence, while An. funestus showed delayed activity, reaching a similar mating rate by Day 8. The introduction of artificial red light significantly accelerated copulation in An. funestus but did not affect An. arabiensis. Dissection confirmed successful sperm transfer and mating plug delivery in over 92% of copulating pairs for both species. Mating occurred primarily at night, with distinct peaks at 22:00 for An. arabiensis and 23:00 for An. funestus. In conclusion, our findings reveal species-specific differences in reproductive behavior, which could improve the colonization of An. funestus, a species historically challenging to rear in captivity. These insights may also inform the development of new vector control technologies, such as sterile insect techniques and genetic-based approaches, that exploit mosquito mating behaviors.

Encarsia tabacivora Viggiani 1985 (Hymenoptera: Aphelinidae), is an endoparasitoid of whiteflies, including Bemisia tabaci (Gennadius) MEAN 1 (Hemiptera: Aleyrodidae), reported from southern USA, Caribbean, and Brazil. In field surveys, the natural occurrence of E. tabacivora was confirmed in the Canete valley of Peru. Its biology and development was studied under laboratory conditions at 20{degrees}C and 70-75% RH. The results showed that fertilized females lay their eggs inside nymphs of the 3rd and early 4th instar of B. tabaci MEAN 1. The stages of development are egg, three larval instars, prepupa and pupa. Development from egg to adult lasted 19.3 (SE{+/-}0.17) days for females. No males were produced, which indicates that E. tabacivora exhibits thelytokous parthenogenesis. Parasitized host nymphs exhibited multiple oviposition punctures and several L1-instar wasps were observed in individual host nymphs, indicating superparasitism. However, from the L2-instar onwards only one individual developed to adult stage. E. tabacivora might be a prospective biological control agent for B. tabaci in sweet potato also for minimizing the spread of whitefly-induced plant viruses in Peru and other regions of the world. The knowledge gained might be helpful in establishing mass rearing protocols of E. tabacivora for inoculative releases.