Medical and Veterinary Entomology

Diapause is a critical adaptive strategy that enables temperate mosquito species to survive adverse environmental conditions and maintain population persistence across seasons. In Culex pipiens, diapause plays a key role in overwintering and influences the seasonal dynamics of arbovirus transmission. However, diapause expression is often assessed using single traits, limiting our understanding of its integrated physiological basis and variation among populations. In this study, we investigated the behavioural, morphological, and reproductive signatures of diapause across three laboratory strains of Culex pipiens (Mogden, Pirbright, and Pirbright Hybrid) reared under diapause-inducing (10 {degrees}C), cold (14 {degrees}C), and control (26-27 {degrees}C) conditions. We quantified blood-feeding behaviour, wing size as a proxy for somatic growth, and spermatheca size as an indicator of reproductive development. Diapause-inducing conditions resulted in a coordinated phenotype characterised by strong suppression of blood-feeding, increased somatic size, and marked inhibition of reproductive development. Mosquitoes reared at 10 {degrees}C exhibited near-complete feeding inhibition and significantly reduced spermatheca size, consistent with reproductive arrest, while those reared at 14 {degrees}C showed intermediate phenotypes. In contrast, control mosquitoes displayed active feeding and fully developed reproductive structures. Wing size increased progressively with decreasing temperature, with the largest individuals observed under diapause-inducing conditions. When analysed together, wing size and spermatheca development exhibited opposing responses across temperature treatments, revealing a strong negative association and indicating a trade-off between somatic growth and reproductive investment. This integrated response supports the interpretation of diapause as a coordinated life-history strategy involving resource reallocation towards survival. Additionally, diapause expression varied among strains, with the Mogden strain showing reduced sensitivity compared with Pirbright and hybrid populations, highlighting the role of genetic background in diapause plasticity. These findings demonstrate that diapause in Culex pipiens is a multi-trait, plastic phenotype with important implications for overwintering success and the seasonal dynamics of arbovirus transmission in temperate regions.

IntroductionChanges in vector control strategies alter the selection pressures exerted on natural populations of Anopheles gambiae s.l. and may influence the dynamics of insecticide resistance mechanisms. However, the consequences of discontinuing indoor residual spraying (IRS) campaigns on the evolution of Ace-1-mediated resistance remain poorly documented under natural conditions. This study aimed to assess the spatiotemporal evolution of resistance to pirimiphos-methyl following the cessation of IRS and to investigate evidence consistent with the existence of a biological cost associated with Ace-1-mediated resistance. MethodsNatural populations of An. gambiae s.l. were collected between 2017 and 2023 in three districts in Burkina Faso that had undergone IRS campaigns (Kampti, Solenzo, and Kongoussi). Susceptibility tests with pirimiphos-methyl (0.25%) were conducted out following WHO protocols, and a subsample of exposed mosquitoes was genotyped to detect the Ace-1 G119S mutation. Spatiotemporal trends in mortality, allele frequencies and genotypes were analyzed according to the pre-IRS, IRS, and post-IRS periods. The association between the Ace-1 genotype and survival following exposure to pirimiphos-methyl was assessed using logistic regression, while the concordance between phenotypic and molecular indicators of resistance was examined using Spearmans correlation. ResultsThe susceptibility of An. gambiae s.l. populations to pirimiphos-methyl was gradually restored after the discontinuation of IRS at all sites. At the same time, the frequencies of the Ace-1 119S resistance allele declined sharply, particularly in Kampti and Solenzo, while they remained low in Kongoussi throughout the study period. Mosquitoes carrying resistant genotypes had a significantly higher probability of survival after exposure to pirimiphos-methyl than susceptible homozygotes, with resistant homozygotes (RR) exhibiting the greatest survival advantage (OR = 27.62; 95% CI: 6.91-110.45; p < 0.001). A significant negative correlation was observed between the frequency of the Ace-1 119S allele and phenotypic mortality ({rho} = -0.48; p = 0.033), indicating a concordance between the two indicators of resistance. The progressive decline in allele frequencies, the decreasing prevalence of resistant genotypes, and the concomitant restoration of susceptibility are field observations consistent with the existence of biological costs associated with Ace-1-mediated resistance. ConclusionThis study provides field evidence consistent with the existence of biological costs associated with Ace-1-mediated resistance in natural populations of An. gambiae s.l. These results underscore the value of an adaptive resistance management strategy based on alternating selection pressures and could guide future vector control strategies, particularly if indoor residual spraying campaigns or other interventions relying on organophosphates were reintroduced.

BackgroundResistance of the arbovirus vector Aedes albopictus to pyrethroid insecticides is an emerging threat to vector control programs in Europe. Three knock-down resistance (Kdr) mutations affecting the voltage-gated sodium channels targeted by pyrethroids are known to confer resistance: V1016G, I1532T and F1534C. As these Kdr mutations are actively circulating in Europe, their monitoring is crucial for resistance surveillance programs. However, current Kdr genotyping methods are labor-intensive and costly, limiting their applicability for large-scale high-throughput surveillance. MethodologyNovel digital droplet PCR (ddPCR) TaqMan assays were developed, allowing the quantification of these Kdr mutations from pooled mosquito samples from field populations. The specificity of these assays was validated by using mosquitoes of known genotypes together with synthetic DNA constructs carrying haplotype combinations previously unseen in the field. The assays accuracy was assessed by comparing Kdr frequencies measured from pooled mosquitoes to those derived from individual genotypes. These assays were then used in a pilot surveillance study in mainland France, integrating deltamethrin bioassays, pooled Kdr mutation tracking and vector control interventions data. FindingsThe developed ddPCR assays demonstrated high accuracy and specificity, with matching Kdr mutation frequencies between pooled samples and individual genotyping. The pilot surveillance study confirmed the low prevalence of Kdr V1016G and I1532T mutations in most French populations, though some populations exhibited a moderate decrease in susceptibility to deltamethrin. Deltamethrin spraying intensity was weakly correlated with Kdr I1532T mutation frequency while no correlation was observed with deltamethrin susceptibility, suggesting that insecticide selective pressure from curative vector control activities is at most a minor driver of resistance. ConclusionThese novel ddPCR assays provide a simple, cost-effective and high-throughput method for quantifying the frequency of Kdr mutations in Ae. albopictus populations. Their implementation in routine country-wide surveillance programs will enhance the detection of emerging resistance, and inform vector control strategies, preventing arboviral disease transmission. Author summaryAedes albopictus, the Asian tiger mosquito, is an emerging global threat due to its ability to transmit the dengue, chikungunya and Zika viruses among others. Recurrent use of insecticides to prevent infections selects resistant mosquitoes. Kdr (knock-down resistance) mutations confer resistance to pyrethroid insecticides, like deltamethrin. In order to track the arrival of these mutations in a population, individual PCR tests are routinely used, which is very inefficient and costly. The digital droplet PCR tests presented in this study can instead be performed on pools of mosquitoes from the field, greatly increasing the output while reducing the costs. This makes them ideal for use in the surveillance of these mutations in field populations. We first confirmed that the tests are as efficient in pools as they are on single mosquitoes, then we applied these tests in a pilot study on French field mosquitoes to demonstrate their applicability for use in surveillance. Finally, we explored the link between deltamethrin sprayings, bioassay mortality and Kdr mutations frequency, which is non-existent. This means the current management of mosquito populations via insecticide use is sensible. We believe these tests have a place in streamlining the future surveillance programs of these mutations in the field.

IntroductionProgress against malaria has stalled since 2015, with insecticide resistance a key driver. Spatial emanators release volatile insecticides into the air, exposing mosquitoes through a route distinct from the tarsal contact used by treated nets, indoor residual spraying, and standard bioassays. Transfluthrin is currently monitored using the WHO bottle bioassay, which combines contact and vapour exposure and cannot isolate airborne effects. A scalable, vapour-only method is needed to characterise susceptibility to volatile pyrethroids. MethodsWe adapted the WHO tube bioassay to deliver non-contact transfluthrin vapour exposure without contacting treated papers. Non-blood-fed female Anopheles were exposed to acetone control or transfluthrin papers at 0.005, 0.1 and 2 mg/paper. Knockdown was recorded over 60 minutes and mortality at 24 hours. Testing spanned three laboratories (LSTM, AIRID and KEMRI) using susceptible reference and resistant lab colonies. Dose response models estimated EC{square}{square} for knockdown (EC{square}{square}KD) and mortality (EC{square}{square}Mort), with resistance ratios (RR) against the local susceptible reference. ResultsThe assay generated clear concentration- and time-dependent responses. At LSTM, Kisumu gave an EC{square}{square}KD of 0.052 and EC{square}{square}Mort of 0.054 mg/paper; Siaya was comparable (RR{square}{square}KD 1.17, RR{square}{square}Mort 0.89), whereas Tiassale 13 and KDR showed reduced susceptibility (RR{square}{square}KD 2.42 and 2.85; RR{square}{square}Mort 5.56 and 6.65). At AIRID, the resistant Cove strain did not reach 50% knockdown or mortality. At KEMRI, Siaya showed reduced susceptibility (RR{square}{square}KD 3.36/RR{square}{square}Mort 5.65). Kisumu at AIRID and KEMRI was comparable to LSTM (EC{square}{square}KD 0.007/EC{square}{square}Mort 0.030; EC{square}{square}KD 0.044/EC{square}{square}Mort 0.023). Despite inter-laboratory variation, susceptible strains remained distinguishable from resistant ones. Lower RRs than for contact pyrethroids suggest contact-based phenotypes may not predict vapour susceptibility. ConclusionThe assay provides a practical benchtop method using standard WHO hardware, distinguishing susceptible from resistant strains across three laboratories. Twenty-four-hour mortality is the recommended endpoint, 60-minute knockdown secondary. Not yet validated, it provides a foundation for further validation and adaptation to other actives.

Insects can experience a loss of fertility under acute heat stress, but impacts vary among species and depend on life stage and sex. We examined the effects of sublethal heat shocks on the fertility of three mosquito species (Aedes aegypti, Aedes notoscriptus and Culex quinquefasciatus) when males or females were exposed at the pupal and adult stages. Reproductive performance was assessed through egg-laying success, fecundity, egg hatchability and viable offspring production. Female heat shock had limited effects on fertility in all three species, though exposure of adult females to 41 {degrees}C for 1 hr reduced viable offspring production in Ae. notoscriptus. Male heat shock reduced egg hatchability in Cx. quinquefasciatus and viable offspring production in Ae. aegypti depending on the life stage exposed. We investigated several other aspects of heat shock exposure on Ae. aegypti fertility. In this species, male fertility partially recovered within 24-48 hours following heat shock exposure, while effects of heat shocks on female fertility occurred regardless of whether heat exposure occurred pre- or post-mating. In addition, maternal heat shock exposure enhanced offspring fecundity and viable offspring production under subsequent sublethal heat stress. Overall, our findings demonstrate that mosquito fertility is largely robust to short-term heat shocks, but there are also complex trait, sex, stage, temporal and species-specific effects, with implications for predicting mosquito population persistence and species persistence under future climate extremes.

Japanese encephalitis virus (JEV) is an emerging mosquito-borne flavivirus with potential for geographic expansion, yet the risk of establishment in North America remains poorly characterized. We assessed vector competence of three North American Culex species (Cx. pipiens, Cx. quinquefasciatus, and Cx. tarsalis) for the JEV Nakayama strain, isolated from human brain in 1934 in Japan, across five constant temperatures (15, 20, 25, 30, and 33{degrees}C) at 4, 7, and 14 days post-feeding, quantifying infection, dissemination, and transmission rates. Vector competence was low but non-zero across all species. Cx. pipiens showed higher infection rates than the other species, whereas Cx. quinquefasciatus and Cx. tarsalis were minimally susceptible under these experimental conditions. Temperature had limited effects on infection and no detectable effects on dissemination or transmission. These findings suggest limited transmission potential of JEV Nakayama in North America, with Cx. pipiens as a relatively permissive vector.

BackgroundThe invasive mosquito Aedes koreicus has established populations in several European countries during the past decade, raising increasing public health concerns due to its potential role as a vector of pathogens. While species identification is primarily based on morphological characters, Ae. koreicus exhibits distinct morphological variants originating from mainland Korea and Jeju Island, which complicates surveillance and may lead to misidentification, particularly in regions where closely related species co-occur. To date, the genetic basis and population-level relevance of these morphological forms in Europe remain poorly understood. MethodsWe investigated the co-occurrence of two morphological forms of Ae. koreicus in Hungary, representing the first confirmed European location where both forms were detected sympatrically and even within the same breeding sites. Adult mosquitoes were morphologically characterised using diagnostic traits, and individuals representing both morphotypes were subjected to comprehensive genetic analyses. We sequenced multiple mitochondrial markers (COX1, COX2, COX3, ATP6, ND1, ND3) and the nuclear ITS2 region using Oxford Nanopore long-read sequencing. Phylogenetic reconstructions and haplotype network analyses were applied to assess genetic differentiation between morphotypes and to compare them with Aedes japonicus as a closely related reference species. ResultsAcross all analysed mitochondrial and nuclear markers, no genetic differentiation was detected between specimens identified as the "mainland" or "Jeju-do" morphological forms of Ae. koreicus. Phylogenetic and haplotype network analyses consistently grouped individuals independently of their morphotype, indicating a shared genetic background at the population level. In contrast, Ae. japonicus formed a clearly distinct genetic lineage, confirming the robustness of the applied markers for interspecific discrimination. ConclusionsOur results demonstrate that the observed morphological variability in Ae. koreicus populations are not underpinned by detectable genetic differentiation using commonly applied mitochondrial and genomic markers. These findings highlight the limitations of using morphology alone to infer population origin or structure and emphasise the need for heightened awareness of intraspecific variability in routine surveillance. Accurate morphological identification remains critical, particularly in citizen science-based monitoring programmes and AI-based automatization platforms, such as Mosquito Alert, to avoid confusion with morphologically similar invasive species. Further studies integrating genomic and ecological approaches are required to elucidate the mechanisms underlying morphological variability in this emerging vector species.

Bugtok disease remains a major bacterial constraint of cooking banana production in the Philippines and is characterized by vascular discoloration, fruit browning, and progressive decline associated with members of the Ralstonia solanacearum species complex that infect banana inflorescences and fruits. This study investigated whether insects visiting Saba banana flowers in Indang, Cavite harbor R. solanacearum, with emphasis on the possible role of Thrips hawaiiensis as a possible candidate vector under field conditions. Destructive sampling was conducted three times in a Saba-monoculturing farm with high reported bugtok incidence, targeting flowers present at the time of collection and prioritizing insects recovered directly from banana inflorescences. Field-collected insects were surface sterilized, subjected to bacterial isolation, and confirmed by PCR using RSSC-specific and phylotype-specific primers; representative thrips were then morphologically identified. Preliminary acquisition assay with infected flowers for 1 and 3 days was conducted using field collected T. hawaiiensis from a non-Bugtok infested farm in Laguna. Among the insects recovered, thrips and stingless bees (Tetragonula spp.) were the most prominent flower visitors, but only thrips yielded internal detection of R. solanacearum after surface sterilization and homogenization, supporting the presence of the pathogen within the insect body rather than simple external contamination. Morphological characters of the positive specimens were consistent with T. hawaiiensis, including a pale antennal segment III, bicolored body, paired pronotal posteroangular setae, discal setae on abdominal sternite VII, and a complete comb on abdominal tergite VIII. In preliminary acquisition tests, healthy T. hawaiiensis exposed to infected flowers acquired the pathogen at mean positivity values of 1.75 {+/-} 1.50 after 1 day and 2.67 {+/-} 4.72 after 3 days, whereas control thrips remained negative. These findings provide field-based evidence that T. hawaiiensis can acquire R. solanacearum from infected Saba flowers and should be considered in Bugtok epidemiology and integrated disease management in Luzon.

Despite extensive efforts to understand the population biology and ecology of the African malaria mosquitoes questions regarding their movement pattern, survival, and population size persist, reflecting methodological limitations. Site fidelity, in which mosquitoes return to feeding sites, resting sites, or oviposition sites remain debated. Mark release recapture (MRR) studies are vital to address such questions. Using locality- and date-specific DNA tags in fluorescent spray, we carried out a continuous MRR in a Malian village from September to December 2019 with three days interval between capture and release across seven zones. A total of 12,937 Anopheles gambiae s.l. (7,455 females) were captured during 35 indoor collections. Handling related mortality was 3.4%., A. coluzzii predominated (89.7%), followed by A. gambiae (9.4%), and A. arabiensis (0.9%). Overall recapture rate was 1.05% (N=129). Contrary to the site-fidelity hypothesis, the distribution of recaptured mosquitoes across zones (regardless of their zone of release) was similar to the distribution of the captured mosquitoes (r=0.97, P<0.001), with 70% recaptured in a different zone. There was no difference in distance moved between sexes, but males average distance increased over time since release, whereas females distance remained unchanged. Simulated movements (across released points), with equal probability to reach any of the village houses predicted actual distance moved by mosquitoes. The regression of observed distance from each zone over predicted had a slope of 1 (r2=94%, P=0.006), suggesting that the layout of the capture area greatly affected the results. The average days post release (minimum age of wild captured mosquitoes) for recaptures was 6.4 d with the longest being 30 d. No seasonal and sex related difference in minimum age were detected. The corrected probability of daily survival (PDS) was 94% and the daily increase in sporozoite rate was 4.9%. Limiting the recapture duration period showed that PDS increased with recapture duration from 74% to 86% (12 to 30 d, uncorrected). Thus, larger recapture area and longer recapture duration are needed to obtain accurate estimates of movement range and of daily survival.

BackgroundAccurate estimation of mosquito age is critical for malaria surveillance, as only older female Anopheles mosquitoes can transmit the parasite. Traditional age-grading methods, include the Polovodova technique in which dilatations on the ovarian pedicel to tally completed gonotrophic cycles (GC) are counted. Despite their widespread use, these techniques remain poorly validated under realistic operational conditions, with limited evidence on their precision and accuracy across different parity rates, GC, and species. In this study, the accuracy and precision of the Polovodova method was evaluated across multiple age-graders and species under both laboratory and field conditions. MethodsA blinded validation study was conducted using laboratory-reared Anopheles arabiensis and Anopheles funestus (N > 3,600) spanning four gonotrophic cycles, and field-collected mosquitoes from Ulanga district, Tanzania (N = 600). Three blinded researchers performed dissections and readings, while a fourth unblinded researcher served as the reference reader (R0) to obtain ground truth (for parity) or assumed truth (for GC) for laboratory mosquitoes. Accuracy and precision for parity and gonotrophic cycle classification were assessed using pairwise inter-rater comparisons, percent agreement, Fleiss kappa and Cohens kappa O_SCPCAP(C_SCPCAPO_SCPLOWKC_SCPLOWO_SCPCAP)C_SCPCAP statistics. For field mosquitoes, which had no age-referenced group, only precision was assessed. FindingsThe Polovodova method accurately classified parity in both species, correctly identifying 98-100% of nulliparous and 94-96% of parous mosquitoes compared with the reference dataset. Inter-rater reliability for parity was almost perfect in An. arabiensis and An. funestus (k = 0.81-0.83), with 82-96% pairwise agreement. GC classification was highly accurate for early stages (GC0-GC2: 85-100%) but declined at later stages GC4(34-46%), reflecting systematic underestimation of mosquito age. Overall GC reliability was moderate (k = 0.56-0.59). Field classifications showed almost perfect agreement (k = 0.87-0.88), supporting operational applicability. ConclusionsThe Polovodova method provides robust estimates of parity and early gonotrophic-cycle status in Anopheles mosquitoes, but late-cycle classifications are more prone to reader disagreement and systematic underestimation. Operational use should therefore rely on trained multiple-reader workflows, including adjudication of discordant or late-cycle specimens, rather than single-reader classification. These findings provide empirical benchmarks for quality-assured Polovodova age-grading in malaria vector surveillance and intervention evaluation.

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.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

The convergent lady beetle Hippodamia convergens is widespread in the Americas and considered an important beneficial insect due to its use in pest control. Early work on this species characterized the beetles as freeze-avoidant (freeze-intolerant), suggesting they survive low winter temperatures by physiologically preventing ice formation to temperatures as low as -15{degrees}C. Here, we show that H. convergens can be freeze-tolerant if ice formation occurs at relatively high temperatures. There was 100% survival following inoculative freezing at -0.5{degrees}C and exposure to -3{degrees}C for 20 hours, as well as freezing that spontaneously occurred in fed beetles exposed to -4{degrees}C for 4 hours. Males exposed to 0{degrees}C or -4{degrees}C for 4 hours had similar mating behaviours (latency to first mating, copulation duration) as control beetles exposed to 4{degrees}C, although sample sizes were too small to determine whether freezing itself had an effect on these behaviours. Several putative cryoprotectants were detected in fat body tissue of H. convergens: glycerol, proline, trehalose, and myo-inositol - in order of abundance. Exposure to -3{degrees}C for 20 hours, whether frozen or unfrozen, did not statistically affect cryoprotectant accumulation, although there was a trend towards increased glycerol following freezing. This study is the first to describe inoculative freeze tolerance in a lady beetle and establishes a baseline for future studies that examine the mechanisms underlying this freeze tolerance and the effects of freezing on reproductive behaviour. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/744689v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c0382org.highwire.dtl.DTLVardef@12a5932org.highwire.dtl.DTLVardef@145f4adorg.highwire.dtl.DTLVardef@1c2cb22_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHippodamia convergens can tolerate freezing at high subzero temperatures C_LIO_LIMating behaviour of males is normal after chilling or freezing C_LIO_LICryoprotectant accumulation may support overwintering survival C_LI

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

Insecticide treated nets (ITNs) remain one the frontline tool in malaria control in Africa. The synergist piperonyl butoxide (PBO) has been incorporated into ITNs to combat pyrethroid resistance and these pyrethroid-PBO ITNs are now in widespread use across Africa. PBO is known to inhibit the degradation of pyrethroids and other insecticide classes, but little is known about other biological or behavioural effects of PBO exposure. This work assessed the impact of PBO on Anopheles gambiae s.l. male longevity as well as female host-seeking and blood feeding behaviours using laboratory assays. PBO had an immediate impact on blood feeding of female An. gambiae s.l. whereas the effect was not observed 24h post-exposure. Kaplan-Meier survival curves showed a significant difference in mosquito longevity post-exposure to PBO with male mosquitoes living nearly three times as long if exposed to control papers compared to PBO. In olfactometer assays, after exposure to control papers, control mosquitoes were significantly more attracted to host-odours, however, this attraction preference was reversed following PBO exposure. This study shows that in experimental assays, PBO has other effects on mosquito physiology and this is likely to contribute to the impact of pyrethroid - PBO ITNs in vector control programmes.

BackgroundA female Aedes aegypti mosquito typically mates only once, receiving all of the reproductive materials required for her lifetime from a single male. During mating, males transfer chemical signals that permanently suppress female receptivity to subsequent mating attempts. Successful mating can be disrupted by satyrization, a form of interspecific reproductive interference in which Aedes albopictus males inseminate Aedes aegypti females without producing viable offspring, effectively rendering them sterile. Aedes aegypti populations from areas where both species co-occur can develop resistance to satyrization, which may arise through premating mechanisms including altered mating interactions and/or postmating mechanisms including reduced female sensitivity to male-derived chemical compounds. Methodology/Principal FindingsWe compared two Aedes aegypti strains derived from field collections in Lee County, Florida: one from a site where Aedes albopictus is established (Fort Myers) and one from a site with no known history of Aedes albopictus co-occurrence (Upper Captiva Island). Using laboratory mating assays, fluorescent dye transfer experiments, and male reproductive homogenate injections, we show that females from the co-occurring population exhibit reduced susceptibility to satyrization. This resistance is associated with two distinct mechanisms: reduced physical interactions with heterospecific males and reduced physiological sensitivity to Aedes albopictus homogenate following injection. Notably, co-occurring females retain sensitivity to signals from conspecific males, indicating that resistance mechanisms are species-selective rather than a generalized reduction in mating responsiveness. ConclusionsAedes aegypti females from a population co-occurring with Aedes albopictus show reduced susceptibility to satyrization under laboratory conditions, mediated by both premating and postmating mechanisms. The species selectivity of these responses suggests that co-occurring populations can discriminate between conspecific and heterospecific signals. Understanding how this discrimination is achieved and how it varies across populations will be important for interpreting interspecific mating dynamics and for evaluating vector control strategies that depend on mating interference, including sterile male release programs.

Mosquito-borne diseases, such as malaria, remain an important public health problem globally, including on the island of Madagascar. Malaria vector control strategies are typically aimed at adult Anopheles mosquitoes in predominantly rural settings. However, very little information is available on larval populations of Anopheles and other vector genera, such as Aedes and Culex, in urban areas, where urbanization creates a multitude of potential larval habitats. Here, the objectives are to identify different types of existing larval habitats and the diversity of vector species in the capital city of Antananarivo, and to study the temporal dynamics and spatial distribution of mosquito vectors of public health interest. From 2022-2023, longitudinal larval surveillance was carried out across all six districts of Antananarivo and consisted of prospecting 2,856 potential larval habitats, of which 653 (23%) were found to contain Aedes, Culex, or Anopheles larvae. Anopheles larvae were reared and morphologically identified using taxonomic keys, and molecular identification by PCR and Sanger sequencing was used for Anopheles sibling species determination. A total of 27,418 larval specimens were collected from eight species: Culex quinquefasciatus (53%), Aedes albopictus (26%), Cx. pipiens (14%), Anopheles gambiae s.l. (3%, all molecularly identified as An. arabiensis), Cx. univittatus (1%), Cx. antennatus (1%), Cx. poicilipes (1%), and An. coustani (1%). Geospatial analysis reveals that 55% of larvae came from artificial larval habitats within the city, which harbor lower species diversity than natural larval habitats. Twenty-one different types of larval habitats were observed, and Cx. quinquefasciatus, a vector of West Nile virus, was present in all of them. Aedes albopictus, vector of arboviral pathogens, prefers artificial habitats such as tires and flower pots, whereas the common rural malaria vector, An. arabiensis preferred natural sites like brick pits and rice fields. An. coustani, often considered a livestock-associated rural vector, was found to thrive in brick pits, wells, ponds, and in aquatic agriculture. Notably, of the 1,270 Anopheles larvae collected, none were found in an artificial container. Culex quinquefasciatus, Cx. pipiens, and Ae. albopictus reached peak abundance in March during the rainy season, while malaria vectors An. arabiensis and An. coustani showed unexpected abundance peaks during the dry periods, in September and April. Two districts (arrondissements 2 and 5) showed higher abundance and diversity of mosquito vectors than the other districts. The data presented here reveal the spatiotemporal dynamics of malaria and arboviral mosquito vectors in urban Madagascar and highlight the heterogeneity of habitats across the urban ecosystem, with important practical implications for the surveillance and control of mosquito-borne diseases.

Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. Kisumu is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae. In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC50 values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

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.