Medical and Veterinary Entomology

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.

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.

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

Personal insect repellents can offer an effective means of preventing mosquito bites. Despite a wide variety of products that claim to repel mosquitoes, relatively few studies have systematically compared their performance through standardized tests. In Australia, chemical-based repellents must be registered with the Australian Pesticides and Veterinary Medicines Authority (APVMA); however, many unregistered products with unverified effectiveness remain commercially available. Here, we tested 55 insect repellents and 5 non-repellent perfumes and deodorants sold in Australia for their protection against Aedes aegypti mosquito landings (as a proxy for bites) using arm-in-cage assays across eight human subjects. We tested 43 topical repellents (containing 18 different primary active ingredients), four wristbands, three clothing stickers, three ultrasonic devices, one garment, one repellent applied to fabric, two perfumes, two deodorants and one antiperspirant. Thirteen repellents, including all wristbands, stickers and ultrasonic devices did not provide complete protection against mosquito landings for any human subject, while a further sixteen provided complete protection for 1 hr or less on average. Repellents containing DEET, oil of lemon eucalyptus, picaridin and IR3535 as the primary active ingredient were the only products that provided more than 2 hours of complete protection, but with significant variation among products with the same active ingredient. Only 7/30 essential oil-based topical repellents and perfumes provided complete protection for greater than 1 hr. Nearly half (25/52) of the repellents subject to registration with the APVMA were unregistered despite being advertised or labelled as mosquito repellents, and these tended to be less effective than registered products. Only 5/28 repellents met or exceeded their claimed protection time. This study highlights the prevalence of unregistered and ineffective mosquito repellents on the Australian market while providing evidence for products and active ingredients that do protect against mosquito bites under laboratory conditions.

Parasitic infections pose multifaceted threats to farmed fish, extending beyond direct pathogenicity to facilitate infections of bacteria, viruses, and microparasites. This synergistic interaction often leads to co-infections that significantly exacerbate disease outbreaks and mortality, presenting a severe challenge to aquaculture sustainability. Recently, a novel trypanosomiasis caused by the Trypanosoma carassii spectrum has emerged in cage-cultured Larimichthys crocea along the southeast coast of China, resulting in widespread prevalence and high mortality rates. Although this pathogen is hypothesized to originate from freshwater fish, its transmission route in marine environments has remained elusive. In this study, we investigated potential vectors and intermediate hosts of T. carassii spectrum, including leeches and monogenean in natural marine settings, and simulated transmission pathways using an established laboratory model involving T. carassii spectrum, Micropterus salmoides and the leech Poecilobdella manillensis. First, our field surveys in the coast of Ningde, Fujian Province, revealed a nearly 100% co-infection rate of T. carassii spectrum and the monogenean Neobenedenia girellae in diseased juvenile L. crocea. PCR analysis detected T. carassii spectrum traces in some N. girellae specimens, and subsequent experiments confirmed that N. girellae ingests the trypanosome while feeding on host blood. Furthermore, bacterial co-pathogens, such as Vibrio harveyi, were also detected within N. girellae. We also document two fatal leech infestations: Zeylanicobdella arugamensis in hybrid groupers (Epinephelus moara [female] X Epinephelus lanceolatus [male]) in Zhangpu, and Limnotrachelobdella okae in E. lanceolatus and E. fuscoguttatus in Raoping. These leeches tested negative for trypanosomes but carried pathogenic bacteria that co-infected the host fish; nonetheless, they are established vectors for trypanosome transmission. In a laboratory cohabitation model simulating T. carassii spectrum transmission, infected M. salmoides were housed with healthy conspecifics under three conditions: Group A (with the leech P. manillensis), Group B (no leeches), and Group C (no leeches, with physical separation between infected and healthy fish). After 14 days, blood smear microscopy and PCR analysis revealed infection rates in healthy fish of 58.33% in Group A, 40.00% in Group B, and 0% in Group C. Conclusively, T. carassii spectrum can be transmitted via leeches (with higher efficiency) and may also spread through direct contact under high-density aquaculture conditions, whereas N. girellae may act as an incidental vector, further research is warranted to clarify transmission dynamics in natural marine ecosystems. Additionally, our findings highlight the role of ectoparasites, including N. girellae and leeches, as potential reservoirs and vectors for bacterial pathogens of fish. In high-density intensive aquaculture, this vectorial capacity transforms parasites from primary pathogens into key drivers of polymicrobial disease outbreaks.

BackgroundMalaria control is increasingly challenged by the urban-adapted vector Anopheles stephensi, yet molecular and cellular tools for this species remain scarce, restricting functional genomic studies and the development of genetic control strategies. To help address this gap, we established a new embryo-derived Anopheles stephensi cell line. ResultsWe generated and characterised a novel embryo-derived Anopheles stephensi (SDA-500) cell line capable of sustained growth in vitro. Species identity was confirmed by mitochondrial COI barcoding, and karyotypic analysis revealed a diploid chromosome complement with the presence of a Y chromosome, confirming that at least some cells are of male origin. Transfection conditions were optimized, with TransIT-PRO showing higher efficiency than Lipofectamine-based reagents. Using a dual-luciferase reporter assay, of several promoters tested the Anopheles gambiae polyubiquitin promoter exhibited the strongest and most consistent transcriptional activity in SDA-500 cells. ConclusionsThe SDA-500 cell line provides a stable and genetically validated in vitro platform that supports efficient transgene expression. This resource provides a useful system for functional genomics and molecular manipulation in Anopheles stephensi and is expected to facilitate studies of mosquito biology and contribute to the development of novel malaria control strategies.

Background Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into distinct genetic groups known as Discrete Typing Units (DTUs), each with distinct genetic traits that influence epidemiology and transmission dynamics. Several triatomine species serve as potential vectors of T. cruzi in the United States. However, despite the growing number of Chagas disease cases in the country, little is known about the genetic diversity and population structure of T. cruzi in natural vector populations. Methodology/Principal Findings We applied a multilocus metabarcoding approach to improve DTU resolution and characterize the genetic diversity and structure of T. cruzi in triatomines collected across five states of the southern United States. Five single-copy nuclear markers and one mitochondrial marker were amplified and processed by high-throughput sequencing to assess genetic diversity. We recovered 35 nuclear and 15 mitochondrial haplotypes from 70 infected specimens. Overall, genetic diversity was low ({pi} < 0.01 at all nuclear loci), with DTUs TcI and the North American lineage of TcIV detected, TcI being the most prevalent. Geographic structuring was particularly evident in TcI strains, which exhibited a distinctive haplotype profile in Florida populations, potentially linked to the recently revalidated vector species Triatoma ambigua. Mitochondrial introgression from TcIV into TcI suggests inter-DTU genetic exchange in these populations. Multiple haplotypes within individual insects detected across single-copy nuclear markers, support multiclonal infection as common feature of T. cruzi in natural vectors. Conclusions/Significance These findings provide new insights into the genetic landscape and evolution of T. cruzi in the United States. Evolutionary connectivity through mitochondrial introgression and frequent multiclonality highlights the importance of deep sequencing approaches for resolving T. cruzi genetic diversity, with direct implications for understanding for transmission dynamics, disease monitoring and control.

The global expansion of Black Soldier Fly (BSF), Hermetia illucens (L.), production systems for organic waste management has increased the need to better understand its reproductive biology in order to optimize mass-rearing programs. In this study, we macroscopically characterized chronological morphological changes in the internal genitalia of adult H. illucens reared on a sustainable alternative larval diet based on coffee pulp and corn bran. Morphological assessments and dissections were conducted on female and male reproductive tracts at 3, 6, and 9 days after adult emergence. Overall, the general organization of both reproductive systems was consistent with previous descriptions. A notable observation was the presence of a tripartite fertilization chamber in females, composed of three distinct compartments apparently associated with the three spermathecae. The functional significance of this anatomical organization remains to be determined. Females showed progressively advanced ovarian development and reached the clearest morphological indicators of reproductive maturity at 9 days, while males showed the greatest testicular distention and opacity at the same age. Compared with maturation times reported in previous studies using conventional larval diets, these observations suggest a later pattern of reproductive maturation under the coffee pulp-based diet. The observed differences may be associated with the nutritional composition and carbohydrate-to-protein balance of the larval diet. These results provide chronological and iconographic information that may contribute to the optimization of laboratory rearing protocols and the use of coffee by-products in H. illucens bioconversion systems.

Predicting species distributions under climate change typically relies on thermal limits for survival. However, recent evidence suggests that sublethal temperatures that reduce fertility can constrain distributions more strongly than temperatures causing mortality alone. Despite the importance of mosquitoes as vectors of human disease, thermal fertility limits remain largely uncharacterized in these insects. Here, we show that fertility in Aedes aegypti is highly sensitive to thermal stress and exhibits distinct sex-specific responses. Adult males were more vulnerable to both heat and cold exposure than females. At 38{degrees}C, survival remained high in both sexes despite significant reproductive impairment, indicating that sterility can occur independently of mortality. Similarly, exposure to 2{degrees}C induced male sterility whereas females maintained reproductive function. Morphological analyses of reproductive organs showed temperature-associated damage consistent with the observed fertility loss. Incorporating these fertility thresholds into ecological niche models generated different predictions of climatic suitability compared with models based solely on lethal temperature limits. Our findings demonstrate that sublethal effects on reproduction can substantially influence estimates of mosquito climatic suitability and highlight the importance of incorporating fertility based thermal limits into projections of vector distributions and disease risk under future climate change.

Mosquitoes in the Toxorhynchites genus (Theobald, 1901) are specialized predators of container-breeding mosquito larvae, including major Aedes disease vectors. Despite their biological control potential, species boundaries and phylogenetic relationships remain poorly understood. Utilizing country-wide sampling across Panama, we evaluated the taxonomic status and diversity of local Toxorhynchites species. We collected 147 specimens from artificial and natural containers, yielding 97 cytochrome c oxidase subunit I barcodes from 101 sequenced individuals. A Panama-only Neighbor-Joining analysis revealed four distinct, well-supported clusters (I - IV) exhibiting high mean inter-cluster genetic divergence (4.8 - 20.2%), alongside morphological and ecological segregation. A subsequent global Neighbor-Joining analysis incorporating BOLD and GenBank sequences recovered Toxorhynchites as a strongly supported monophyletic group (99.0% bootstrap support) and revealed deep phylogenetic divergence separating Old World Toxorhynchites (Toxorhynchites) (Clades A and B) from New World Toxorhynchites (Lynchiella) (Clades C - G) lineages. Panamanian Clusters I and II formed two distinct groupings within Clade G, nesting as sisters to Subclade G1 characterized by severe taxonomic discordance involving sequences labeled as Tx. moctezuma, Tx. theobaldi, and Tx. rutilus. Cluster III nested within Tx. hypoptes (1.5% divergence) in Clade E, whereas Cluster IV associated with Tx. haemorrhoidalis s.l. (>5.0% divergence) in Clade C. The high degree of taxonomic uncertainty or cryptic diversity uncovered within Clades C, D, and G underscores an urgent need for formal taxonomic revision to accurately delimit species boundaries. Due to its widespread peri-urban distribution, Cluster I (within the Tx. moctezuma s.l. complex) shows the greatest promise for mass-rearing and Aedes biocontrol applications in Panama.

Monogenean ectoparasites, particularly Neobenedenia species, cause severe economic losses in mariculture. Here, ectoparasites isolated from cultured hybrid groupers (Epinephelus fuscoguttatus[female] x E. lanceolatus[male]) were confirmed as Neobenedenia melleni based on ITS1 phylogeny. In vivo screening of six structurally diverse compounds identified compound D (CAS No. 206111-37-7), a 5,6-dihydropyridine derivative, as the most effective antiparasitic agent, achieving 76.54% efficacy at 0.5 mg/L in a 90 min bath treatment. Dose-response assays demonstrated that 0.7 mg/L compound D achieved 95.23% antiparasitic efficacy without causing evident tissue damage or cytotoxicity to GF-1 cells. Ultrastructural observation by scanning electron microscopy revealed marked tegumental alterations, including deep fissures and extensive surface folding, in treated parasites. Molecular docking against ten candidate proteins identified {beta}-tubulin as the most favorable docking target, with a binding energy of -6.53 kcal/mol and three hydrogen-bond interactions, suggesting that {beta}-tubulin may be involved in the antiparasitic activity of compound D. Overall, these findings highlight compound D as a promising lead candidate for short-bath therapy against N. melleni and suggest that cytoskeletal disruption through {beta}-tubulin interaction represents a plausible mechanism of action.

Rising global temperatures are reshaping species interactions and the ecological conditions governing vector-borne disease transmission. Although previous studies show that West Nile virus (WNV) infection alters mosquito longevity, fecundity, blood-feeding behavior, and the thermal performance of these traits, trait-based R models largely rely on data from uninfected mosquitoes, implicitly assuming homogeneous vector populations. This overlooks infection-induced trait variation that may influence transmission dynamics. Here, we examined how temperature, infection status, and viral strain interact to shape transmission potential for WNV in Culex pipiens. Life-history traits of WNV-exposed and unexposed mosquitoes were measured across constant temperatures ranging from 10{degrees}C to 33{degrees}C, as well as under a fluctuating temperature regime of 25{degrees}C {+/-} 5{degrees}C. These data were used to generate thermal performance curves and estimate temperature-dependent relative R across treatments. Infection altered the thermal performance of mosquito life-history traits, vector competence, and overall transmission potential. We also found evidence for a bimodal effect of temperature on vector competence, potentially driven by tradeoffs between viral replication and mosquito immune responses. Incorporating infection-sensitive traits into relative R calculations reduced estimated transmission intensity across much of the thermal range without shifting thermal optima or limits, suggesting that current models may overestimate transmission.

Short-chain dehydrogenases/reductases (SDRs) constitute a large enzyme superfamily involved in endogenous metabolism and xenobiotic biotransformation. In the parasitic nematode Haemonchus contortus, SDRs may catalyze the carbonyl reduction of a benzimidazole anthelmintic flubendazole (FLU), whose increased reduction is associated with FLU resistance. This study thus investigated the constitutive expression of SDRs and their inducibility by FLU in drug-susceptible and benzimidazole-resistant strains of H. contortus. The expression of 23 sdr genes was analyzed by quantitative PCR, while targeted proteomic assays enabled the quantification of 15 SDR proteins. In adult nematodes, pronounced sex-dependent differences were detected at both transcript and protein levels. Resistance-associated alterations were less pronounced and were observed predominantly in males, with SDR9, SDR12, SDR15, and SDR20 displaying increased protein abundances in the resistant strain. Exposure to FLU induced only minimal transcriptional responses in juvenile stages, whereas adult nematodes exhibited marked sex- and strain-specific responses in the expression of SDRs. The strongest transcriptional effects were detected in resistant males, while significant protein-level changes following FLU treatment were observed exclusively in adults of the drug-susceptible strain. Notably, SDR9 and SDR20 combined resistance-associated expression patterns with responsiveness to FLU exposure. Taking together, the first targeted proteomic characterization of SDRs in H. contortus revealed several SDR isozymes with constitutive overexpression in resistant nematodes and/or inducibility by FLU, suggesting a potential role in adaptation to anthelmintic exposure.

IntroductionDengue, one of the most important arboviral infections worldwide, is transmitted primarily by the mosquito Aedes aegypti, a vector closely associated with human habitations. Because vector control is the primary prevention strategy for this disease novel tools are urgently needed. We evaluated an Attractive Lethal OviTrap (ALOT) that targets epidemiologically relevant gravid female mosquitoes for public health impact against dengue disease. MethodsWe conducted a proof-of-concept field efficacy trial in the Amazonian city of Iquitos, Peru, to quantify the reduction of vector density, parity, and sex ratio and symptomatic human dengue infection over a 3.5 year follow-up period. After three baseline entomological surveys in core (753 houses), and buffer (1,549 houses) areas where traps were placed in and around homes, and a control (1,233 houses) area without traps, entomological surveys were carried out every 2 months, and household residents were monitored for dengue disease 3 times a week. Trap maintenance was conducted at 2-3 week intervals by study staff during the first 2.5 years and by residents in the final year. ResultsTotal and female Ae. aegypti abundance demonstrated a strong initial effect with 52% and 47% fewer mosquitoes after trap placement, respectively (Total: RR = 0.48, 95% CI: 0.40 - 0.58, p < 0.001; Females: RR = 0.53, 95% CI: 0.43 - 0.66, p < 0.001). The effect diminished significantly, however, over the study period (interaction coefficient = 0.035 per month, p < 0.001; interaction coefficient = 0.030 per month, p < 0.001). Female-to-male ratio decreased after trap deployment and impact on non-Aedes abundance was like that of Aedes. Cumulative symptomatic dengue incidence was 1.40% (95% CI: 1.00%-1.70%) in the ALOT area versus 3.20% (95% CI: 2.60%-3.80%) in the control area, a 56% relative reduction (log-rank {chi}{superscript 2}=30.0, p<0.001). Restricted mean survival time analysis showed participants in the intervention area on average remained dengue-free for 15.5 additional days (95% CI: 11.1-20.0, p<0.001). ConclusionsThe ALOT strategy functioned as predicted, decreasing but not eliminating female vector densities, with a clear public health impact, lowering dengue disease in areas where traps were deployed. Our study also illustrates the challenges associated with conducting large-scale vector control trials and the need to consider programmatic implementation early in the process of bringing a new product to market. Author SummaryDengue is one of the most important mosquito transmitted infections worldwide. Aedes aegypti, the most important species involved, has a unique biology living in containers associated with households. New methods to control this mosquito are needed and we present our evaluation of a novel trap called the Attractive Lethal OviTrap (ALOT) that targets older female mosquitoes that are the most important for dengue disease transmission. We conducted a trial in the Amazonian city of Iquitos, Peru, to measure reductions in mosquito numbers, age, and sex ratio and symptomatic human dengue infections over a 3.5 years. After baseline entomological surveys prior to placing ALOT traps in homes in a treatment area, which included a central core area with 753 houses and a protective buffer area with 1,549 houses areas. We also had a control area without any traps in 1,233 houses. Mosquito surveys were carried out every 2 months, and household residents were monitored for dengue disease 3 times per week. Trap maintenance was conducted at 2 - 3 week intervals by study staff during the first 2.5 years and by residents in the final year. The number of all Ae. aegypti were initially reduced by 52% after trap placement whereas females were reduced by 47%. The effect diminished significantly, however, over the study period. Female-to-male ratio decreased after trap deployment and impact on other mosquito species was like that of Aedes. Dengue incidence was 56% less in the area with traps compared to the control area, estimating that participants in the intervention area on average remained dengue-free for 15.5 additional days. The ALOT strategy functioned as predicted, decreasing but not eliminating female mosquitos and had a clear public health impact, lowering dengue disease in areas where traps were deployed. Our study also illustrates the challenges associated with conducting large-scale vector control trials and the need to consider programmatic implementation early in the process of bringing a new product to market.

Invasive mosquito species pose substantial risks to human and animal health. Since 2004, Culex coronator, a mosquito vector species of public health concern, has shown rapid range expansion within the United States, spreading from a historically limited distribution in southern Texas to across the Gulf Coast region and into eastern and mid-Atlantic states. However, changes in environmental suitability associated with this expansion across historical, contemporary, and future climate conditions have not been evaluated. Here, we used species distribution models (SDMs) to compare predictions of abiotic suitability for Cx. coronator under historic (1960-1989) and recent (2000-2024) climate conditions calibrated on the historical range in the United States. We also created a contemporary SDM based on occurrence records prior to and following species range expansion (1960-2024), and further, to predict potential distributions under current and future climate conditions. Models calibrated on the historical range predicted only modest changes in suitability along the Gulf Coast region and failed to identify large areas of the humid subtropical eastern United States that are now occupied. In contrast, the contemporary model predicted widespread suitability across much of the southern and eastern United States. Future projections under the mid-range SSP3 scenario predicted increasing suitability at higher latitudes and elevations. Across all models, suitability was consistently low in arid and semi-arid regions, including along the historical western range limit, suggesting that moisture availability may constrain Cx. coronator distributions. Together, these results highlight the need to incorporate updated occurrence records when modeling invasive mosquito species to strengthen surveillance and control strategies.

BackgroundMalaria caused by Plasmodium knowlesi and lymphatic filariasis caused by Brugia malayi are mosquito-borne infections with non-human primates as reservoirs. P. knowlesi has emerged as a significant cause of human malaria in Southeast Asia over the past two decades. Belitung district, Indonesia, was until recently assumed to have eliminated B. malayi until infections were detected in humans and long-tailed macaques. To investigate whether the local reservoir of B. malayi is also a reservoir for malaria we screened macaques from 4 areas in Belitung for malaria parasites. Methods and findingsBlood samples from 163 long-tailed macaques (Macaca fascicularis) that had been tested for B. malayi were examined by quantitative PCR assays specific for Plasmodium spp., P. knowlesi, P. inui, P. coatneyi and P. cynomolgi. A total of 130 macaques (79.8%) tested positive in the pan-Plasmodium qPCR assay. Plasmodium inui was most prevalent (41.7%), followed by P. knowlesi (38.7%), P. coatneyi (24.5%) and P. cynomolgi (13.5%). Multiple species infections, with 2-3 Plasmodium species were detected in 37% of macaques. Notably, 20 (91%) of 22 B. malayi-positive macaques were co-infected with at least one Plasmodium species. We sequenced the complete mitochondrion from 9 samples diagnosed by qPCR as mono-infections. Phylogenetic analysis confirmed 7 as P. knowlesi, and the other two as P. inui and P. coatneyi. Phylogenetic and pairwise analysis revealed that P. knowlesi isolates from Belitung were closely related to each other and to P. knowlesi from humans and monkeys from Thailand, Malaysia and Indonesia. ConclusionsMolecular evidence shows high prevalence of zoonotic malaria parasites in macaques from Belitung, emphasizing the risk of human transmission. Increased surveillance, improved diagnostics, and targeted interventions are needed to prevent zoonotic spillover of P. knowlesi as it has been observed for B. malayi in Belitung. Author summaryWe examined macaques for Plasmodium parasites in Belitung Island, Indonesia, a region classified as free of locally transmitted human malaria. Plasmodium knowlesi is a monkey parasite that commonly infects humans and has emerged as a concern in Southwest Asia over the last 20 years. In Belitung, macaques are infected with Brugia malayi, a filarial nematode, that causes lymphatic filariasis in humans. Blood samples from 163 macaques were screened for Plasmodium DNA and 80% carried malaria parasites. Plasmodium inui was most common, followed by P. knowlesi, P. coatneyi, and P. cynomolgi. Many macaques were infected with multiple parasites simultaneously, but no host was infected with all four Plasmodium species. Ninety one percent of B. malayi-positive macaques were co-infected with malaria parasites, including P. knowlesi, indicating multi-parasite infections that could potentially spread to humans. Analysis of the mitochondrial genome of 7 P. knowlesi isolates from Belitung showed that they were most similar to each other and both human and monkey samples from Malaysia, Thailand and Indonesia. The study highlights the need for enhanced malaria monitoring and prevention, given the complex epidemiology of co-infecting parasites and risk to humans and animals.

Experimental hut trials (EHTs) are WHO-recommended for the entomological evaluation of insecticide-treated nets (ITNs), but several hut designs are in operational use, and structural differences between them may confound efficacy predictions and limit cross-site comparability. We developed a Bayesian hierarchical framework comprising a host-seeking model, which jointly estimates biting deterrence and preprandial mortality while accounting for night-to-night variation and overdispersion, and a postprandial mortality model, which expresses hut and net effects as hazard ratios through a complementary log-log link. We applied it to a comparative trial of four hut designs (East African, West African, Ifakara and Rapley) conducted at a single site in Tanzania, evaluating eight ITNs when new and after twenty washes. Posterior estimates parameterise a vectorial capacity framework to predict reductions in transmission potential. Hut design influenced baseline mosquito behaviour and all three modes of action. Relative to the Rapley reference, baseline feeding rates were substantially lower in the East African and West African huts and closer to Rapley in the Ifakara hut. Preprandial mortality was amplified in the Ifakara hut. Comparing to previous analysis provides evidence that combining mortality before and after feeding into a single endpoint does not reliably reflect impact, supporting the decomposition of entomological outcomes into separate modes of action. Expressing modes of action as mechanism-specific parameters allows the estimates to be carried directly into transmission models. For every net, the predicted reduction in vectorial capacity was greatest in the Ifakara hut and smallest in the West African and Rapley huts. The effect of hut design on predicted impact exceeded that of washing the nets twenty times. Results indicate that the hut design under which trial data were collected should be considered when forecasting population-level effect.

Catenulids are free-living flatworms, common in eutrophic freshwaters such as ponds, ditches, or peatbogs, with most of the diversity described from tropical regions to date. Although the majority of the species have been described from warmer climates, most molecular studies have been done on specimens from temperate zones in Europe. We addressed this gap by sampling for exotic species in localities available in a temperate climate. In this study, we investigated catenulid diversity in the greenhouses at the University of Warsaw Botanic Garden and recorded two species known only from tropical areas (Stenostomum paraguayense and Suomina evelinae) and one exotic species recorded previously from a greenhouse in Poland (Stenostomum corderoi). Additionally, in the latter species, we provide evidence for environmentally induced coloration of sensory pits, which has not been reported thus far. We placed the collected species on a phylogeny using barcoding of 18S, 28S, and COI genes and retrieved paraphyly of the family Catenulidae, with S. evelinae forming a sister group to the genus Paracatenula, and hence we propose a revision of its systematic position. In total, we recorded six species, including three with a wide cosmopolitan distribution (C. turgida, S. grande and S. tuberculosum), and provided sequences for five of them, three of which had no previous molecular records (S. paraguayense, S. evelinae and S. corderoi). Thus, we confirm that greenhouses represent an important source of exotic species for taxonomic work on microscopic invertebrates.

The black-legged tick (Ixodes scapularis), a key vector of Lyme disease, anaplasmosis, and babesiosis, exhibits regionally distinct patterns of seasonal activity driven by climate. Consequently, the relative timing of larval and nymphal activity varies across geographic locations, influencing pathogen transmission dynamics. Early-emerging nymphs may increase pathogen transmission, whereas early-emerging larvae may reduce transmission. In addition, synchrony between the two life-stages facilitates co-feeding transmission, which contributes to pathogen maintenance and coinfection risk. Temperature is thought to be an important driver of tick phenology, but existing mechanistic models that incorporate temperature fail to accurately capture the timing of larval and nymphal tick activity. To address this limitation, we developed a mechanistic model that includes two additional factors: humidity-dependent questing and low rates of overwinter development. To assess the value of these factors for explaining real-world patterns, we fitted alternative models to tick collection data from the National Ecological Observatory Network. In doing so, we found that explicitly incorporating humidity is necessary to reproduce observed tick phenology, with larval ticks being especially sensitive to relative humidity compared to other life stages. In addition, we found that accounting for humidity had a larger effect at Mid-Atlantic sites than at Northeastern sites, underscoring the importance of region-specific interactions between temperature and humidity in shaping I. scapularis phenology. By more accurately capturing tick seasonality compared to existing mechanistic models, our model illustrates the importance of accounting for factors beyond temperature for investigating how climate variability influences seasonal tick activity and pathogen transmission.