Parasites & Vectors

BackgroundAedes vittatus, an emerging invasive species and arboviral vector with high ecological plasticity and dispersal capacity, is expanding in the Caribbean, being detected in the Dominican Republic, Cuba, and Jamaica and recently in Yucatan, Mexico. ObjectivesThis study aimed to provide evidence of the establishment of Ae. vittatus in Haiti. MethodsLarval surveys were done across 12 localities around Cap-Haitien, in the North-East department of Haiti, the specimens were identified morphologically and confirmed by COI DNA barcoding. FindingsWe report, for the first time, the detection of the invasive vector Aedes (Fredwardsius) vittatus in Haiti, confirmed morphologically and molecularly, and being established in half of the municipalities sampled. Phylogenetic analyses group Haitian specimens with Caribbean and American populations, closely related to Mexico, indicating regional expansion. Main conclusionGiven its role as a vector of chikungunya, dengue, Zika and yellow fever, strengthened entomological surveillance and early detection in North America and the Caribbean are urgently needed.

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.

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.

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.

In many leishmaniasis foci, reservoirs that maintain infection remain unknown. Here, we developed a field-applicable toolkit based on the analysis of individual blood fed sand flies (IBF) to identify reservoirs. Sand flies were given a Leishmania donovani-infected first blood meal (iBM1) by feeding artificially on a membrane or naturally on a clinically ill animal followed by two subsequent uninfected blood meals (BMS+). Bulk-RNAseq was used to identify two target parasite genes, sherp and a novel hypothetical gene (HPB), which exhibited a significantly higher expression in BMS+ compared to iBM1 sand flies. DNA and RNA were co-extracted from IBF. DNA was used to detect Leishmania infection and the blood meal source; RNA was used to assess expression of target genes by qRT-PCR. Linear discriminant analysis (LDA) of target gene expression classified sand fly specimens based on their iBM1 or BMS+ status. Co-extraction yielded a mean of >800ng per IBF for DNA and RNA. We detected [≥]1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. LDA identified iBM1 parasites with a predictive accuracy of [~]87% and [~]82%, in membrane or naturally fed sand flies, respectively. This toolkit provides an innovative approach to identification of leishmaniasis reservoirs informing targeted control strategies.

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.

The effectiveness of malaria vector control is increasingly threatened by widespread insecticide resistance, particularly to pyrethroids. In response, the World Health Organization recommends dual-active ingredients (dual-A.I.) insecticide-treated nets (ITNs) in resistant settings. This study evaluated the efficacy of several dual-A.I. ITNs in two experimental hut trial sites an area with high insecticide resistant malaria vectors in Tanzania with differing vector species composition and bionomics. Five ITNs were evaluated: a pyrethroid-only net (MAGNet(R)), three pyrethroid-piperonyl butoxide (PBO) nets (OlysetTM Plus, Veeralin(R), and PermaNet(R) 3.0), one dual-A.I. net (Interceptor(R) G2; chlorfenapyr + alpha-cypermethrin), and an untreated control net. Mosquitoes were collected from huts, morphologically identified, and monitored for mortality up to 72 h. WHO cone and tunnel tests using susceptible An. gambiae s.s. were conducted for quality control, while field-collected mosquitoes were used for bio-efficacy monitoring. Molecular species identification was performed using PCR assays, and genomic DNA was extracted from An. funestus s.l. and An. gambiae s.l. collected from the trials. All ITNs met WHO bio-efficacy criteria in cone and tunnel tests. An. gambiae s.l. predominated in Mwagagala (77%) but was rare in Magu (4%), where An. funestus complex was dominant. In Magu, Interceptor(R) G2 and Veeralin(R) achieved significantly higher mortality than MAGNet(R) at 24 h (53% and 45% vs 24%) and 72 h (61% and 53% vs 24%). In contrast, no significant mortality differences were observed among ITNs in Mwagagala. High resistance intensity was detected in both vector groups (mortality <20% in bioassays). In An. funestus from Magu, the 4.3 kb-SV resistant genotype was fixed, while G454A-CYP9K1 resistance was highly prevalent (RR: 0.70-1.00). Interceptor(R) G2 and Veeralin(R) outperformed the pyrethroid-only reference net against An. funestus complex but not against predominantly An. arabiensis populations.

Laboratory models are invaluable tools for studying parasite biology and pathogenesis, especially for helminth infections. However, the complex life cycles and frequently narrow host specificity of helminths present challenges to maintaining access to critical parasite material in a laboratory setting. This is especially true of Necator americanus, the most common species of hookworm that infects humans globally. Here we report the successful laboratory adaptation of an African strain of N. americanus, originally isolated from infected individuals in Beposo, Ghana. The Beposo strain has been successfully passaged across 9 generations in Golden Syrian hamsters maintained on oral dexamethasone. Differential susceptibility to mebendazole and albendazole was evaluated using an egg hatch assay, and DNA sequencing of the beta-tubulin isotype 1 gene did not identify known resistance-associated mutations in the endemic strain. Sequencing of the mitochondrial COX1 gene revealed that specimens of N. americanus from Ghana, along with reported sequences from Togo, are distinct from those from South America and Asia. Complementary microsatellite-based population analysis revealed substantial genetic variation in the founding parasite population. To further characterize the novel Beposo strain, a draft hybrid genome assembly was generated from genomic DNA extracted from a single adult male worm via an optimized Oxford Nanopore Technologies MinION library preparation approach tailored to low-input sample types. This high-quality assembly, including a complete mitogenome, is 202.8Mb in 950 contigs with an N50 >449 kb. It contains >95% of conserved nematode orthologs in complete single copy and is estimated by homology-based gene prediction to contain 12,804 genes. This study represents the first comprehensive characterization of a strain of N. americanus originating in Africa that has been successfully adapted to a laboratory animal model.

Entomological surveillance is essential for the control of lymphatic filariasis (LF). This xenomonitoring study, conducted in 2022-2023, evaluated the effectiveness of three mosquito trapping methods to identify the most suitable tool for surveillance following the discontinuation of mass drug administration. The surveys took place in two Malian villages: Konfra (seasonal watercourse) and Siradjouba (permanent watercourse). Three techniques were compared: Pyrethrum spray catch (PSC) conducted at the end of each visit inside 30 randomly selected houses, and different collection points were chosen based on the characteristics of the study area to place the Ifakara Type C tent (Ifakara) and the gravid trap (GT). Collections were conducted simultaneously at both sites. All Anopheles gambiae and Culex spp. collected in 2022-2023 were morphologically identified and then analyzed in the laboratory for their infectious status. Data was processed using SPSS v25; Fishers exact test was used to compare proportions, with a significance threshold of p < 0.05. A total of 4,732 mosquitoes were captured (95.05% in 2022). In 2022, the Anopheles were predominant in Konfra (91.38%), while Culex predominated in Siradjouba (63.41%). In 2023, Anopheles was found only in Konfra (3 specimens), while Culex was predominant in Siradjouba (64.07%). In 2022, PSC collected 90.57% of Anopheles, Ifakara 9.43%, and GT 0%. For Culex, GT collected 53.67%, PSC 36.62%, and Ifakara 9.71%. In 2023, the few Anopheles (3) came from PSC; Culex were mainly collected by the Ifakara tent (43.29%). For FL xenomonitoring, the combination of PSC and the GT appear to be effective collection methods.

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.

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.

Wildlife hosts play important roles in the ecology and transmission of vector-borne parasites, yet information on host associations remains scarce in many biodiverse tropical regions. Within a One Health framework, characterizing parasite diversity in wildlife can improve understanding of ecosystem health and disease emergence. Road-killed animals provide a non-invasive opportunity to investigate host-parasite interactions while minimizing disturbance to natural habitats. We screened 127 liver and intestinal tissue samples obtained from 76 road-killed vertebrates collected near protected areas in two Ecuadorian biodiversity hotspots, the Tropical Andes and Choco-Darien, for trypanosomatids and other vector-borne microorganisms. Molecular analyses targeted the 18S rRNA and cytochrome b genes of trypanosomatids and included additional screening for Trypanosoma cruzi, Trypanosoma rangeli, Rickettsia spp., and piroplasmids. Twenty-nine samples were positive for kinetoplastids. We detected diverse trypanosomatids representing the genera Leishmania, Porcisia, Trypanosoma, Phytomonas, Blastocrithidia, and Obscuromonas, as well as free-living kinetoplastids of the order Neobodonida. The most frequently detected species was Leishmania amazonensis, identified in 17 samples from at least 13 species of birds, reptiles, and caecilians, predominantly in liver tissue, suggesting previously unrecognized host associations. We also identified a putatively novel species of Porcisia and three potentially undescribed avian trypanosomes belonging to the subgenus Ornithotrypanum. No evidence of T. cruzi, T. rangeli, Rickettsia spp., or piroplasmids was found. Our findings identify birds, reptiles, and caecilians as potential reservoir hosts of L. amazonensis. In addition, we substantially expanded current knowledge of kinetoplastid diversity in Ecuadorian wildlife. This study demonstrates the value of road-killed animals as a practical, non-invasive resource for wildlife pathogen surveillance and highlights the importance of integrating biodiversity research into One Health approaches to better understand parasite transmission dynamics in rapidly changing tropical ecosystems. Author summaryMany parasites that affect humans circulate naturally in wildlife, but identifying their animal hosts is often difficult in remote, biodiverse regions. We used road-killed animals as a non-invasive source of biological material to investigate parasites in wildlife from two biodiversity hotspots in Ecuador. By analyzing tissues from birds, reptiles, amphibians, and mammals, we found a remarkable diversity of kinetoplastid flagellates, a group that includes the agents of Chagas disease and leishmaniasis. Although we did not detect human-infective trypanosomes, we repeatedly identified Leishmania amazonensis (a species causing human disease) in birds, reptiles, and caecilians. These vertebrate groups have not previously been recognized as potential hosts of this parasite. We also discovered several undescribed trypanosomatid species, emphasizing how little is known about parasite diversity in tropical wildlife. Our results show that road-killed animals can provide valuable information on host-parasite interactions without disturbing living populations. Such surveillance contributes to One Health efforts by improving our understanding of how environmental change, wildlife, and human health are interconnected.

ObjectivesTo estimate the pooled prevalence of Wolbachia infection in biting midges (Ceratopogonidae) across Southwest Asia and to evaluate ecological and biological factors associated with infection patterns. Study DesignSystematic review and meta-analysis. MethodsA comprehensive search of international and regional databases (PubMed, Scopus, Web of Science, Embase, SID, MagIran) was conducted without date restriction. Eligible studies included those using molecular techniques to detect Wolbachia in Ceratopogonidae collected from Southwest Asia. Pooled prevalence was calculated using a random-effects model. Subgroup and meta-regression analyses were performed to assess variations by country, species, altitude, habitat type, and sex. Heterogeneity and publication bias were evaluated using I{superscript 2}, Cochrans Q, and Eggers tests in accordance with PRISMA guidelines. ResultsTwenty-four studies comprising 14,832 midges from six countries were included. The pooled prevalence of Wolbachia infection was 32.6% (95% CI: 28.4-36.9%; I{superscript 2}=78.3%). Iran showed the highest prevalence (38.2%), and Culicoides imicola was the most frequently infected species (36.8%). Higher prevalence was associated with lower altitudes (<500 m; P=0.012), rural habitats (P=0.034), and female midges (P=0.008). Limited evidence suggested the presence of cytoplasmic incompatibility and reduced bluetongue virus competence in infected midges. ConclusionsWolbachia infection is common among Ceratopogonidae in Southwest Asia and is influenced by ecological and biological factors. These findings highlight the potential of Wolbachia as a biocontrol tool in regional vector management, underscoring the need for further experimental and strain-level studies.

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.

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.

Background: Climate change is a major driver of increasing mosquito populations and mosquito-borne disease (MBD) risks across Europe. In Germany, rising temperatures and shifting precipitation regimes are creating more favourable environmental conditions for mosquito establishment and pathogen transmission. However, the extent to which regional climate variability shape spatial patterns of mosquito community composition remain insufficiently understood. Methods: Mosquito specimens were trapped using Biogents CO2-baited traps at several locations in Germany between 2016 and 2025. Using k-means cluster analysis applied to a set of mosquito-relevant bioclimatic indicators, we developed a data-driven ecoclimatic regionalisation to identify seven mosquito ecoclimatic regions (MERs). To validate the regionalisation, we conducted community analysis on nationwide mosquito surveillance dataset. Mosquito richness and diversity metrics were estimated per MER. Non-metric multidimensional scaling (NMDS) and Permutational Multivariate Analysis of Variance (PERMANOVA) were used to visualise and test differences in community composition between MERs. The robustness of the regionalisation was further evaluated by comparing within- and between-region Bray-Curtis beta diversity. Results: A total of 288,689 mosquito specimens were collected using 5,840 trap deployments. Results revealed varying mosquito habitat suitability and community composition across MERs in Germany. Pronounced regional differences in mosquito richness (S) and Simpson diversity (D) were observed among MERs. The identified MERs include: Alpine (S=1.00, D=1.16), Low Mountains (S=7.99, D=2.04), Northwest Cool (S=21.67, D=1.99), Continental-Dry (S=31.99, D=2.39), Warm Continental (S=32.49, D=2.55), Southeast Foothill (S=24.39, D=2.77) and Coastal Maritime (S=24.00, D=4.39) regions. Future MER projections reveal expansion in the spatial distribution of dry and warm conditions in Germany under the RCP8.5 scenario. Community composition differed significantly among MERs (NMDS: stress=0.133; PERMANOVA: p < 0.05, F=6.68, R2=0.16, d.f.=4). Beta-diversity analyses showed that mosquito communities were more similar within than between MERs (Mann-Whitney-Wilcoxon test: p < 0.0001; Z=23.26). Across regions, Culex pipiens s.l. was the dominant native vector while Aedes albopictus and Aedes japonicus were observed in several MERs. Conclusions: Identifying ecoclimatic regions that explain spatial variability in mosquito habitat suitability and community composition is essential for targeted vector surveillance and to improved early warning of climate-sensitive MBDs.

BackgroundHuman head lice (Pediculus humanus capitis de Geer) are known to harbor diverse maternally inherited bacterial symbionts. These endosymbiotic bacteria may contribute to insecticide degradation, potentially helping lice withstand particular environmental pressures. Using next-generation sequencing (NGS), this study investigated the bacterial symbionts present in wild head-louse populations and characterized their phylogenetic relationships in mutant and permethrin-susceptible strains. MethodsHead lice specimens were collected from 10 locations across Fars Province, Iran. Following DNA extraction, the samples were analyzed using polymerase chain reaction (PCR), and the resulting amplicons were sequenced to detect mutations in specimens from each location. Lice were then classified according to the presence or absence of mutations and subjected to NGS to characterize the symbiotic bacterial communities in mutant and putatively permethrin-susceptible strains. ResultsMutant strains were detected at only three sampling stations. Bioinformatic analysis of the nucleic acid sequences revealed three exons and two introns, with an expected amplicon length of 582 bp. NGS analysis showed that Candidatus Riesia pediculicola (Arsenophonus), belonging to the phylum Proteobacteria, was the predominant bacterial genus. Actinobacteria and Firmicutes were the second- and third-most abundant phyla, respectively. Most of the remaining 25 bacterial taxa were associated with the mutant strain. Additionally, two previously unreported bacterial genera were deposited in GenBank. ConclusionsThe distinct distribution of Arsenophonus species between susceptible and mutant head-lice strains--along with the greater abundance of Escherichia, Shigella, Lawsonella, and Megamonas in mutant strains--highlights the need for advanced metagenomic analyses to determine how these endosymbionts may help their hosts withstand specific environmental disturbances.

The Culex pipiens complex comprises globally distributed mosquito species that serve as vectors for various arboviruses and filariasis. Female mosquitoes within the Cx. pipiens complex are morphologically very similar, making species identification based on morphologically characteristics challenging. To date, Cx. pallens and Cx. pipiens f. molestus have been reported to inhabit the Republic of Korea (ROK). Conversely, Cx. quinquefasciatus, predominantly distributed in tropical regions, has not been documented in the country since a single record in the 1950s. Recently, however, Cx. quinquefasciatus was reported from the Jeju region of the ROK based on molecular identification. This study aimed to re-evaluate the occurrence of Cx. quinquefasciatus in the ROK by conducting molecular identification using the ace-2 and CQ11 markers on Cx. pipiens complex specimens collected nationwide, including from Jeju, where Cx. quinquefasciatus had previously been reported. Our analysis identified only Cx. quinquefasciatus-Cx. pallens hybrid individuals among the Cx. pipiens complex specimens from the Jeju region, highlighting the need to re-evaluate the analytical methods used in previous studies that reported the presence of pure Cx. quinquefasciatus. These findings provide essential evidence for future surveillance of the distribution, introduction, and establishment of Cx. quinquefasciatus and underscore the need for continuous monitoring.

BackgroundLymphatic filariasis (LF), caused by Wuchereria (W) bancrofti, Brugia malayi and Brugia (B) timori, remains a major public health problem known for its disfiguring and debilitating effects despite notable progress under the Global Program to Eliminate Lymphatic Filariasis (GPELF). Following recent national directives (2024) restructuring implementation units (IUs) to the block level, all TAS-cleared blocks now require re-evaluation using night blood surveys (NBS) or filarial test strip (FTS) kits. Molecular Xenomonitoring (MX) is recommended as a post-validation surveillance method for detecting active LF transmission in vectors. Therefore, an exploratory study was conducted in two coastal districts of Odisha, namely Jagatsinghpur and Puri, which cleared TAS-III, to investigate the presence of B. malayi and W. bancrofti infections in filaria-transmitting vectors. Methodology/Principal FindingsAdult female mosquitoes were collected from three villages across Jagatsinghpur and Puri districts during March-April 2025. Species were identified morphologically, segregated and pooled. The pooled samples were screened for the presence of filarial DNA using species-specific PCR assays. Of 1,518 collected adult female mosquitoes, 22.6% were Culex quinquefasciatus, 18.3% were Mansonia (Mn) annulifera, and 4.3% were Mansonia uniformis. Among 33 pools screened, six pools were positive for B. malayi DNA (18.18% pool positivity; infection rate 2.88%, 95% CI: 1.23-5.74), in Mn. annulifera, whereas W. bancrofti was not detected. Conclusions/SignificanceThe study provides the first molecular confirmation of B. malayi infection in Mn. annulifera, indicating a persistent Brugian transmission in Odisha even after the implementation of mass drug administration. The Study highlights MX as a sensitive, non-invasive surveillance tool for detecting residual infection in post-MDA and post-TAS settings. The study underscores the need for a standardized MX protocol and integration with entomological monitoring to sustain national LF elimination targets by 2027. Author SummaryLymphatic filariasis is a mosquito-borne disease that can cause severe, disfiguring swelling of the limbs and other lasting disabilities, and it remains a serious public health problem in India. National programmes in India are actively working to eliminate it by giving preventive medicines to communities, conducting surveys to confirm that transmission has ceased, and once an area passes, this treatment is withdrawn. However, it is not always clear whether the parasite has truly disappeared or is still lingering at very low levels. In this study, we searched for the parasite directly inside mosquitoes, an approach that does not require collecting blood from people. We sampled mosquitoes from two coastal districts of Odisha, India, that had already passed the required surveys and stopped treatment. We found the parasite Brugia malayi in Mansonia mosquitoes, the first time this has been shown using this molecular method in the region, although the more common filarial parasite was not detected. Our findings show that transmission can smoulder on even after an area is judged free of disease. Because India has no available rapid field test for this particular parasite, we argue that examining mosquitoes should become a routine safeguard for the countrys elimination effort.