Parasites & Vectors

BackgroundTegumentary leishmaniasis (TL) caused by Leishmania infantum has re-emerged in northern Italy, raising questions about the genetic diversity and population structure of circulating parasites and their potential role in shaping different clinical outcomes. Methodology/Principal findingsMultilocus microsatellite typing (MLMT) based on 15 polymorphic loci was applied to 44 L. infantum strains obtained from TL cases diagnosed between 2013 and 2024 in the Emilia-Romagna region. These strains were compared with sympatric isolates from VL cases, dogs and sand flies. MLMT revealed a considerable genetic variation among TL-associated strains, with 43 distinct multilocus genotypes identified. Population structure analyses using Bayesian clustering, multivariate approaches and phylogenetic reconstruction consistently identified three highly differentiated genetic populations (Fst >0.25). TL strains were divided into two main populations: one shared with VL-associated strains (PopB; 9/44) and a second population found exclusively among TL cases (PopC; 28/44). In contrast, the canine-associated population (PopA) showed no overlap with TL cases in this region. Populations also displayed divergent heterozygosity patterns, as indicated by positive and negative Fis values. Conclusions/SignificanceThese findings revealed previously unknown diversity within L. infantum in the study area and demonstrated that inclusion of tegumentary strains is essential to uncover hidden components of parasite population structure. The identification of a TL-associated population supports the existence of multiple evolutionary pathways and emphasises the importance of integrated One Health surveillance, which combines data from humans, animal hosts and vectors to improve understanding of the epidemiology of leishmaniasis in Italy. Author summaryLeishmania infantum is a parasite transmitted to humans through the bite of infected insect vectors. It can cause different forms of leishmaniasis, ranging from a systemic disease known as visceral leishmaniasis to a less common form that affects the skin and mucous membranes, called tegumentary leishmaniasis. Dogs are the main reservoir of the parasite and play a key role in maintaining its circulation in endemic areas. In recent years, cases of tegumentary leishmaniasis have re-emerged in northern Italy. This unexpected increase has raised questions about how the parasite is spreading and whether genetic differences among the parasites could explain these new patterns. To explore this, we examined parasitic DNA obtained from tegumentary leishmaniasis cases and compared it with DNA from patients with visceral leishmaniasis, from dogs and insect vectors from the same area. By examining multiple genetic markers, we found that parasites causing the tegumentary form of the disease are genetically diverse and belong to different groups. Notably, one parasite group was found only in cases of tegumentary leishmaniasis and not in visceral infections nor in infected dogs, suggesting that some parasite lineages may be more closely associated with skin and mucosal disease. Overall, our findings show that studying parasites from cutaneous and mucosal lesions provides important information that would otherwise remain hidden. By combining data from humans, animals and insect vectors, our study highlights the importance of integrated surveillance systems for improving our understanding of disease spread and supporting effective public health strategies.

BackgroundOviCol has recently been proposed as a disruptive strategy for the surveillance and control of synanthropic Aedes mosquitoes, vectors of dengue, Zika, and chikungunya viruses. The approach integrates monitoring and control through ultra-low-cost ovitraps ([~]0.2 USD), bioattractants, and egg inactivation using hot water. However, large-scale ovitrap surveillance generates thousands of egg substrates that require time-consuming manual counting, creating a major operational bottleneck. To address this limitation, we developed Col-Ovo, an artificial intelligence-based tool for automated counting of Aedes aegypti eggs from real field samples, together with OviLab, a digital platform for annotation, curation, and management of entomological image datasets. Methodology/Principal FindingsThe detection model was trained using YOLOv11m on a dataset of 275 oviposition substrates (20.5 cm strips) collected under routine operational conditions. Images were captured in situ without preprocessing and included substrates heavily stained by bioattractants such as blackstrap molasses and dry yeast (Saccharomyces cerevisiae), as well as sand and particulate debris, reflecting realistic field conditions. The system was designed to operate with standard smartphone images and tolerate compression artifacts produced by messaging platforms such as WhatsApp. Performance was evaluated by comparing automated egg counts with expert manual counts and with virtual-human counts conducted in OviLab using >200% image magnification. Col-Ovo achieved >95% agreement with expert counts and 88% agreement with OviLab while reducing processing time from approximately 15 minutes to <3 seconds per sample. Conclusions/SignificanceCol-Ovo enables rapid, scalable quantification of Ae. aegypti eggs from smartphone images, addressing a critical operational barrier in ovitrap-based surveillance. The system requires no image preprocessing or specialized hardware and is accessible through a lightweight web interface supported by an AI architecture that allows retraining for new ecological contexts or additional Aedes species. Integrated with OviLab, this platform provides a flexible digital infrastructure that can strengthen routine vector surveillance and community-level control programs across regions where Aedes mosquitoes continue to expand. Author SummaryMosquitoes that transmit dengue, Zika, and chikungunya are expanding in many parts of the world. Monitoring their populations is essential for guiding prevention and control actions. A common surveillance method uses small traps where female mosquitoes lay their eggs. By counting the eggs collected in these traps, health programs can estimate mosquito abundance and detect increases in risk. However, the eggs are extremely small: about 0.065 mm{superscript 2}, and are usually counted manually under magnification. This process is slow, requires trained personnel, and limits how many samples can be analyzed in routine surveillance. In this study, we developed a digital tool that automatically counts mosquito eggs from photographs taken with a smartphone. The system was trained using images collected under real field conditions, including samples with stains, dirt, and other materials commonly found in mosquito traps. The tool can analyze images even when they are compressed and shared through WhatsApp. By reducing counting time from 15 minutes to only a 25 seconds, this approach can help strengthen mosquito surveillance and support faster responses to mosquito-borne disease risks.

Helminthiases remain a major global health burden, and limitations of current anthelmintic therapies highlight the need for new pharmacological targets. In this study, we examined the effects of ion-channel and cytoskeletal modulators on bovine lung protoscoleces (PSCs) of Echinococcus granulosus sensu lato. Compounds acting on ion channels (praziquantel, amiloride, and amlodipine) and cytoskeletal components (albendazole and cytochalasin D) were evaluated using a semi-automated motility assay, methylene blue exclusion to assess viability, and scanning electron microscopy (SEM) to characterize structural damage. All compounds produced concentration-dependent reductions in PSCs motility. Amlodipine was the most potent inhibitor of motility, whereas praziquantel and cytochalasin D produced pronounced tegumental alterations and strong correlations between motility impairment and parasite death. In contrast, amiloride markedly reduced motility with comparatively minor effects on viability, indicating a primarily paralytic effect. Cytoskeletal disruption induced severe structural damage and parallel declines in motility and viability. SEM analysis revealed extensive tegumental collapse, loss of glycocalyx, and microtrichial damage in PSCs exposed to cytoskeletal and calcium-modulating agents. These findings highlight cytoskeletal organization and calcium-dependent ion fluxes as key physiological vulnerabilities in E. granulosus. Comparative analysis of these pharmacological targets provides mechanistic insight into how disruptions in cytoskeletal dynamics and cation homeostasis compromise parasite motility and survival.

Accurate species identification is crucial to assess the medical and veterinary relevance of a mosquito specimen, but it requires high experience of the observers and well-equipped laboratories. This study aimed to evaluate whether low-cost imaging in combination with geometric wing morphometrics can provide accurate identification of invasive, morphologically similar Aedes species. The right wings of 670 female specimens covering 184 Ae. aegypti, 156 Ae. albopictus, 166 Ae. j. japonicus and 164 Ae. koreicus, were removed, mounted and photographed with a professional stereomicroscope (Olympus SZ61, Olympus, Tokyo, Japan) and a macro lens (Apexel-24XMH, Apexel, Shenzhen, China) attached to a smartphone. The coordinates of 18 landmarks on the vein crosses were digitalized by a single observer for each image. In addition, the landmarks of 20 specimens per species and imaging device were digitalized by six different observers to assess the degree of the observer error. The superimposed shape variables were used to compare the species classification accuracy of linear discriminant analysis (LDA), support vector machine (SVM), Random Forest (RF), and XGBoost. In the single-observer landmark data, the LDA achieved the best classification results with a mean accuracy of 95 % for landmarks from microscope images and 92 % for those obtained from smartphone images. In the multi-observer landmark data, LDA consistently performed worse than the other three classifiers, and the reduction in the accuracy was more pronounced for smartphone images than for microscope images. This pattern was associated with a higher degree of observer error for smartphone images, as confirmed by a landmark-wise comparison across all landmarks. Geometric wing morphometrics provides a reliable method to distinguish the most common invasive Aedes species in Europe. Thereby, the image quality obtained by smartphones equipped with a macro lens is sufficient and represents a cost-effective alternative to professional microscopes. However, due to the greater degree of observer variation for smartphone images, landmark coordinates for such images should ideally be collected by a single observer.

BackgroundAnopheles mosquitoes vector pathogens responsible for more than 600,000 human deaths annually. Ecological studies of these insects are important to guide effective vector-control campaigns and to understand their broader ecological consequences. Molecular ecology methods, particularly qPCR, provide a valuable tool in such studies. By detecting trace DNA of a taxon of interest within mixed or environmental samples, qPCR can facilitate identification of prey taxa of interest in the diets of consumers. However, no protocol for the detection of An. gambiae complex mosquitoes in dietary samples has been available. MethodsWe introduce a new set of qPCR primers (Agam_CO1_F1 and Agam_CO1_R1) and a probe-based assay for detection of Anopheles gambiae-complex mosquitoes, even with short reads common in dietary and environmental samples. The primers were tested in vitro for their specificity and sensitivity, and in silico using Primer-BLAST to assess potential off-target amplification. ResultsThe qPCR primers amplified An. gambiae DNA even at low starting concentrations (5 copies {micro}l-1). The primers did not amplify any non-target DNA in either the in vitro or in silico tests, but consistently amplified An. gambiae complex DNA. The primers can therefore provide reliable tests for the presence or absence of An. gambiae complex in dietary or eDNA samples. ConclusionsThe new qPCR primers should allow advances in research into mosquito ecology by allowing detection of even trace amounts of An. gambiae DNA in dietary and environmental samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/707393v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1fcce3corg.highwire.dtl.DTLVardef@47e5f5org.highwire.dtl.DTLVardef@4a7063org.highwire.dtl.DTLVardef@1188d60_HPS_FORMAT_FIGEXP M_FIG C_FIG

Insecticide spraying is a common strategy for controlling dengue outbreaks, but its effectiveness is compromised by the development of resistance in mosquito populations. In this study, we subjected a strain of Aedes aegypti known for its exceptional ability to develop resistance to controlled permethrin and lambda-cyhalothrin insecticides pressure using two different concentrations. We analyzed resistance mechanisms that are enhanced at each concentration and used RNA sequencing to identify transcripts specifically associated with these exposure levels. Our objective was to uncover the molecular mechanisms triggered by different insecticide concentrations and to distinguish responses between type I and type II pyrethroids, which differ in chemical structure. Our results showed that kdr mutations confer only moderate levels of resistance, as do detoxifying enzymes. For lambda-cyhalothrin, we identified genes involved in the electron transport chain, mitochondrial function, and overall responses to oxidative stress. tRNA transcripts were also upregulated, along with mitochondrial and stress-response transcripts, suggesting a metabolic shift, particularly toward maintaining homeostasis under oxidative stress. These changes point to mechanisms that sustain resistance to this type II insecticide beyond direct detoxification in this population. On the contrary, permethrin induced marked overexpression of cuticle genes, CYP450 genes (especially CYP4), and Odorant Binding Proteins. These expression patterns, together with metabolic enzymes, point to detoxification, reduced penetration, or even sequestration of insecticide, all of which intensify with increasing concentrations. This overregulation of genes suggests an integrated response complemented by classical metabolic detoxification and accompanied by overregulation of mitochondrial complexes. We showed that despite the shared mode of action of the insecticides permethrin and lambda-cyhalothrin, they elicit distinct responses in this Ae. aegypti population. We also showed that the transcriptomic response depends on insecticide concentration and may modulate insecticide tolerance. This article advances understanding of the complexity of pyrethroid resistance in Aedes aegypti and underscores the importance of considering both the insecticide type and the concentration used in vector control programs. Author summaryAedes aegypti mosquitoes transmit dengue and other arboviruses, being a major public health problem in tropical regions like Colombia, where control relies on pyrethroid insecticide spraying. Based on reports of inconsistent results in the field due to different effects of insecticide concentrations, we recreated variable doses by exposing a resistant Colombian Aedes aegypti strain to low (LC25) and high (LC75) concentrations of permethrin (type I) and lambda-cyhalothrin (type II) to identify concentration-dependent resistance mechanisms. Using genetic mutation analysis, enzyme activity assays, and RNA sequencing, we identified the molecular mechanisms these mosquitoes use to survive. Knockdown resistance (kdr) and detoxification enzymes contributed to some extent to resistance but varied by insecticide type and concentration. RNAseq identified that lambda-cyhalothrin upregulated genes for mitochondrial energy production, oxidative stress defense, immune signaling, and transfer RNAs, facilitating homeostasis under chemical stressors. Permethrin instead upregulated genes for cuticle thickening, cytochrome P450 enzymes, and odorant-binding proteins, which are associated with improved penetration barriers, and metabolic breakdown that intensified with higher concentrations. This reveals pyrethroid resistance as complex beyond classic mechanisms, as even low field doses favor stress tolerance or physical defenses to evade sprays. We detected transcripts that improve survival at high concentrations and could be selected in these mosquitoes. Carefully selecting the type of pyrethroid to be used and the dose should be an important factor in vector control. This optimizes current interventions, prolongs their efficacy, and aids researchers in modeling resistance to protect communities.

BackgroundTreatment response in canine leishmaniosis is driven by the dog host, the Leishmania parasite, and pharmacological factors, with drug resistance increasingly undermining the effectiveness of therapy. A direct quantitative PCR test (LeishGenR) was applied to 104 clinical samples from 95 dogs in the Mediterranean area diagnosed with leishmaniosis in veterinary clinical settings and testing positive for Leishmania infantum by PCR. The assay enabled rapid detection of genetic drug-resistance biomarkers for allopurinol (metk), meglumine antimoniate (mrpa), and miltefosine (LdMT), providing a clinically relevant, timely alternative to culture-based approaches by directly analyzing circulating Leishmania infantum amastigotes. ResultsThe assay (LeishGenR) showed high specificity (100%) and sensitivity (>87.5%) for genetic drug-resistance profile assignment and a strong correlation with whole-genome sequencing for gene copy number assessment (metk: r = 0.878; mrpa: r = 0.943 and LdMT = 0.691). Genetic drug-resistance biomarkers were detected in 24.3% of L. infantum DNA from clinical samples analyzed (20/82; 95% CI 16.3-34.6)), most commonly for allopurinol (13.4%; 95% CI 7.6-22.4), then meglumine antimoniate (9.4%; 95% CI 4.6-18.2), and for miltefosine (5.4%; 95% CI 1.8-14.8). Prevalence was higher in dogs previously treated for leishmaniosis. ConclusionThis study demonstrates the ability to detect genetic biomarkers of drug resistance in L. infantum directly from clinical samples of dogs with leishmaniosis. This method enables rapid, precise detection of genomic biomarkers, circumventing delays associated with culture-based methods and supporting more effective clinical management and surveillance. Among dogs with high parasitemia referred to clinics in Mediterranean regions sampled in this study, the findings reveal a significant prevalence of circulating L. infantum strains carrying genomic drug resistance biomarkers to standard treatments for canine leishmaniosis.

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

BackgroundIn 2018, Samoa was the first country to distribute nationwide triple-drug mass drug administration (MDA) for lymphatic filariasis (LF) elimination. Prevalence of filarial antigen (Ag) is the main programmatic indicator used to define elimination target thresholds; however, anti-filarial antibodies (Ab) may provide more sensitive measures of transmission compared Ag. We aimed to investigate the utility of Ag and Ab to measure the impact of one round of triple-drug MDA on LF transmission in Samoa after 7-9 months. MethodsTwo community-based cross-sectional serosurveys of [&ge;]5-year-olds were conducted in 2018 (1-3 months post-MDA) and 2019 (7-9 months post-MDA) in 35 primary sampling units. Ag was detected by Alere Filariasis Test Strips (FTS). Multiplex bead assays (MBA) were used to detect Ab-seropositivity (Bm14 Ab, Wb123 Ab) using Ab-specific mean fluorescence intensity minus background (MFI-bg). Thresholds of seropositivity were determined using finite mixture models (FMM) for log-transformed MFI-bg, defined as the mean of the presumed seronegative class plus three standard deviations. Seroprevalence was adjusted for study design, age, and gender. FindingsA total of 3795 participants (mean age: 20.7; 49% male) were surveyed in 2018 and 4052 (mean age: 20.4; 48% male) in 2019. Between surveys, adjusted Bm14 Ab prevalence decreased (31.2% [95% CI=27.0-35.8] vs 26.3% [95% CI=22.5-30.4]; p=0.02). In 2019 vs 2018, lower odds of Bm14 Ab-positivity (aOR=0.76 [0.64-0.90]), Wb123 Ab-positivity (aOR=0.70 [0.55-0.89]), and dual positivity (aOR=0.68 [0.54-0.87]) were seen. Ag-positive, microfilariae (Mf)-positive participants had significantly higher mean MFI-bg for Wb123 Ab and Bm14 Ab compared to Ag-positive, Mf-negative participants. ConclusionSignificant reductions in Bm14 Ab seroprevalence suggest reduced LF transmission following one round of triple-drug MDA. Thus Abs may provide a more sensitive indicator of change compared to Ag. High MFI values could help identify persistent transmission in the absence of Ag testing. Author summaryLymphatic filariasis is a mosquito-borne disease that can cause lifelong disability and stigma. The main global strategy to eliminate this disease is to treat entire at-risk communities with preventive medicines, aiming to stop transmission altogether. Progress is usually measured using rapid tests that detect parasite antigens in blood. However, as infection becomes rarer, these tests may miss remaining cases, raising concerns that ongoing transmission could go undetected. In this study, we examined whether measuring antibodies in blood could provide earlier or more sensitive signals of infection following treatment. We analysed blood samples collected from communities in Samoa at two time points after a nationwide treatment campaign using a three-drug combination. We found clear reductions in some antibodies over time, even though antigen levels remained largely unchanged. People with active infection had much higher antibody levels than those without, suggesting antibodies may help identify areas where transmission is still occurring. Our findings show that antibody testing can add valuable information to current monitoring approaches and may help programmes better judge whether treatment is working. This is especially important at a time when funding for disease-specific surveillance is limited, and integrated approaches are increasingly needed to protect elimination gains.

There is empirical evidence that biophysical factors determine the spatio-temporal distribution of mosquito vectors, and identifying the variables that shape their ecology allows decision-makers to design effective surveillance and control strategies. This study evaluated the spatiotemporal distribution of Aedes albopictus in relation to environmental and biotic variables in the Iguazu Department, Misiones Province, Argentina, within the tri-border region shared with Brazil and Paraguay. Environmental characterization integrated field data and remotely sensed biophysical variables, and vector occurrence was analyzed at micro- and meso-spatial scales using generalized linear mixed models. Eleven sampling sessions were conducted between April 2019 and February 2020 at 81 sites representing urban, periurban, and wild environments. A total of 1,614 Ae. albopictus and 4,358 Ae. aegypti specimens were identified. Rainfall, minimum temperature, exposure days, and land cover were the main predictors of Ae. albopictus presence, showing nonlinear responses to precipitation and vegetation. The selected model explained 67% of the variance. The species exhibited clear spatiotemporal stratification, with periurban and disturbed wild areas functioning as ecotones favorable to its establishment. These findings provide key insights to guide preventive actions and strengthen integrated vector management strategies in the region.

BackgroundOnchocerciasis elimination programs increasingly rely on tests that detect antibodies to the Ov16 antigen, yet the performance of currently available rapid tests remains uncertain. The existing Ov16 rapid test shows inconsistent sensitivity when used on whole blood and does not consistently meet international thresholds for mapping or stopping mass drug administration. Two new Ov16 rapid tests have recently been developed, but their accuracy has not been fully evaluated using methods that account for the lack of a true reference standard. Methodology/Principal FindingsWe pooled data from three field studies conducted in Mozambique, Ghana, and Benin in 2023 to evaluate two novel Ov16 rapid tests alongside the existing test. A Bayesian latent class model was used to estimate sensitivity and specificity without assuming that any test was perfect. Across all model specifications, the GADx rapid test showed the highest sensitivity, consistently exceeding the 89% threshold recommended for decisions to stop mass drug administration (posterior medians: 92.0%-92.8%). The SD Bioline test met this threshold in most models but showed lower sensitivity in one model (83.2%). The DDTD test demonstrated lower sensitivity overall (86.6%-88.4%). Specificity estimates were uniformly high for all tests but remained below the recommended threshold of 99.8% (highest median: 98.8%). Alternative definitions of a positive result for the multi-antigen DDTD test did not materially change its performance. Conclusions/SignificanceThe two novel rapid tests showed improved sensitivity relative to the existing test, particularly the GADx test. However, none of the tests achieved the specificity level needed to support decisions to stop mass drug administration on their own. These findings highlight the need for improved diagnostic tools or confirmatory testing strategies as countries work toward onchocerciasis elimination. Author SummaryIn this study, we evaluated three rapid tests used to detect exposure to the parasite that causes river blindness. These tests are important tools for country programs that are trying to eliminate the disease and decide when to stop giving communities preventive treatment. One challenge is that there is no perfect test to compare new tools against. To address this, we combined data from studies in Mozambique, Ghana, and Benin and used a statistical approach that estimates test accuracy without relying on a single reference test. We found that one of the new tests, made by GADx, identified people with exposure more accurately than the others. The existing test and the DDTD test showed lower accuracy. All three tests correctly identified most people who were not exposed, but not at the very high level needed for programs to confidently stop treatment. Our results show that while new rapid tests are improving, more work is needed to create tools that are accurate enough for elimination decisions. This information can help guide public health programs as they work toward ending river blindness.

Three molecularly undescribed filarial species were co-detected, while screening animals for Brugia malayi, the agent of lymphatic filariasis. Single microfilariae (Mf) isolated from blood samples of crab-eating macaques (Macaca fascicularis) from Belitung, Indonesia, and from pet dogs and cats in Sabah, Malaysia, were analyzed. Among 163 macaques, 33 (20.2%) were positive for large Mf (mean length 498.9 {micro}m) similar to Dirofilaria ( Belitung I). One macaque was infected with small Mf (mean length 150.4 {micro}m) ( Belitung II), with a high density of 17,150 Mf/mL. In two cats co-infected with B. malayi, Mf of a Dirofilaria species ( Sabah) with an average length of 299.1 {micro}m were detected. Morphometric analysis of Mf showed distinct differences between these three species and other Mf described in the area. Whole genome amplification and genome sequencing of 24 individual Mf enabled phylogenetic analysis of mitochondrial genomes, and analysis of specific mitochondrial and nuclear barcode regions. The three Mf groups formed distinct clusters and did not match any currently available reference sequence. Cluster Belitung I from macaques formed a sister group to all other Dirofilaria. Cluster Belitung II included bird filariae and primate filariae of the genus Mansonella as close relatives. The cluster Sabah formed a monophyletic group with the zoonotic species D. asiatica and Dirofilaria sp. Thailand. DNA of Wolbachia endobacteria was detected in Mf of Belitung I and Sabah, but not in Belitung II. These findings highlight the limited understanding of filarial diversity in macaques and cats in Asia and underscore the need for a more comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites. Author summaryFilarial worms are parasitic nematodes that infect humans and animals and are often transmitted by the same vector mosquito. We identified three molecularly undescribed filarial species while investigating animals as reservoirs for the agent of lymphatic filariasis, Brugia malayi on Belitung Island, Indonesia, and in Sabah, Malaysia. Blood samples were collected from Indonesian macaques and Malaysian pet cats. Out of 163 macaques, 20.2% tested positive for exceptionally large microfilariae (Mf) of an unclassified Dirofilaria-like species (Belitung I). Another filarial species ( Belitung II) with very small Mf, but with a remarkably high density of 17,150 Mf/mL was detected in one macaque. Two cats harbored medium sized Mf of a Dirofilaria species (Sabah). Genetic analysis revealed unique phylogenetic clusters that did not match any reference sequence. Dirofilaria sp. Sabah was closely related to the zoonotic D. asiatica complex, whereas Belitung I clustered as a sister group to Dirofilaria. Belitung II Mf clustered next to but not within the Mansonella spp. cluster. DNA of Wolbachia endobacteria was only detected in Mf of Belitung I and Sabah. These findings highlight the limited understanding of filarial diversity in animals and underscore the need for a comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites.

Ultra-low volume (ULV) insecticide spraying with deltamethrin as the active ingredient is widely used in mosquito control programs, yet its effectiveness against target mosquitoes and its ecological side effects remain poorly quantified under field conditions in Central Europe. Here, we experimentally evaluated the short-term impact of ground ULV spraying on both mosquito populations and non-target flying insects in Hungary using a paired before-after-control-impact (BACI) design. Mosquitoes were sampled with BG Sentinel traps, while non-target insects were collected using malaise traps. ULV treatment resulted in a significant reduction in mosquito abundance at treated sites, with an average decline of approximately 45%. Native and invasive mosquito species, including Aedes albopictus and Aedes koreicus, showed similar proportional decreases. However, treatment effectiveness varied substantially among sites and was influenced by initial mosquito abundance and wind conditions. In parallel, malaise trap samples revealed a marked decline in non-target flying insects, with reductions exceeding 40% across multiple taxonomic groups, particularly among small- and medium-sized insects, and also when considering pollinator taxa together. Our results indicate that while ULV spraying can temporarily reduce mosquito abundance, it also imposes considerable short-term impacts on non-target insect communities, highlighting trade-offs between vector control and insect conservation within mosquito management programs.

Background: Household insecticides are widely used for domestic pest control, yet exposure patterns in traditionally underserved populations remain poorly characterized. In the Brazilian Amazon, data on use patterns among older adults living in riverine communities are particularly scarce. Objective: To describe the prevalence, frequency, duration, application practices, and types of household insecticides used by older adults living in near-urban riverine insular communities in the Brazilian Amazon. Methods: Cross-sectional, population-based door-to-door survey conducted from August 2022 to July 2025 in four islands (Cotijuba, Mosqueiro, Outeiro, and Combu) in the city of Belem, Brazil. All residents aged 60 years or more registered in the primary care system were invited to participate. Trained interviewers administered an in-person standardized questionnaire to participants on current household insecticide use, frequency, duration, self-application, protective equipment, insecticide types, and product brands. Results: Among 1,101 screened individuals, 1,084 were included (median age at evaluation: 68 years). Overall, 78.4% reported current use of household insecticides. Weekly or more frequent use was reported by 58.9%, and 33.4% reported use for more than 5 years. Self-application was common (57.5%), whereas use of protective equipment was rare (8.2%). Aerosol sprays were the most frequently reported type (39.4%). Commonly recalled aerosols contained pyrethroid mixtures including cypermethrin, imiprothrin, prallethrin, and transfluthrin. A substantial proportion of participants reported using unregulated products and veterinary-only insecticides for household purposes. Conclusions: Household insecticide use is highly prevalent and frequent in Amazonian riverine communities, with minimal use of protective equipment and substantial irregular practices, underscoring the need for targeted risk communication and surveillance.

Spatial emanators (SE) are a promising complement to existing tools for preventing mosquito transmitted diseases. In 2025, the WHO updated the WHO Guidelines for Malaria to include a conditional recommendation for the indoor use of prequalified SE products in malaria control. Both prequalified, and many other SE products contain the volatile pyrethroid transfluthrin, which shares the same target site as other (contact/solid phase) pyrethroids. Therefore, an assessment of cross resistance is critical to predict effectiveness against mosquitoes with existing pyrethroid resistance. Our results show that resistance to solid phase pyrethroids is correlated with resistance to transfluthrin in Anopheles and Aedes species. Moreover, commonly-selected resistance mechanisms including target site mutations and over-expression of P450 detoxification enzymes can confer resistance to transfluthrin. Furthermore, we show that resistant mosquitoes are less impacted by transfluthrin in terms of flight activation (irritancy) and reduced blood feeding inhibition, with the response correlating with resistance strength. Transfluthrin did not elicit an electroantennography response in Anopheles gambiae and surgically ablating mosquitoes antennae did not result in differences in flight activation upon transfluthrin exposure, suggesting the antennae are not required for transfluthrin to elicit behavioral responses. These results provide new insight regarding the mode of action of transfluthrin and the risk of resistance reducing transfluthrins efficacy in vector control interventions.

Vector-borne diseases represent a growing public health issue nationwide. Nebraska reports a sustained burden of mosquito-borne diseases and expanding tick-borne disease risk. This study aims to assess trends in vector abundance, vector infection rates, and human vector-borne disease reports using retrospective surveillance data and to examine the relationship between vector factors and human risk across the state. Vector abundance and pathogen infection rates were mapped alongside presence and incidence of key vector-borne diseases. Mosquito surveillance and mosquito-borne disease data were available from 2012-2024, while tick surveillance and associated tick-borne disease data were available from 2021-2024. Statistical models and comparative tests were used to explore associations between vector factors and disease reports. Overall, the current vector-borne disease surveillance suggests that mosquito-borne disease remains the primary concern in Nebraska, with notable geographic variation in mosquito species distribution and human cases. Tick surveillance indicates established populations of clinically relevant tick species in distinct regions of the state, with pathogen detections generally aligning with areas where human cases have been reported. Differences in the level of human case reporting and data availability affected interpretation of long-term trends and limited strong conclusions regarding direct relationships between vector factors and human disease. Nebraska maintains comprehensive vector-borne disease surveillance systems that support understandings of vector abundance, vector pathogen infection rates, and human risk. Continued integration of entomological surveillance with human case information may help clarify patterns of vector-borne disease risk and inform efforts to address current and emerging threats in Nebraska.

Male mosquitoes are mass-reared around the globe for use in mosquito control programs like Sterile Insect Technique and Incompatible Insect Technique. During larval development, mosquitoes co-exist with complex microbial communities that serve as food and also form the internal microbiota of the organism. The microbiota can impact multiple larval and adult life history traits including development rate and male body size. In the present study, we investigated how male wing length and adult male longevity is impacted by the addition of a single bacterial isolate to otherwise conventional larval rearing water. Of three isolates tested, we found that larval exposure to one, Cedecea sp., resulted in adult males with significantly reduced wing length and longevity. Our findings suggest that minor modifications of the microbial community during larval development can have life-long effects on mosquitoes which, like many organisms, acquire their microbiota from the environment. Moreover, they suggest that mosquito life history traits can be influenced by small shifts in the microbial rearing community, which could impact the efficacy and efficiency of mosquito mass rearing for vector control.

BackgroundThe malaria transmission potential and the vulnerability of Anopheles mosquitoes to different vector control methods depend, among other factors, on the endophily, endophagy, anthropophagy and survival of each species. Local information on these bionomic parameters is generally unavailable. MethodsTo address this, we estimated species-specific values of these parameters using an augmented version of the global database of bionomics data by Massey et al. (2016). We applied inclusion and exclusion criteria to select eligible studies with relevant experimental designs that minimise bias from collection methods for parous, sac, endophagy, and endophily rates as well as for the resting duration. For the human blood index (HBI), we separated data from indoor and outdoor collections. We fitted hierarchical Bayesian models with levels based on Anopheles taxonomy to estimate these quantities. Based on the estimated bionomics, we quantified the expected vectorial capacity reduction after the introduction of a pyrethroid-pyrrole insecticide-treated net (ITN) for 57 Anopheles species. ResultsWe identified 26 eligible studies for endophagy and 61 for the parous rate, leading to a Bayesian posterior average for the Anopheles genus of 42% (95% credible interval: 18-70) and 55% (32-77) respectively. HBI values widely varied depending on the location of collection, except for some species showing strong anthropophilic behaviours. Resting duration was estimated to be 2.1 days (1.2 - 4.8) at the genus level. Few studies were available to estimate the sac and endophily rates, which prevented us from deriving precise estimates for the whole Anopheles genus. Our estimates of the vectorial capacity reduction following the introduction of a pyrrole-pyrethroid ITN ranged between 48% and 76% across species, highlighting the important differences among mosquito species in vulnerability to vector control interventions. ConclusionThis work demonstrates how data from both Anopheles species complexes and individual species can be leveraged to generate species-specific estimates of bionomic parameters, capturing the local characteristics and behaviour of malaria vectors. The dataset is readily updatable as new data become available. However, more frequent and standardised field surveys are still needed to accurately characterise local vector behaviour.

Insects are associated with diverse RNA viruses, including vertically transmitted viruses that form persistent infections without apparent symptoms. One of the first documented vertically transmitted viruses is sigmavirus (Rhabdoviridae) affecting fitness of Drosophila. Sigmaviruses and related rhabdoviruses have also been detected in pest fruit flies and other arthropods. However, their prevalence, transmission, tissue localisation and fitness effects remain poorly known, despite their potentially common infections in diverse hosts. We investigated Sigmavirus tryoni (BtSV) prevalence, load, transmission across multiple generations and host effects in Queensland fruit fly (Bactrocera tryoni), Australias most significant horticultural pest, which carries BtSV at low prevalence (13.7%) across field populations. We detected BtSV in 6 of 12 laboratory populations (prevalence 12.5% to 80.4%) where it was transmitted biparentally within embryos. Although incomplete, maternal transmission was more reliable and resulted in higher BtSV load than paternal transmission. Paternally transmitted BtSV was almost entirely lost after two generations. BtSV became detectable in most uninfected individuals cohabiting with infected flies, but this resulted in a low load that was subsequently transmitted to only few offspring. BtSV occurred across developmental stages, digestive and reproductive tissues, albeit its viral load was lower in reproductive tissues when received paternally than maternally, and lower in testes than ovaries. Furthermore, BtSV-infected individuals suffered paralysis and mortality when exposed to high CO2 concentrations, a Rhabdoviridae effect previously reported for several Drosophila species, a muscid fly and mosquitoes. Our study suggests that sigmavirus transmission dynamics and fitness effects may apply broadly to arthropod hosts and affect their management.

BackgroundOnchocerciasis remains a public-health challenge in the Democratic Republic of the Congo (DRC). The Kakoi-Koda focus, Ituri Province, exhibited high endemicity in the early 2000s and received community-directed treatment with ivermectin (CDTI) in some health zones (e.g., Nyarambe), but not in others (e.g., Logo). Moxidectin clinical trials were conducted in these health zones, alongside onchocerciasis-associated epilepsy studies. MethodologyWe synthesised epidemiological (including nodule prevalence), entomological and CDTI programmatic data. We collated anti-Ov16 serological data from epilepsy-related studies (community, cohort, case-control designs, 2015-2021) and skin-snip microscopy results from two moxidectin trial screenings (2009-2011; 2021-2023) and epilepsy-related studies (2015-2017). Geospatial analyses were used to describe land-cover change relevant to vector ecology and to identify areas with recent transmission. Principal findingsOnchocerca volvulus transmission declined markedly over time. In CDTI-naive Logo villages, microfilarial prevalence fell from 69-79% (first trial, 2009-2011) to 9% (second trial, 2021-2023), and mean infection intensity from 17-26 to 1 microfilariae per skin snip, similar to declines observed in Nyarambe villages under CDTI (72% to 3% and 11 to 0.4, respectively). Anti-Ov16 seroprevalence among children aged 3-10 years was low (0-5%) from 2016 onwards, and seropositivity was geographically circumscribed, mirroring contemporary skin-snip results. Human landing catches and breeding-site prospections (2015-2017) identified Simulium dentulosum and S. vorax as the current anthropophagic species, with no evidence of S. neavei after 2009. Progressive deforestation and canopy opening provide a plausible mechanism for a shift from crab-associated S. neavei habitats towards more open-habitat vectors. SignificanceConsistent parasitological, serological, entomological and geospatial evidence indicates substantially reduced transmission across Kakoi-Koda, with spatially-circumscribed residual transmission. Whether the current simuliid species can sustain transmission above elimination thresholds remains uncertain. Targeted, integrated surveillance is warranted to guide CDTI and stop-CDTI decisions. The dataset assembled here can be used to inform transmission modelling of these dynamics. Author SummaryOnchocerciasis, also known as river blindness, is a parasitic disease of public health concern in sub-Saharan Africa, transmitted by blackfly vectors. The disease is responsible for skin and eye clinical manifestations and is associated with neurological complications. We investigated an area in north-eastern Democratic Republic of the Congo called the Kakoi-Koda onchocerciasis focus, where the infection was once common. We reviewed and assembled data from past studies on infection in humans and blackflies, and analysed satellite imagery to assess the loss of tree cover that can affect where blackflies live and breed. We found that the prevalence of onchocerciasis in Kakoi-Koda has declined markedly in recent years. This decline appears linked to the disappearance (by deforestation) of the habitat suitable for some blackfly species, and to ivermectin distribution to treat onchocerciasis in parts of the focus. Our findings help to understand why onchocerciasis has decreased across the Kakoi-Koda focus and highlight a small number of fast-flowing river segments where other blackfly species may allow small pockets of local transmission. These results support continued, targeted monitoring to determine whether the disease is still transmitted in specific locations where elimination interventions may be needed.