Parasites & Vectors

Malaria is transmitted when Anopheline mosquitoes ingest Plasmodium parasites during blood-feeding. Artificial feeding assays allow mosquitoes to take up blood from membrane feeders, and are widely used to study malaria transmission. These assays require large quantities of mosquitoes; insectaries optimize their rearing procedures to generate high yields of permissive, homogeneous mosquito populations. Rearing of Anopheline stephensi mosquitoes was protocolized at the Radboudumc in the 1980s, yet infection outcomes remain heterogeneous. This study explores possible improvements in mosquito rearing to improve homogeneity of the resulting mosquito populations. It compares the current mass-rearing standard with an adapted alternative approach from another institute that optimizes larval density per tray and applies a diet that has previously been reported. Differences between procedures were assessed by measuring mosquito size by proxy of wing-length and imbibed blood meal volume. To assess receptiveness to P. falciparum infection mosquitoes were fed cultured gametocytes. We observed a slight decrease in mosquito size when applying the alternative rearing procedure, but generated equally parasite-receptive P. falciparum mosquitoes compared to the standard procedure. We conclude that both rearing protocols can be used to generate susceptible mosquitoes for conducting malaria research.

BackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, resulting in worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 {degrees}C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi infected Ae. aegypti. ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produce emerging third-stage larvae. Once D. immitis third-stage larvae emerge at 13 days post infection, the proportion of mosquitoes producing them, and the number produced per mosquito remain stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 days post infection. Increased uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae. ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious L3 for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

In extensive surveillance programmes of Culicoides biting midges (Diptera: Ceratopogonidae), morphological identification can be time-consuming and difficult, while DNA barcoding, although highly accurate, may not be cost-effective or suitable for rapid analysis, as it requires individual specimen processing. To address these limitations, we developed a rapid screening method using real-time PCR assays with either SYBR Green or hydrolysis probe-based detection chemistries. Species-specific primers and, where necessary, hydrolysis probes were designed based on the updated sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assays were validated both in silico and through real-time PCR testing on target and non-target Culicoides species, tested individually and as mixed-species samples. The new real-time PCR assays detect vector species including C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi, C. pulicaris, C. punctatus, and C. impunctatus in pools of individual specimens, with single-specimen sensitivity. The molecular techniques developed in this study provide a valuable tool for accurate and high-throughput Culicoides surveillance, which can be used for year-round monitoring of adult midges in traps and larvae in environmental samples, potentially revealing novel insights into the spatial and temporal turnover of Culicoides species. These methods can be applied to large-scale vector screening programmes involving pooled samples, addressing the limitations of previously described methods used in midge surveillance.

Treatment of livestock with endectocides such as ivermectin is viewed as a complementary vector control approach to address residual transmission of malaria. However, efficacy of this treatment may vary between animal species. Hence, our purpose was to investigate the effects of ivermectin treatments of common livestock species on life history traits of the opportunistic malaria vector Anopheles coluzzii. Sheep, goats and pigs were treated using injectable veterinary ivermectin formulation at the species-specific doses (recommended dose for all species and high dose in pig). Mosquito batches were exposed to treated and control (not injected) animals at different days after treatment. Daily mosquito mortality was recorded and fecundity assessed through the count of gravid females and the number of eggs they developed. The recommended dose of ivermectin induced a significant decrease in mosquito survival for up to 7 days after injection (DAI), with a decrease of 89.7%, 66.7%, and 48.4% in treated pigs, goats and sheep, respectively, compared to control animals. In treated pigs, the triple therapeutic dose decreased mosquito survival of 68.97% relatively to controls up to 14 DAI. The average number in gravid females Anopheles that survived after feeding on treated animals were reduced when blood-meals were taken on sheep (2.57% and 42.03% at 2 and 7 DAI), or on goats (decrease of the 28.28% and 73.64% respectively at 2 and 7 DAI). This study shows that ivermectin treatments to animals negatively impacts An. coluzzii life history traits and could reduce vector densities in areas where livestock live near humans. However, due to short-term efficacy of single dose treatments, repeated treatments and potentially increased dosages would be required to span the transmission season. The use of long-acting ivermectin formulations is discussed as a mean for extending efficacy while remaining cost effective.

Flies are among the first insects to arrive at a corpse to lay eggs. The developing larvae can be used to determine post-mortem interval (PMI) using species specific growth models. The larvae can be difficult to identify due to minute morphological characteristics and rearing larvae into adults is time consuming and may be unsuccessful. We investigated the potential of coupling Environmental Scanning Electron Microscopy (ESEM) and DNA barcoding to enable the acquisition of high resolution images while obtaining DNA in a minimally destructive manner. This study focused on the widespread and forensically relevant species Chrysomya rufifacies as a model. Specimens were prepared in three different drying treatments: unaltered, dried, dried and imaged. Following the imaging process, three different DNA extraction methods with different levels of sample destruction were tested. ESEM imaging showed that there were no visual differences between specimens that underwent drying treatments and ones that were not dried. No significant differences in DNA yield were observed for drying treatments and extraction methods post extraction. However, extraction methods demonstrated significant differences in yield of PCR products. Sequenced PCR products aligned with 100% identity to the C. rufifacies mitochondrial genome reference sequence. Lower nucleotide identities between the sequenced PCR products and other forensically relevant fly species demonstrated that combining ESEM and DNA barcoding is a promising approach to rapid and reliable identification of insect larvae recovered from forensic samples.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to address a lack of knowledge of lancet flukes in the Himalaya ranges of Pakistan by characterising Dicrocoelium species collected from the Chitral valley. The morphology of 48 flukes belonging to eight host populations was examined in detail and according to published keys, they were identified as either D. dendriticum or Dicrocoelium chinensis. PCR and sequencing of fragments of ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) and NADH dehydrogenase-1 (ND-1) mitochondrial DNA from 34, 14 and 3 flukes revealed 10, 4 and 1 unique haplotypes, respectively. Single nucleotide polymorphisms in these haplotypes were used to differentiate between D. chinensis and D. dendriticum, and confirm the molecular species identity of each of the lancet flukes as D. dendriticum. Phylogenetic comparison of the D. dendriticum rDNA, COX-1 and ND-1 sequences with those from D. chinensis, Fasciola hepatica and Fasciola gigantica species was performed to assess within and between species variation and validate the use of species-specific markers for D. dendriticum. Genetic variations between D. dendriticum populations derived from different locations in the Himalaya ranges of Pakistan illustrate the potential impact of animal movements on gene flow. This work provides a proof of concept for the validation of species-specific D. dendriticum markers and is the first molecular confirmation of this parasite species from the Himalaya ranges of Pakistan. The characterisation of this parasite will allow research questions to be addressed on its ecology, biological diversity, and epidemiology.

BackgroundMicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the hosts miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod hosts miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. MethodsSmall RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. ResultsThe sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. ConclusionsThe current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

AbstractsO_ST_ABSBackgroundC_ST_ABSDevelopment of insecticide resistance (IR) in sand fly populations is an upcoming issue of public health concern, threatening leishmaniasis mitigation efforts by insecticide- based vector control. There is a major knowledge gap in the IR status of wild populations worldwide, possibly attributed to the unavailability of specialized tools, such as bioassay protocols, species baseline susceptibility to insecticides and molecular markers, to monitor such phenomena in sand flies. Methodology/ Principal findingsSeveral sand fly populations from (semi-)rural regions of Greece, Turkey and Iran were sampled and identified to species, showing populations structure in accordance with previously reported data. Genotyping of known pyrethroid resistance-associated loci revealed the occurrence of voltage-gated sodium channel (vgsc) mutations in all surveyed countries. Knock-down resistance (kdr) mutation L1014F was prevalent in Turkish regions and L1014F and L1014S were recorded for the first time in Iran and in Turkey and Greece, respectively, yet in low frequencies. Moreover, CDC bottle bioassays against pyrethroids in mixed species populations from Greece indicated full susceptibility, using though the mosquito discriminating doses. In parallel, we established a novel individual bioassay protocol and applied it comparatively among distinct Phlebotomus species populations, to detect any possible divergent species-specific response to insecticides. Indeed, a significantly different knock-down rate between P. simici and P. perfiliewi was observed upon exposure to deltamethrin. Conclusions/SignificanceIR in sand flies is increasingly reported in leishmaniasis endemic regions, highlighting the necessity to generate additional monitoring tools, that could be implemented in relevant eco-epidemiological settings, in the context of IR management. Our molecular and phenotypic data add to the IR map in a macroarea with otherwise limited data coverage. Author SummaryPhlebotomine sand flies vector leishmaniasis to human and animals, a neglected tropical disease of the (sub-)tropics, displaying an expanding epidemiological tendency towards previously non-endemic regions. As leishmaniasis eradication largely relies on vectors insecticidal control, regular monitoring of insecticide resistance (IR) is a core element of integrated vector management. IR data are limited in sand fly populations worldwide though, among others due to the unavailability of robust molecular diagnostic and phenotyping tools. Here, we aim to characterise the pyrethroid resistance (PyR) status of populations originating from countries of the Mediterranean basin and the Middle East; i.e. Greece, Turkey and Iran. PyR-associated mutations were detected in all three countries, while the populations from Greece exhibited susceptibility upon exposure to deltamethrin in CDC bioassays. We established an individual bioassay protocol to enable IR monitoring in settings with multi- species population structure, and applied it comparatively among three distinct Phlebotomus species, revealing that they respond differently to insecticide treatment. Our study will facilitate systematic surveillance and evidence-based sand fly control in the endemic regions.

BackgroundParasitic helminths are highly diverse and ubiquitously distributed in various environments and hosts. Their vast species diversity renders morphological and molecular species delimitation challenging, due to phenotypic and genotypic variations. Currently used approaches to species delimitation are generally computationally intensive. Here, using genetic distances, we developed and validated a simple and easy-to-use application, Applying taxonomic Boundaries for species Identification (ABIapp), to aid in helminth species delimitation. Methodology/Principal FindingsABIapp uses a database of cut-off genetic distances obtained using the K-means algorithm to determine helminth taxonomic boundaries for ten genetic markers: The nuclear 18S and 28S rRNA genes, ITS1 and 2 regions, and the mitochondrial 12S and 16S rRNA, COI, COII, cytB, and ND1 genes. ABIapp was written in R, and the Shiny framework was used to produce an interactive and user-friendly interface. ABIapp requires just three types of input (genetic distance, genetic marker, helminth group) that are easily generated through basic morphological and molecular analysis. To validate ABIapps accuracy and robustness for use, validation was performed both in silico and with actual specimens. Prior to validation, ABIapps database of genetic distances and species used was increased to broaden the apps applicability. In silico validation was conducted by obtaining 534 genetic distances from 91 publications and inputting these into ABIapp. Using confusion matrices, an overall classification accuracy of 79% was achieved, demonstrating the robustness and accuracy of ABIapp. Using sequences of the 12S, 16S, COI, and 18S rRNA genes obtained from ten representative helminth specimens, an overall classification accuracy of 75% was achieved. Conclusions/SignificanceOur results demonstrate the applicability and robustness of ABIapp for helminth species delimitation using ten common genetic markers. With a user-friendly interface, coupled with minimal and simple data input and robust classification accuracy, ABIapp provides helminth researchers with a convenient tool for helminth species delimitation. Author summarySpecies delimitation of organisms is often an issue of debate, with varying criteria used to determine species boundaries. Helminths are no exception, and their vast species diversity renders species delimitation challenging due to both physical and genetic variations. Moreover, as climate change progresses, helminths are also adapting to the changing environment through morphological and molecular changes. These variations render it challenging for helminthologists to determine whether a particular helminth belongs to the same or a different species. We have developed an application, ABIapp, a simple tool to aid helminth species delimitation using genetic distances; this app is readily available for a wide audience. Encompassing ten genetic markers for the three parasitic helminth groups (nematodes, trematodes, and cestodes), ABIapp uses cut-off genetic distances generated via machine learning to define species boundaries at each taxonomic level. To use ABIapp, just three types of information are needed, requiring only basic morphological and molecular expertise. We validated ABIapp using both mathematically modeled genetic distances and actual specimens and demonstrated a classification accuracy of 79% and 75%, respectively. This new, convenient, and validated application for helminth species delimitation will aid species identification applicable in the fields of helminth taxonomy, disease diagnosis, and biodiversity.

BackgroundSchistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma. The pathogen is transmitted via freshwater snails. These snails indirectly benefit from agricultural pesticides which affect their enemy species. Pesticide exposure of surface waters may thus increase the risk of schistosomiasis transmission unless it also affects the pathogen. MethodologyWe tested the tolerance of the free-swimming infective life stages (miracidia and cercariae) of Schistosoma mansoni to the commonly applied insecticides diazinon and imidacloprid. Additionally, we investigated whether these pesticides decrease the ability of miracidia to infect and further develop as sporocysts within the host snail Biomphalaria pfeifferi. Principal findingsExposure to imidacloprid for 6 and 12 hours immobilized 50% of miracidia at 150 and 16 g/L, respectively (nominal EC50); 50% of cercariae were immobilized at 403 and 284 g/L. Diazinon immobilized 50% of miracidia at 51 and 21 g/L after 6 and 12 hours; 50% of cercariae were immobilized at 25 and 13 g/L. This insecticide tolerance is lower than those of the host snail B. pfeifferi but comparable to those of other commonly tested freshwater invertebrates. Exposure for up to 6 hours decreased the infectivity of miracidia at high sublethal concentrations (48.8 g imidacloprid/L and 10.5 g diazinon/L, i.e. 20 - 33 % of EC50) but not at lower concentrations commonly observed in the field (4.88 g imidacloprid/L and 1.05 g diazinon/L). The development of sporocysts within the snail host was not affected at any of these test concentrations. ConclusionsInsecticides did not affect the performance of S. mansoni at environmentally relevant concentrations. Accordingly, pesticide exposure is likely to increase the risk of schistosomiasis transmission by increasing host snail abundance without affecting the pathogen. Our results illustrate how the ecological side effects of pesticides are linked to human health, emphasizing the need for appropriate mitigation measures. Author summarySchistosomiasis is a major public health problem in 51 countries worldwide. Transmission requires human contact with freshwater snails that act as intermediate hosts, releasing free-swimming life stages of the trematodes. The host snails are highly tolerant to agricultural pesticides used in plant protection products. Pesticides enter freshwaters via drift and runoff, and indirectly foster the spread of host snails via adverse effects on more sensitive competitor and predator species in the water. Increasing the abundance of intermediate hosts raises potential contact with the human definitive host while transmission of the pathogen is not affected. Here we show that pesticides do not affect the ability of the trematode Schistosoma mansoni to infect and develop within its host snail Biomphalaria pfeifferi at environmentally relevant concentrations. Consequently, risk of schistosomiasis increases when pesticide pollution favours the proliferation of snail hosts whilst not negatively affecting the free-living parasites nor their development in their snail hosts. Measures to mitigate pesticide pollution of freshwaters should be a concern in public health programs to sustainably roll back schistosomiasis. Intersectional collaborations are required to bridge the gap between the agricultural and the public health sector in search of sustainable and safe methods of crop production.

Background and aimTo prevent the spread of infectious diseases, successful interventions require early detection. The timing of implementation of preventive measures is crucial, but as outbreaks are hard to anticipate, control efforts often start too late. This applies to mosquito-borne diseases, for which the multifaceted nature of transmission complicates surveillance. Resilience indicators have been studied as a generic, model-free early warning method. However, the large data requirements limit their use in practice. In the present study, we compare the performance of multivariate indicators of resilience, combining the information contained in multiple data sources, to the performance of univariate ones focusing on one single time series. Additionally, by comparing various monitoring scenarios, we aim to find which data sources are the most informative as early warnings. Methods and resultsWest Nile virus was used as a case study due to its complex transmission cycle with different hosts and vectors interacting. A synthetic dataset was generated using a compartmental model under different monitoring scenarios, including data-poor scenarios. Multivariate indicators of resilience relied on different data reduction techniques such as principal component analysis (PCA) and Max Autocorrelation Factor analysis (MAF). Multivariate indicators outperformed univariate ones, especially in data-poor scenarios such as reduced resolution or observation probabilities. This finding held across the different monitoring scenarios investigated. In the explored system, species that were more involved in the transmission cycle or preferred by the mosquitoes were not more informative for early warnings. ImplicationsOverall, these results indicate that combining multiple data sources into multivariate indicators can help overcome the challenges of data requirements for resilience indicators. The final decision should be based on whether the additional effort is worth the gain in prediction performance. Future studies should confirm these findings in real-world data and estimate the sensitivity, specificity, and lead time of multivariate resilience indicators. Author summaryVector-borne diseases (VBD) represent a significant proportion of infectious diseases and are expanding their range every year because of among other things climate change and increasing urbanization. Successful interventions against the spread of VBD requires anticipation. Resilience indicators are a generic, model-free approach to anticipate critical transitions including disease outbreaks, however the large data requirements limit their use in practice. Since the transmission of VBD involves several species interacting with one another, which can be monitored as different data sources. The information contained by these different data sources can be combined to calculate multivariate indicators of resilience, allowing a reduction of the data requirements compared to univariate indicators relying solely on one data source. We found that such multivariate indicators outperformed univariate indicators in data-poor contexts. Multivariate indicators could be used to anticipate not only VBD outbreaks but also other transitions in complex systems such as ecosystems collapse or episodes of chronic diseases. Adapting the surveillance programs to collect the relevant data for multivariate indicators of resilience entails new challenges related to costs, logistic ramifications and coordination of different institutions involved in surveillance.

Culex pipiens complex is an important vector of epizootic and zoonotic pathogens, including West Nile virus. Chicago, Illinois and its suburbs have suffered high incidence of human West Nile virus infections in the past. This makes abatement programs in and around the Chicago area an essential service. The control of Cx. pipiens is often complicated by rapidly evolving resistance to pyrethroids, which are the most widely used chemical class in US mosquito abatement programs. The present study assessed Sumithrin(R) resistance in Cx. pipiens collected from five locations around Cook County, Illinois, neighboring the city limits of Chicago. According to CDC guidelines, samples from all five locations demonstrated some resistance to Sumithrin(R). When assessed with Anvil(R), a formulated product made of Sumithrin(R) synergized with piperonyl butoxide, susceptibility was rescued in mosquitoes from three out of the five locations, suggesting involvement of mixed-function oxidases and/or carboxylesterases in Sumithrin(R) resistance at these locations. Not all locations had susceptibility rescued by Anvil(R), but these locations had relatively low knockdown resistance allele frequencies, suggesting that mechanisms other than knockdown resistance may be involved. Enzyme activities did not reveal any marked trends that could be related back to mortality in the bottle bioassays, which highlights the need for multiple types of assays to infer enzymatic involvement in resistance. Future directions in pyrethroid resistance management in Chicago area Cx. pipiens are discussed.

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.

BackgroundAedes aegypti is an urban mosquito vector of Dengue and other arboviruses. During epidemic periods, pyrethroid insecticides are used for the control of adult mosquitoes; the worldwide distributed resistance to these insecticides is a cause of failures in vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel; point mutations on this channel, called kdr mutations, are associated with pyrethroid resistance. Two kdr mutations, called V1016I and F1534C, augmented in frequency in natural populations of Ae. aegypti from the Americas in the last decade. The diagnostic of kdr polymorphisms allows an early detection of insecticide resistance spreading, which is critical for timely decisions on vector management. Given the relevance of resistance management, high-throughput methods for kdr genotyping are invaluable tools for resistance monitoring programs. These methods should also be cost-effective, to allow regional-scale surveys. Despite the extended presence of Ae. aegypti and the incidence of dengue in Argentina, the presence, abundance and distribution of kdr mutations were not reported in this country up to date. Methodology and findingsWe report a multiplex high-throughput assay based in High Resolution Melting PCR for the simultaneous genotyping of 1016 and 1534 sites in voltage-gated sodium channel gene. We used this method for the study of individual mosquito samples collected in localities which received different selection pressure with pyrethroids. Compared to other genotyping methods, multiplex High Resolution Melting was high-throughput, cost-efficient, sensitive and specific. We demonstrate for the first time the presence of kdr mutations in Argentina in regions under different selection pressure with pyrethroids. Conclusions and SignificanceWe have developed a high-throughput method for the genotyping of alleles associated with pyrethroid resistance in Ae. aegypti from the American continent. The method developed here is comparable in its sensitivity and reliability with other genotyping methods, but reduces costs and running time. It could be incorporated in control campaigns for control the presence and spreading of resistance-associated alleles. We report here for the first time the presence of kdr mutations in distant populations from Argentina, with different epidemiological situations and different history of mosquito control efforts. Authors summaryAedes aegypti is a mosquito vector of viruses such as dengue, causing millions of infections yearly worldwide. Emergence and distribution of insecticide resistance in this mosquito is a challenge for control campaigns. In this context, the implementation of resistance management strategies became a requisite for successful and sustainable mosquito management. To achieve this objective, it is important to count with early genetic markers of resistance, such as kdr mutations, which are associated with pyrethroid resistance phenotype. Kdr markers can be detected by molecular genotyping diagnostic at early stages in the emergence of resistance; this information should be considered in rational design of control campaigns. Here we present a high-throughput cost-effective method to detect kdr mutations in Ae. aegypti. Also, we demonstrate for the first time the presence of kdr mutations in Argentina.

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

BACKGROUNDThe Sierra Nevada de Santa Marta rainforest has diverse fauna due to its position in northern Colombia, a Caribbean region with predominantly tropical, dry, and rainforest ecosystems in which there is a high diversity of mosquito species that may act as arbovirus vectors. OBJECTIVESThe present study reports the molecular characterization of select mosquito species in this rainforest. METHODSManual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. The COI marker was used for species confirmation, and phylogenetic analysis was performed, using the neighbor-joining method, with the Kimura-2-Parameters model. FINDINGSAedes serratus, Psorophora ferox, Johnbelkinia ulopus, Sabethes cyaneus, Wyeomyia aporonoma, Wyeomyia pseudopecten, Wyeomyia ulocoma and Wyeomyia luteoventralis were identified and intra-species variation >2% for most species. MAIN CONCLUSIONSWe report the first records on the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed for identification at the species level, and the corroboration by means of classical taxonomy suggested complementarity of both methods, which may be employed when morphological or molecular data are poor or not available. The genetic and morphological characterization of jungle mosquito populations will help to understand their biology.

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.

Anopheles stephensi is an invasive malaria vector in Africa. To determine the status of the mosquito in Yemen, An. stephensi vector surveillance and molecular confirmation was conducted in Al Hudaydah Governorate in 2021 and 2022. Mosquito larvae were collected in suspected man-made breeding habitats in Ah Dahi and Zabid city in 2021 and 2022, respectively. Mosquitoes morphologically identified as An. stephensi underwent molecular confirmation through PCR assays, sequencing, and phylogenetic analysis of the cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 locus (ITS2). Analysis confirmed An. stephensi identification for the majority of samples (39/41), with two COI haplotypes detected: one newly reported haplotype and one haplotype common to Northeast Ethiopia and Somaliland. No clustering with An. stephensi from the Arabian Peninsula was observed. These findings provide preliminary insight into the diversity of An. stephensi in Yemen and the connection between An. stephensi in Yemen and East Africa.

BackgroundControlling Aedes mosquitoes remains as a primary means through the application of synthetic insecticides in Malaysia, but its efficacy is undermined by the evolution of resistance. Aedes albopictus as one of the dominant and competent arboviral vectors has an elusive insecticide resistance status in different geographical regions of Northern Peninsular Malaysia. This underscores the importance of assessing the diverse types of insecticides used and their association with target-site resistance mechanism in this species, which forms the basis of the present study. MethodsWHO-bioassays were performed on Ae. albopictus larvae and adults from four localities (Penang and Perlis), towards 0.034 ppm temephos, 0.25% permethrin, 0.03% deltamethrin, 0.25% primiphos methyl and 0.1% propoxur. The partial voltage-gated sodium channel (vgsc) gene domain (DIIS6, DIIIS6, and DIVS6) of pyrethroid-exposed samples were subsequently genotyped through direct sequencing for any diagnostic single-nucleotide mutations, together with genetic variations and haplotype networks analysis. The predicted protein structures for the mutated regions and their binding affinities to pyrethroids were also evaluated using in-silico docking. Results and discussionsVarying degrees of resistance were observed in all Penang and Perlis strains to all tested insecticides. Moreover, the detection of the F1534L mutation and newly discovered non-synonymous mutations (A1022S/P, E1041K, P1585R, and F1695L) suggest the progression of resistance alleles dissemination in other strains. The analysis of genetic variations, resistance allele distribution patterns, and haplotype networks showed evidence for multiple origins of these mutations. Data also revealed the discovered mutations affect the affinity of vgsc-binding proteins to pyrethroids. ConclusionThis study highlights the genotype-phenotype associations in Ae. albopictus and their genetic links to pyrethroid resistance, offering insights to strengthen vector control strategies in Malaysia. Author summaryIn Malaysia, as in many other countries, dengue epidemic control primarily relies on managing the main vector, Ae. albopictus through commercially available insecticide applications. Vector control strategies have been extensively implemented by local health authorities, often without comprehensive information on insecticide resistance mechanisms in vector populations that could pose a major drawback of insecticide used. In an effort to assess the susceptibility status of Ae. albopictus populations, and determine the most effective insecticide for reducing Malaysian vector populations from different geographical settings, we conducted bioassays towards pyrethroid, organophosphate, and carbamate insecticide classes. Altogether, molecular-based assays were incorporated with phenotypic assay to elucidate the mechanisms modulating insecticide resistance and to unravel their genetic dynamics. Such screening offers detailed insights into specific mechanisms involved in conferring resistance for distinct adopted insecticides. This evidence aims to guide local health authorities in developing vector control strategies and exploring alternative solutions.

BackgroundDengue is one of the leading causes of morbidity worldwide. Wolbachia-mediated dengue biocontrol has emerged as a novel strategy in recent decades and depends on a lack of Wolbachia in the natural populations of Aedes mosquitoes. Through a systematic review of the published literature, this study sought to estimate the natural occurrence of Wolbachia among field-collected Aedes mosquitoes worldwide. MethodsWe conducted hand and systematic searches from PubMed, the Cochrane Library, and Google Scholar for all relevant published articles on Wolbachia infection in Aedes mosquitoes published before September 12, 2024. The prevalence estimates were analysed using a random effects meta-analysis, and a random effects meta-regression was performed to determine sources of heterogeneity in prevalence estimates. ResultsTwenty-three studies with 16,997 field-collected Aedes mosquitoes from different countries worldwide were included. The meta-analysis revealed a global pooled prevalence of natural Wolbachia infection in Aedes mosquitoes of 57.7% (95% CI: 41.0-72.8%), which was six times higher in Ae. albopictus than Ae. aegypti (p <0.001) and ranged from 6.0% (95% CI: 2.6-13.1%) in Ae. aegypti to 87.1% (95% CI: 78.0-92.8%) in Ae. albopictus. Continentally, Asia had the highest percentage of Wolbachia infection in Ae. aegypti (7.1%), followed by Europe (5.0%), North America (1.9%), and Africa (0.7%). Similarly, Asia had the highest prevalence of Wolbachia in Ae. albopictus (95.5%), followed by Europe (94.8%), North America (91.6%), South America (85.2%), and Africa (71.6%). Overall, dengue incidence was negatively related to Wolbachia prevalence (B = -0.0006, p = 0.0281). Species wise, infection rates in Ae. aegypti were significantly higher among females than males (OR = 1.72; 95% CI = 1.01, 2.92, p = 0.046), while there was no difference between males and females in Ae. albopictus (p = 0.098). Furthermore, Wolbachia infection rates in Ae. albopictus were inversely correlated with dengue incidence ({beta} = -0.0013, p <0.01) but not in Ae. aegypti (p = 0.0984). In contrast, higher temperature was negatively associated with Wolbachia prevalence in Ae. aegypti but not in Ae. albopictus. In contrast, higher temperature was negatively associated with Wolbachia prevalence in Ae. aegypti ({beta} = --2.5736, p <0.001) but not in Ae. albopictus (p = 0.7050). ConclusionAedes mosquitoes had a high and variable prevalence of naturally occurring Wolbachia, and this was negatively correlated with dengue incidence across countries. While the natural infection of Ae. albopictus is more common, detection in Ae. aegypti may reflect contamination and require high-quality multicentre studies to verify the above findings.