Acta Tropica

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

This data paper presents a curated, georeferenced dataset of the frequencies of the two main target site mutations (V1016G and F1534C) associated with resistance to pyrethroid insecticides in Aedes albopictus in Italy. Populations were collected in 102 out 107 Italian provinces between 2023 and 2025. Specimens were sampled by members of the Mosquito Insecticide Resistance Italian Network (MosqIRIT) as part of RN2 activities within the INF-ACT project. Genotyping was performed on 3,517 individuals by specific allele-specific PCR assays. Each record includes metadata on sampling site, administrative location, developmental stage, collection method, and mutation-specific genotype frequencies. To support spatial analysis modelling effort, the dataset integrates geographic, eco-climatic, and demographic data. This resource will support mosquito control programs, pyrethroid resistance monitoring and managing, as well as ecological modelling, and is compliant with the FAIR data program.

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

Introduction: Malaria, primarily transmitted by Anopheles mosquitoes, remains a major public health concern in Maiduguri, Borno State, Nigeria. While conventional control methods (e.g., ITNs) face challenges due to insecticide resistance and accessibility constraints, many communities rely on locally sourced natural products. This study aimed to assess the prevalence, usage patterns, and associated factors of these natural alternatives. Methods: A cross-sectional survey was conducted across three purposefully selected communities in Maiduguri (Mairi, Furi, Lagos Street). A total of 450 household heads were interviewed using a structured questionnaire, collecting data on socio-demographics, specific natural products used, method of application, frequency, and perceived efficacy. Data were analyzed using descriptive statistics and binary logistic regression. Results: Overall usage prevalence of natural products was high at 68.4%. The most common products identified were Neem (Azadirachta indica) extract (45.9%) and burnt Lemon Grass (Cymbopogon citratus) (31.2%). Usage pattern was predominantly indoor fumigation (burning), and over 70% of users prepared the products crudely at home. Logistic regression revealed that rural residence (Odds Ratio (OR): 2.1; p<0.01) and low education level (OR: 1.8; p<0.05) were significant independent predictors of higher natural product reliance. Conclusion: Natural products constitute a widely adopted, community-driven vector control method in Borno State. The high prevalence and association with vulnerable populations suggest an urgent need to standardize the preparation and application of these products for potential integration into regional malaria control programs. Keywords: Anopheles, Adulticides, Borno State, Malaria, Natural Repellents, Vector Control, Usage Pattern.

West Nile virus (WNV; Flaviviridae) and Eastern Equine Encephalitis virus (EEEV; Togaviridae) represent the two most significant mosquito-borne zoonoses in the southeastern United States. While both viruses utilize avian amplifying hosts and mosquito vectors, it is unclear if they occur within distinct ecological niches. This study assessed the cumulative effects of both landscape composition and weather variables on the spatial-temporal distribution of WNV and EEEV outbreaks in Georgia over a twenty-four-year period (2001-2025). We used a modeling framework that directly accounts for both spatial and temporal effects but additionally integrates key EO variables (Earth observation). These environmental effects were investigated using Bernoulli generalized linear mixed-effects models (GLMMs) and executed with an integrated nested Laplace approximation (INLA). Our findings revealed disproportional distribution ranges of Culex quinquefasciatus, Aedes albopictus and Culiseta melanura in highly urbanized Georgian counties such as Chatham, Dekalb, and Fulton. Results showed that WNV transmission was heavily influenced by urban land cover (Posterior Mean: +0.78, 95% CrI: [0.61,0.95]) but negatively associated with lagged precipitation (Posterior Mean: -0.18, 95% CrI: [-0.29, -0.07]), confirming drought-driven amplification. Conversely, EEEV transmission was strongly influenced by wetland cover (Posterior Mean: +1.12, 95% CrI: [0.94,1.30]) and precipitation (Posterior Mean: +0.51, 95% CrI: [0.39,0.63]). These results underscore the need for improved mosquito surveillance across all Georgian counties in the face of growing vector-borne disease risks.

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

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

Background Onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus and transmitted by Simulium blackflies, remains a public health challenge in sub-Saharan Africa. Following the transition from vector control to large-scale ivermectin distribution, microfilariae (mf) prevalence has declined substantially across Ghana. The World Health Organization targets onchocerciasis elimination, defined as sustained mf prevalence below 1%. This requires identifying areas of persistent or resurgent transmission to guide targeted mapping surveys and interventions. Methods A Bayesian spatiotemporal, multilevel, generalized additive model with a zero-inflated beta-binomial outcome was fitted to mf prevalence data from 1353 surveys across 671 villages and environmental and socio-economic predictors. Temporal prevalence trends for ecozones within operational transmission zones (OTZ) were modelled using a 2D spatial smooth to account for residual spatial autocorrelation. Temporal autocorrelation in prevalence was accounted for using a random effect for village nested within ecozone. Results Runoff probability and surface wetness were positively associated with prevalence, consistent with Simulium breeding in fast-flowing water. Despite high initial mf prevalence, by 2015 the median prevalence across all OTZs in Ghana was estimated to be between 0.43 and 1.97%. The probability that prevalence was <1% in 2015 ranged from 0.15 to 0.87. Assuming testing rates and mass drug administration have remained consistent (twice a year and >80% coverage), linear extrapolation predicted that the three OTZs have some probability of exceeding the elimination threshold by 2030, while two were predicted to have near-zero prevalence. The Tano-Ankobra OTZ, which includes both savanna and forest agro-ecological zones, was a persistent hotspot. Conclusions This first comprehensive spatio-temporal analysis of onchocerciasis in Ghana reflects the well-documented challenge of onchocerciasis control in forested riverine environments. Persistent transmission foci in eastern and southern Ghana, particularly in forest zones with sustained vector habitat, warrant targeted investigation and intensified intervention to meet WHO elimination thresholds.

Ivermectin is an important broad-spectrum anthelmintic used to treat nematode parasites including gastro-intestinal infections of humans and animals. The mode of action for Ivermectin is understood to involve activation of inhibitory glutamate-gated chloride channels (GluCls). Ivermectin has also been reported to inhibit the release of extracellular vesicles (EVs). We found that EVs are released from the whole intestine of the gastro-intestinal parasite, Ascaris suum. Proteomic analysis identified 1,574 proteins within these intestinal EVs, including 96 nematode proteins with putative immune-associated functions based on homology to proteins involved in host immune processes and 130 proteins with predicted digestive functions. Comparative analysis following ivermectin exposure revealed 38 differentially abundant proteins that included the putative immune-related proteins: transthyretin-like proteins, a small heat-shock antigen, a phospholipase A2, and the NF-{kappa}B subunit p105. Thus, ivermectin modulated the potential immune-related cargo of intestinal EVs. The ivermectin inhibition of intestinal EV release was concentration-dependent with an IC50 of 64 nM. We also identified the expression of GluCl subunit receptor genes in the Ascaris intestine. The potent inhibitory effect of ivermectin on the release of these EVs from the nematode intestine and the expression of GluCl channel subunits sheds further light on the site and mechanisms of action of this important anthelmintic.

Beef cattle farming plays a crucial role in the livestock-related economies of both Italy and Argentina. Although both countries have grazing systems of meat production, the heterogeneity of those systems leads to different parasitological scenarios regarding epidemiology and the presence of resistance to commonly used anthelmintic drugs. Considering ivermectin (IVM) is the most widely used anthelmintic for treating gastrointestinal nematode (GIN) infections in cattle, the current studies evaluated the efficacy of ivermectin (IVM) given subcutaneously (SC) to both young calves and adult cows in ten (10) commercial cattle farms from Italy (A to E) and Argentina (F to J). The work was complemented with the assessment of the IVM plasma exposure and disposition kinetics both in young and adult animals. While adult cows and young calves were included in the study performed in Italy (Campania region), only calves aged 8-14 months old were selected to perform the efficacy trials in Argentina (central area of the Buenos Aires province). Fifteen (15) calves/cows naturally infected with GINs were treated with IVM (0.2 mg/kg) in each of the Farms. The therapeutic response (efficacy) was determined at 14 days after treatment by the faecal egg count reduction test. Six (6) adult cows and eight (8) young calves treated with IVM were randomly selected to perform the pharmacokinetic (PK) study with blood samples being taken 2 h and 21 days post-treatment. Drug concentrations were measured by HPLC. Similar IVM PK trends were obtained for both adult and young cattle. The IVM systemic exposure (expressed as AUC) obtained for adult cattle (380{+/-}158 ng.d/mL) was similar to that observed in calves group (313{+/-}85.5 ng.d/mL). No statistical differences between animals from both ages were observed for the most representative PK parameters (P>0.05). Surprisingly, while in Argentina IVM resistance was present in all the farms evaluated (90% CI = 0%-95%), in Italy a fully susceptible nematode population was presented in three farms (90% CI = 96%-100%) and only a very low level of IVM resistance was detected in the rest of the farms (90% CI = 92%-98%). Whereas Cooperia spp. and Haemonchus spp. were identified as the principal genera exhibiting resistance to IVM in Argentina, Cooperia spp., Ostertagia spp., and Oesophagostomum spp. were reported to display low levels of resistance under the Italian field scenario. In conclusion, IVM can be safely and effectively used in both young and adult cattle with similar disposition kinetic patterns. While its therapeutic response is failing under the conditions investigated in the Argentinian farms, its efficacy remains notably high in the Italian region under study. However, the use of IVM, as well any other active antiparasitic principle, should always be preceded by a diagnostic assessment of the nematode populations resistance status.

Locally acquired arboviral infections are an increasing public health concern in the United States. Aedes aegypti and Culex quinquefasciatus, vectors of dengue virus and West Nile virus, respectively, are established in regions where local transmission has been reported. Seasonal variation in mosquito abundance affects vector density, human-vector contact, and arbovirus transmission risk. The aim of this study is to investigate seasonal variations in Ae. aegypti and Cx. quinquefasciatus population dynamics in Miami-Dade County, Florida; Maricopa County, Arizona; and Clark County, Nevada. Monthly relative abundance, average mosquitoes collected per trap-night, normalized abundance ratios, and seasonal-trend decomposition were used to evaluate species- and location-specific seasonal patterns. Mosquito population dynamics differed by species and location. In Miami-Dade County, the two species showed seasonal turnover, with Cx. quinquefasciatus peaking during winter and spring and Ae. aegypti peaking during summer. In Maricopa and Clark counties, both species peaked primarily between August and September. Species dominance also differed by site, with Ae. aegypti more abundant than Cx. quinquefasciatus in Maricopa County and Cx. quinquefasciatus more abundant than Ae. aegypti in Clark and Miami-Dade counties. Seasonal decomposition showed that Ae. aegypti peaked earlier in Miami-Dade County than in Maricopa and Clark counties, whereas Cx. quinquefasciatus showed a spring peak in Miami-Dade County and bimodal seasonal patterns in Maricopa and Clark counties. These results suggest that mosquito population dynamics are species- and location-specific and support the use of local surveillance data to guide the timing of mosquito control and arbovirus preparedness.

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

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

Malaria remains one of the most pressing health problems, especially in Sub-Saharan Africa. Various antimalarial drugs, used to combat this debilitating illness, may directly or indirectly affect blood indices in humans. This study aims to evaluate the toxicological effects of sequential administration of Artemether-lumefantrine and sulfadoxine-pyrimethamine in male Wistar rats. Thirty (30) mature male Albino Wistar rats weighing between 190-280g were randomly divided into five groups comprising six (6) rats each. Group 1 served as control, Group 2 received Artemether-lumefantrine (8 mg/kg/bw) for 3 days, Group 3 received sulfadoxine-pyrimethamine (0.079 mg/kg/bw) for 1 day, Group 4 received a sequential dose of Artemether-Lumefantrine for 3days and sulfadoxine-pyrimethamine for 1 day, while Group 5 received a sequential dose of SP for 1 day and AL for 3 days. Sequential administration of AL and SP resulted in a significant (p < 0.05) elevation of ALT, AST, ALP, serum total and direct bilirubin levels, urea, creatinine, and HDL. There was a significant (pL0.05) decrease in the serum total protein and albumin. Notably, HDL levels increased significantly in the SP [-&gt;] AL group (p < 0.05), while other lipid parameters showed sequence-specific significant changes compared to the control. Sequential administration, particularly the SP [-&gt;] AL sequence, was observed to have more pronounced effects on hepatorenal biomarkers compared to independent administration. These findings are relevant, especially in malaria-endemic regions where unregulated self-medication and drug switching are rampant.

Larval source management (LSM) is a complementary malaria control intervention, yet evidence to guide context-specific implementation remains limited. Nigeria's recent national commitment to LSM scale-up makes the need for operational evidence particularly urgent. Informal settlements embedded within wards of differing dominant settlement archetypes may present distinct Anopheles larval habitat profiles with implications for how LSM strategies should be tailored. We evaluated Anopheles larval habitats within informal settlement areas across wards with contrasting settlement archetypes in Ibadan metropolis, Nigeria, to inform targeted larval source management. Potential breeding habitats were surveyed in dry and wet seasons within informal settlement areas across three wards -- Olopomewa, Challenge, and Agugu -- representing formal, informal, and slum settlement-dominant archetypes respectively. Habitats were characterized and assessed for Anopheles larval presence. Pareto analysis identified habitats accounting for 80% of larval abundance. Breeding habitat density per km{superscript 2} was estimated using a simulated pathway technique. Associations between mosquito dispersal scale and household malaria infections identified through Rapid Diagnostic Testing were evaluated using kernel-based distance-decay weighting. Environmental drivers of habitat suitability were modeled in MaxEnt. Of 420 potential breeding habitats identified, 31 (7.4%) contained Anopheles larvae, predominantly during the wet season (26, 83.9%). Puddles, dug wells, drainages/gutters/ditches and canals accounted for 80% of site-level larval abundance when standardized by sampling effort. Larval and breeding habitat density were highest in Agugu, the slum-dominant ward, across both seasons. Modeled mosquito dispersal scale showed best fit at 30-32m in Challenge (OR 1.41, 95% CI: 1.05-1.89) during the wet season and 16-18m in Agugu (OR 1.29, 95% CI: 1.04-1.60) during the dry season. Habitat suitability in Agugu was higher farther from large water bodies and in areas with higher population density and positive Normalized Difference Water Index values. In Challenge, suitability was higher in areas with lower nighttime light levels, positive Normalized Difference Water Index values, and negative Normalized Difference Moisture Index values. Further studies incorporating multiple wards across diverse urban settings are needed to determine whether differences in larval ecology between settlement archetypes provide a reliable basis for planning larval source management.

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

While emerging diseases pose a global threat to amphibians, the dynamics of understudied vector-borne blood pathogens remain poorly understood. Pathogen occurrence is driven by a combination of environmental, ecological, and phylogenetic factors, yet how these drivers shape blood pathogen communities in tropical amphibians is largely unknown. In this study, we used molecular screening and phylogenetic linear models (PGLMMs) to evaluate how climate and ecomorphology influence the incidence of three blood pathogen groups (Trypanosomatidae, Hepatozoon, and Rickettsia) in wild anurans from a protected area in the Brazilian Atlantic Forest. Among 93 individuals sampled, over 93% were infected with at least one pathogen. Trypanosomatidae was the most common (76.3%), followed by Rickettsia (69.9%) and Hepatozoon (16.1%). Pathogen responses to temperature were contrasting: Hepatozoon occurrence increased in warmer periods, while Trypanosomatidae declined. Furthermore, rheophilic species showed a lower probability of Rickettsia infection, providing the first evidence that microhabitat use influences blood pathogen dynamics in amphibians. Our findings demonstrate that hemoparasites prevalence is driven by a multifaceted interplay of variables, highlighting that conservation strategies must account for these pathogen-specific responses to habitat use and environmental change, even within protected areas.

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

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

BackgroundAedes albopictus is among the worlds most invasive mosquito species and an important vector of dengue, Zika, and chikungunya viruses. Its global spread has been strongly associated with human-mediated transport and international trade, particularly through commodities such as used tires and ornamental plants. However, integrative studies combining population genetics, microbial symbiosis, and trade connectivity remain limited in Latin America, constraining understanding of invasion dynamics and dispersal processes. MethodsAedes albopictus populations from five Colombian departments sampled between 2019 and 2023 were analyzed using eight microsatellite loci and a [~]1.3-kb mitochondrial COI fragment. Wolbachia infection and lineage composition (wAlbA/wAlbB) were evaluated by PCR, and arbovirus detection (DENV, CHIKV, ZIKV) was performed using multiplex RT-PCR. Nuclear and mitochondrial differentiation (FST, {Phi}ST), mito-nuclear discordance, and trade-related connectivity were evaluated in relation to geographic, national transport, and international trade variables derived from customs databases. ResultsMicrosatellite analyses revealed admixed but structured populations consistent with multiple introductions and contemporary gene flow. Colombian populations showed nuclear genetic affinities with Asian, European, and North American populations, supporting a complex invasion history involving multiple geographically distributed lineages. In contrast, mitochondrial COI data showed evidence of regional genetic structure and heterogeneous mito-nuclear discordance among several population pairs. Notably, nuclear and mitochondrial markers captured contrasting dimensions of the invasion process: nuclear differentiation was positively associated with international trade intensity, particularly shipment frequency and used tire importation, whereas mitochondrial differentiation retained stronger regional structure and showed no detectable association with trade-related variables. Wolbachia prevalence ranged from 34% to 100% across departments and showed exploratory patterns consistent with localized mitochondrial differentiation. Natural detection of DENV and CHIKV RNA in larvae provided evidence of local arbovirus circulation. ConclusionsIntegrating population genetics, trade connectivity, and Wolbachia screening supports a scenario in which the Colombian invasion of Ae. albopictus has been shaped by multiple introductions, contemporary human-mediated connectivity, and partially discordant mito-nuclear histories. These findings highlight how different genomic compartments retain complementary signatures of invasion dynamics, with contemporary trade-associated connectivity primarily reflected in nuclear structure and regional lineage persistence retained in mitochondrial variation. Author SummaryThe Asian tiger mosquito, Aedes albopictus, is one of the worlds most invasive mosquito vectors and continues to expand across Latin America through human transportation and trade networks. However, the processes shaping its spread in the region remain poorly understood. We combined population genetics, international trade data, Wolbachia screening, and arbovirus surveillance to investigate the invasion dynamics of Ae. albopictus in Colombia. Our results revealed evidence of multiple introductions and ongoing genetic admixture, with international trade connectivity emerging as an important predictor of contemporary nuclear genetic structure. In contrast, mitochondrial DNA retained stronger regional patterns, generating heterogeneous mito-nuclear discordance among populations. These findings suggest that different genomic compartments retain distinct signatures of the invasion process, with trade-associated connectivity reflected primarily in nuclear variation and stronger regional structure preserved in mitochondrial lineages. More broadly, our study highlights the complex invasion dynamics of Ae. albopictus in Latin America illustrates how integrating genetics and human connectivity data can improve understanding of invasive vector spread.