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.

Abstract Background Chikungunya re-emerged in Mauritius in 2025 after a 19-year period with no outbreak. As precipitation and temperature play a key role in mosquito proliferation, the climatic conditions prevailing in Mauritius in 2025 may have influenced the emergence and spread of the outbreak. Our aim in this study was to investigate the spatial distribution of the 2025 chikungunya outbreak in Mauritius, and to determine the association between chikungunya incidence and selected predictor variables using Geographic Information Systems (GIS). Methods The variables considered were mean altitude, mean air temperature and mean precipitation, population density, road density, number of houses near rivers and the relative deprivation index (RDI) per village council area (VCA) and municipal ward (MW). Rainfall and temperature data were obtained from the climate bulletins of the Mauritius Meteorological Services and were also extracted from the ERA5 Monthly Averaged Data on Single levels available from the platform of Copernicus Climate Data Store. The U.S. Geological Survey Earth Explorer platform was used to access elevation data for Mauritius. In the absence of publicly available, spatially disaggregated official case data, reported chikungunya cases were reconstructed from multiple publicly accessible sources supplemented by communications with professionals involved in the outbreak. Information was collected contemporaneously as the outbreak evolved during 2025 and was supplemented by searches of archived material. Cases were georeferenced and aggregated to VCAs and MWs. All data was mapped using QGIS version 3.40.13.Page 2 of 17 Results The main findings were that the 2025 chikungunya outbreak was significantly associated with elevation and precipitation. Temporal analysis demonstrated that rainfall in the preceding month was the strongest climatic predictor of monthly chikungunya cases. Spatial analysis, however, showed that mean precipitation had a significant (p-value < 0.001) and negative association with chikungunya incidence, indicating lower disease occurrence in areas of high precipitation. Mean elevation was significantly (p-value < 0.001) and negatively associated with chikungunya incidence, indicating lower disease occurrence in higher-altitude areas. Temperature did not have any significant relationship with chikungunya incidence. Conclusions Rainfall had opposite associations depending on the analytical scale: preceding-month rainfall was positively associated with temporal case variation, whereas spatially averaged precipitation was negatively associated with incidence. Thus, rainfall exerted distinct temporal and spatial influences on chikungunya transmission, while higher elevation was associated with lower chikungunya incidence. These findings highlight the importance of integrating climatic and spatial analyses to improve surveillance Keywords Chikungunya; Elevation; Precipitation; Temperature; Geographical Information Systems; Mauritius.

The need to meet the World Health Organization 2030 target for elimination of lymphatic Filariasis necessitates various activities such as mass administration of medicines (MAM), pre-transmission assessment surveys (pre-TAS)/ epidemiological monitoring survey (EMS) and transmission assessment survey. However for this study, pre-TAS/EMS was conducted to validate elimination milestones and elucidate the lingering immunological consequences of chronic infection. This study conducted a comprehensive evaluation across twelve Local Government Areas (LGAs) in Osun State, screening,7388 individuals for circulating filarial antigen (CFA) using Abbott Filarial Test Strips (FTS), while simultaneously investigating immune modulation in patients with clinical elephantiasis through detailed haematological profiling. Results indicated that the prevalence of the disease at sentinel and spot check sites have been lowered below threshold levels thereby qualifying for a first transmission assessment survey (TAS1), evidenced by a 0% antigenemia rate across all surveyed LGAs, a figure significantly below the World Health Organization's <2% threshold. . The average percentage of individual participation in MDA/MAM programs was found to be 56.09%, whereby, Ola-Oluwa LGA recorded the highest at 84.70% while the list MDA program participation was found at Ilesha East with 18.97%, highlighting a potential vulnerability in vector control compliance. Furthermore, haematological assessments of chronic cases revealed a distinct state of immune suppression, characterized by significant reductions in total white blood cell and lymphocyte counts, alongside persistent monocytosis and eosinopenia. These findings suggest that while Osun State has successfully transitioned into a post-transmission phase, the lingering physiological impacts on the chronically ill and the moderate rates of preventive practice necessitate sustained surveillance and enhanced public health interventions to ensure permanent disease eradication

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.

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.

Malaria transmission depends on the survival and mosquito infectivity of mature Plasmodium falciparum gametocytes. Despite malaria febrile episodes reaching 39 to 41.5{degrees}C in infected humans, the impact of febrile temperatures of varying durations on gametocyte viability and transmissibility remains undefined. Here we quantify the effects of exposing mature stage V gametocytes in vitro to febrile temperatures (39{degrees}C, 40{degrees}C, and 41.5{degrees}C) for varying durations (3 to 12 hours). To assess gametocyte morphology, functionality, and mosquito infectivity, we used light microscopy of Giemsa-stained thin blood smears, exflagellation assays, and standard membrane feeding assays (SMFAs). Following exposure to 39{degrees}C, gametocytes retained normal morphology, exflagellation capacity, and mosquito infectivity across all exposure durations. At 40{degrees}C, gametocytes remained morphologically intact and capable of exflagellation but exhibited a time-dependent reduction in mosquito infection, both in prevalence and oocyst intensity, following prolonged exposure. In contrast, exposure of mature gametocytes to 41.5{degrees}C, even for the minimum duration tested of 3 hours, resulted in complete loss of normal morphology, exflagellation capacity and ability to infect mosquitoes. These findings reveal that mosquito infectivity of mature gametocytes is affected both by the magnitude of the febrile temperature and the duration of exposure. This provides insight into how host fever dynamics may influence parasite transmission.

Japanese encephalitis virus is a leading cause of encephalitis in humans and agricultural animals across Southern and Eastern Asia. It is primarily transmitted by mosquitoes of the Culex genus, notably Culex tritaeniorhynchus. In the Philippines, JEV is reported to account for 7% to 18% of clinical cases of meningitis and encephalitis. Therefore, updated and comprehensive data on circulating JEV strains in the country are crucial for developing effective mitigation strategies. This study aimed to optimize an assay for JEV detection using one-step reverse transcription polymerase chain reaction (RT-PCR). Additionally, the presence of JEV in mosquitoes collected from eleven cities and municipalities in Laguna was assessed, and the mosquito carriers were identified based on morphological characteristics. Results demonstrated that the optimized one-step RT-PCR assay successfully detected JEV at various dilutions, with a limit of detection (LoD) of 0.115 ng/L. The assay exhibited 100% specificity, with no amplification observed for any viruses other than JEV. Sequence analysis of the positive control, JEV SA14-14-2 RNA, revealed 96.96% to 98.41% similarity to JEV reference sequences in the NCBI database. Among the 5,522 female mosquitoes collected, Culex tritaeniorhynchus and Culex gelidus were the most prevalent species. The highest mosquito species diversity and the most even distribution were found in the three barangays of Paete, but Siniloan, Laguna had the highest vector density for the three Culex species, Culex tritaeniorhynchus, Culex gelidus, and Culex fuscocephala. RT-PCR analysis of 189 pooled mosquito samples revealed no detectable JEV genetic material. This study is likely the first investigation of JEV presence in mosquito samples from Laguna, Philippines. These findings contribute to proactive JEV surveillance, enhancing our understanding of the virus's presence and distribution. The results underscore the importance of targeted prevention and control measures to mitigate JEV transmission in high-risk areas.

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: Dengue fever has become an epidemiological concern worldwide, with 531,617 cases reported in Argentina during the 2023-2024 season. The El Nino Southern Oscillation (ENSO) phenomenon has been linked to changes in dengue dynamics. This study assessed the relationships among ENSO, local climatic variables, and dengue outbreaks in Argentina from 2018 to 2024. Methods: A nationwide spatiotemporal analysis was conducted using dengue cases and monthly mean temperature, relative humidity, and rainfall. The Southern Oscillation Index (SOI) was used as the ENSO indicator (El Nino phase=< minus0.5, neutral phase= minus0.5 to 0.5, and La Nina phase= > 0.5). We fitted Hierarchical Bayesian models with Integrated Nested Laplace Approximation (INLA). A Negative Binomial distribution of dengue cases was used to account for overdispersion. Spatial dependence was modeled by using the Besag York Mollie 2 (BYM2) structure, adjusted for population. Results: The final model explained 53.7% of the variation in Argentinas departmental monthly dengue cases during the study period. Exposure response curves of posterior probability and 95% credible intervals suggested that dengue risk increased when monthly mean temperatures were between 20 and 30 degrees Celsius during the El Nino or neutral phase, with less consistent effects during the La Nina phase. Humidity >50% was associated with increased dengue risk during the La Nina phase, with less steep or absent associations during El Nino or neutral phases. Negative SOI scores were associated with an increased risk of dengue after controlling for the covariates. Conclusion: Dengue outbreaks in Argentina were independently associated with temperatures between 20 and 30 degrees celsius during the El Nino and neutral phases, humidity > 50% during the La Nina phase, and the El Nino phase of the ENSO. Incorporating ENSO indices into predictive models could enhance early warning systems and timely public health interventions in Argentina.

BackgroundIn Ethiopia as other Sub-Sharan African countries malaria in pregnancy is a major public health threat particularly in the rural communities. Immunosuppression during pregnancy increases the risk of placental malaria; this, in turn, leads to anemia, low birth weight, preterm delivery, and stillbirth, causing severe complications that pose a life-threatening risk to both the mother and fetus. Hence, this study is aimed to assess the prevalence of malaria, and associated factors among pregnant women attending antenatal clinics in Damot Gale district, South Ethiopia. MethodAn institutional-based cross sectional study was conducted from March 1 to May 30, 2024. A multistage sampling technique and pre-tested semi-structured questionnaires were used for data collection. Using standard parasitological methods, blood samples collected from 389 participants were analyzed through microscopic examination of Giemsa-stained thick and thin blood films. Logistic regression was used to assess factors associated with malaria. Adjusted odds ratio with 95% confidence interval was calculated and P-value < 0.05 was considered statistically significant. ResultsIn this study the prevalence of malaria among pregnant women was 10.5% (95% CI: 7.7-13.6%). Among the malaria-positive pregnant women (9%) were P. falciparum infected, followed by P. vivax (1.3) and mixed infections (0.2%). In the multivariate analysis, multigravdae (AOR = 0.358; 95% CI: 0.14-0.92), utilization of LLINs (AOR = 0.286; 95% CI: 0.119-0.687) and use of IRS (AOR = 0.068; 95% CI: 0.027-0.172) were significantly associated with malaria among pregnant women. ConclusionMalaria is still a public health problem among pregnant women in Damote-gale district. The present study indicated that adequate distribution and proper utilization of bed nets In addition seasonal proper spray of IRS might be helpful to prevent and control malaria.

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.

Background: Unexplained spatial heterogeneity in field-observed Human Blood Index (HBI) hinders vector-borne disease control. We synthesized micro-morphological sensory structure densities across primary disease vectors to formulate a novel Proboscis Sensory Index (PSI) from micro-morphological sensory structure densities to evaluate its capacity to predict vector anthropophily, zooprophylactic modulation, and spatial transmission risk across Northern Nigeria. Methods: A systematic review of 42 studies (N = 18,647 blood-fed mosquitoes across 126 georeferenced sites, 2005 to 2025) synthesized baseline species-specific PSI metrics from electron microscopy sensilla counts. A multi-variable binomial Generalized Linear Mixed Model (GLMM) evaluated interactions between PSI, livestock, human density, and environmental covariates. Predictive accuracy was validated against field-derived Entomological Inoculation Rates (EIR) and historical outbreak records. Results: The overall pooled HBI was 0.642 (95% CI: 0.588 to 0.694), exhibiting significant variation across the Sudan (0.728), Guinea (0.615), and Sahel (0.584) savannas. Species specific PSI values graded from Anopheles gambiae s.s. (8.91 {+/-} 0.34), Anopheles funestus (8.12 - 0.38), and Anopheles arabiensis (7.68 {+/-} 0.41) to Culex quinquefasciatus (4.87 {+/-} 0.52). In GLMM analysis, PSI was the strongest predictor of anthropophily ({beta} = 0.89, aOR = 2.44, 95% CI: 2.32 to 2.56, p < 0.001), with fixed effects explaining 78% of total field variance (Marginal R2 = 0.78). High PSI species maintained elevated anthropophily regardless of livestock availability, whereas low-PSI species displayed steep zooprophylactic diversion ({Delta}HBI = -0.71, p < 0.001) under high cattle density. Incorporating PSI reduced localized EIR prediction error by 64.2% (RMSE = 15.0 vs. 41.8 infective bites/person/month), and high-resolution (1 km2) predictive mapping correctly identified 87.1% (27/31) of historically documented outbreak Local Government Areas. Conclusion: Micro-sensory organ density dictates vector host preference and livestock-mediated zooprophylactic potential. Integrating PSI into spatial models resolves localized transmission heterogeneity, guiding targeted vector control prioritizing indoor residual spraying and dual-ingredient nets in high PSI foci, while leveraging endectocides and larval management in low-PSI pastoral corridors.

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.

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.

Barmah Forest Virus (BFV), an arthropod-born virus transmitted by mosquitoes, is of significant public health concern in Australia and regions in the Pacific. Recent climate change and globalization raise the potential for BFV to extend its geographic distribution. Despite the rising importance of BFV, its vector dynamics remain poorly understood, particularly concerning the vector competence of different mosquito species. This study aims to investigate the vector competence of BFV across various mosquito species, beyond those endemic to the Australasian region, especially focusing on global relevant vector species. No transmission was observed for Culex quinquefasciatus and Cx. torrentium as well as Anopheles stephensi. In contrast, both investigated Aedes species, Ae. aegypti as well as Ae. albopictus, exhibited BFV-positive saliva across all four temperature profiles (18{degrees}C, 21{degrees}C, 24{degrees}C or 27{degrees}C) examined. These two invasive mosquito species must therefore be classified as potential vectors for BFV, indicating the potential risk of BFV transmission outside of Australia.

The fieldwork was carried out between December 2023 and June 2025, with the aim, among others, of confirming the possible presence of the North African Gerbil (Gerbillus campestris) in Melilla. To this end, 56 camera trap stations were installed, each equipped with a camera trap. Of the total number of camera trap stations installed, the presence of Gerbillus campestris was detected in six of them, representing a capture success rate of 10.71%. Following the detection of the species, a specimen was captured using cage traps. For identification purposes, samples of epithelial tissue from the ear and faeces were collected. These samples were sent to the Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno, Czech Republic, for analysis. There, DNA was extracted from the samples using the Innu PREP DNA Mini Kit 2.0 (IST Innuscreen GmbH, Germany) according to the manufacturer's protocol. The results obtained confirm, for the first time through records of the species, the presence of Gerbillus campestris in Melilla and, therefore, in Spain and Europe.

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.

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