Parasites & Vectors

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

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

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

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

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

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

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

Plasmodium vivax transmission from humans to mosquitoes depends on the density of gametocytes that in turn depends on asexual parasite replication and gametocyte commitment. These processes are often analyzed separately, despite being biologically linked and measured with substantial uncertainty. We used a joint Bayesian latent-variable model to simultaneously analyze parasite density, gametocyte density, and mosquito infectivity while accounting for measurement error and propagating uncertainty across linked processes. The model was applied to individual-level data from three P. vivax transmission studies conducted in Ethiopia (n = 455). A tenfold increase in gametocyte density was associated with more than a twofold increase in the odds of mosquito infection (odds ratio [OR] = 2.32, 95% credible interval [CrI]: 2.12-2.54). Asexual parasite density was also positively associated with infectivity after accounting for gametocyte density (OR = 1.74, 95% CrI: 1.60-1.90), and inclusion of parasite density improved predictive performance. Pathway decomposition within the joint model indicated that approximately 41% of the parasite-infectivity association operated through gametocyte density. Increasing age was associated with lower asexual parasite density but higher gametocyte density, resulting in minimal overall association with infectivity. Predicted infection probability increased sigmoidally with gametocyte density, remaining low at lower densities before increasing sharply and approaching a plateau at higher densities. Gametocyte density produced the largest predicted changes in the proportion of infected mosquitoes, while asexual parasite density added predictive information not fully captured by measured gametocyte density alone. This approach links molecular parasite measurements with mosquito infection risk while accounting for measurement uncertainty and provides an interpretable framework for studying the P. vivax infectious reservoir. Author SummaryMalaria transmission occurs when mosquitoes ingest sexual-stage parasites, called gametocytes, during a blood meal. In Plasmodium vivax infections, human-to-mosquito transmission depends on linked biological stages, including asexual parasite replication, gametocyte production, and mosquito infection. These processes are closely connected and often measured with uncertainty, making them difficult to study using standard approaches that analyze them separately. In this study, we applied a joint Bayesian model that analyzes parasite density, gametocyte density, and mosquito infectivity together while accounting for uncertainty in laboratory measurements. Using data from three studies in Ethiopia, we quantified how parasite density, gametocyte density, and host characteristics relate to mosquito infection. The analysis showed that measured gametocyte density alone did not fully explain variation in infectivity, and that asexual parasite density provided additional predictive information. We also found that age was associated differently with asexual parasite and gametocyte densities, resulting in little overall association with infectivity. This approach helps link molecular parasite measurements with transmission outcomes and improves understanding of the P. vivax infectious reservoir in endemic settings.

Dengue transmission is strongly influenced by climatic conditions that affect mosquito population dynamics and virus circulation. In Southern Brazil, where dengue historically occurred at low levels, recent climatic anomalies may be contributing to the expansion of Aedes vectors and an increase in local dengue incidence. This study investigated the spatiotemporal association between climatic variables, Aedes mosquito infestation and dengue cases in Porto Alegre (Southern Brazil, 2018 to 2025). Entomological, surveillance and climatic data were analyzed using Morans I and LISA for spatial association, Kendall correlation, polynomial regression and LASSO to identify relevant drivers and develop predictive models of mosquito infestation and dengue incidence. A strong spatial association between Aedes aegypti and Aedes albopictus was observed, with persistent local clusters detected across all years. Annual climatic variables were associated with mosquito abundance in several districts. Overall, rainfall frequency had a stronger effect on Aedes aegypti abundance than accumulated rainfall. Temperature and lagged infestation indices showed strong association with both species and dengue incidence, with effects observed up to four weeks prior. Predictive models demonstrated good agreement between observed and predicted values, particularly within low to moderate infestation levels. Lagged variables were consistently retained in both mosquito infestation abundance and dengue incidence models, highlighting the importance of temporal predictors for anticipating vector dynamics and dengue risk. This approach is generally applicable for predicting Aedes infestation and disease incidence and emphasizes the importance of integrating entomological and climatic surveillance data to improve anticipation and detection of dengue risk periods and support more effective public health interventions.

Mosquitoes in urban environments are considered unpleasant by citizens, and they can represent serious health risks as disease vectors. Invasive Aedes species are continuously spreading in Europe and have been increasingly reported from urban settlements. Some of these mosquito species have been associated with small artificial habitats. Therefore, their presence may also be expected in small plastic garden ponds, which are numerous in urban settings across many parts of Europe. Here, we aimed to determine whether urban waters host a high occurrence of mosquitoes and to identify the potential abiotic, biotic and landscape drivers of mosquito presence and abundance in urban ponds. We sampled 53 urban ponds in the city of Budapest (Hungary) of both natural and anthropogenic origin, and 40 garden ponds, which cover both highly urbanised areas and those with high green index in the suburbs. We collected data on macroinvertebrate communities, pond management, physico-chemical parameters, and pond characteristics (mainly morphology and vegetation cover) measured in situ. Two different mosquito detection techniques were used: dip-net sampling and eDNA. While only one invasive species (Aedes koreicus) was detected, occurring in a single pond, several other species were present, including potential malaria vectors increasingly reported from urban environments: the Anopheles maculipennis complex, An. claviger and An. plumbeus. Fish presence was negatively associated with both the presence and abundance of mosquitoes, regardless of pond type. Contrary to expectations, urbanisation did not play a major role in explaining mosquito presence or abundance. These results highlight the importance of local pond characteristics, and particularly the role of fish presence, in regulating mosquito populations in ponds in the urban landscape, although the broader ecological effects of fish on pond communities should also be considered.

Background: Female genital schistosomiasis (FGS) is a neglected gynaecological manifestation of Schistosoma haematobium (S. haematobium) infection, resulting from the deposition of parasite eggs in the female genital tract. Although urogenital schistosomiasis is highly prevalent in parts of coastal Kenya, including Kilifi County, the burden of FGS among women of reproductive age remains poorly characterised. Routine diagnosis of S. haematobium infection relies largely on urine microscopy, which may underestimate genital involvement. This study aimed to assess the prevalence, diagnostic concordance, and risk factors for FGS among women of reproductive age in Kilifi County, Kenya. Methodology: In this cross-sectional study, 320 randomly selected women aged 15-50 years were recruited from rural Kilifi County; 261 provided complete data for analysis. A structured questionnaire was administered to collect sociodemographic and behavioural information. Urinary schistosomiasis was assessed using triplicate urine microscopy over three consecutive days, and FGS was evaluated using real-time polymerase chain reaction (PCR) targeting the S. haematobium Dra1 gene sequence on self-collected high vaginal swabs. Results: Overall, the prevalence of PCR-confirmed FGS was 36.0% (94/261), while urinary egg excretion was detected in 13.0% (34/261) of participants. Concordance between urine microscopy and genital PCR was 70.9%. Notably, 72% of women with PCR-confirmed FGS had no detectable parasite eggs in their urine. In bivariate analyses, factors such as urinary infection severity, water contact behaviours, haematuria, dysuria, age group, place of residence, and prior history of schistosomiasis were found to be associated with female genital schistosomiasis (FGS). However, in the multivariable logistic regression, only sub-location and urinary infection severity remained independently associated with the infection. Additionally, PCR cycle threshold (Ct) values showed a non-linear relationship with mean urinary egg counts, indicating that the detection of genital parasite DNA does not directly correspond to the urinary egg burden. Conclusion: FGS prevalence among women in Kilifi County was substantially higher than indicated by urine microscopy alone. The majority of women with genital schistosomiasis did not exhibit detectable urinary egg excretion, highlighting the limitations of routine parasitological screening for identifying genital disease. These findings underscore the need to incorporate genital sampling and molecular diagnostics into schistosomiasis control strategies targeting women of reproductive age in endemic settings.

Malaria remains a major global health burden, with traditional control methods facing challenges such as insecticide resistance and high operational costs. Genetic biocontrol offers a promising alternative for mosquito population suppression, but its field efficacy would require assessment. This study evaluates the role that population genomic statistics can play in detecting decreases in population size in the context of a cluster randomized control trial (cRCT), investigating the response of nucleotide diversity ({pi}), Tajimas D, segregating sites, and linkage disequilibrium (LD) under both constant and seasonal demographic scenarios. We simulated 90% and 99% population declines with various degrees of between-cluster heterogeneity, and assessed the detection power of each statistic over time and number of clusters per arm. Results show that Tajimas D is highly sensitive and robust across crash severity, seasonality and heterogeneity scenarios. Segregating sites has similar power to Tajimas D when baseline data are available. We further estimated that cRCTs require approximately 3 to 5 villages per treatment arm to achieve adequate statistical power. These findings provide recommendations for genetic monitoring of vector control interventions in wild populations.

Some Anopheles species that act as malaria vectors are members of species complexes, a concept whereby sibling species cannot be differentiated solely on the basis of morphological characters. Therefore, species complexes represent a major problem in malaria vector control, because within an Anopheles complex, vectors cannot be differentiated from non-vector species, unless molecular techniques are used to identify them. The Anopheles tessellatus species complex is an important potential vector in South, East, and Southeast Asia, including certain regions of Indonesia. However, no in-depth studies have been conducted on this species complex in that country. Therefore, this study investigated the taxonomic status of An. tessellatus from diverse populations across five Indonesian islands (Sumatra, Java, West Nusa Tenggara, East Nusa Tenggara, and Sulawesi) and identified interpopulation genetic variation based on molecular data of the ITS2, COI, and COII genes. Phylogenetic relationships were constructed using the Maximum Likelihood method. Haplotype and network analysis were also conducted. The results indicate that An. tessellatus constitutes a monophyletic group comprising three well-defined lineages that exhibit clear intraspecific genetic differentiation. Cluster 1 corresponds to the population of Sumatra, Cluster 2 represents population from Sulawesi, and Cluster 3 encompasses populations from Java, West Nusa Tenggara, and East Nusa Tenggara. These findings demonstrate high haplotype diversity and low nucleotide diversity within the species. Populations from West Sumatra, Manado, Tojo Una - Una, and North Morowali (Sulawesi) have the potential for speciation with a genetic distance of 0.61 - 0.94% for COI, between 0.81 - 0.95% for ITS2, and between 0.62 - 0.71% for COII. These findings underscore the need for further integrative studies to obtain a more comprehensive understanding of the An. tessellatus complex in Indonesia and its role in malaria transmission.

O_LIAlthough ecological research has long focused on the effects of temperature on population growth, arthropod pests are exposed to a wide variety of environmental factors that affect their performance, such as chemical pesticides targeted against them. Moreover, these environmental factors likely do not act in isolation. Identifying the extent to which abiotic factors interact to affect pest population dynamics can strengthen current and future pest management programs. C_LIO_LIHere, we investigated the extent to which temephos, a common pesticide applied to aquatic environments for mosquito control, influences the thermal performance of juvenile survival and development rate, as well as the intrinsic population growth rate, of the invasive mosquito pest, Aedes aegypti. We implemented a response surface experimental design to measure these traits across seven temperatures and five temephos concentrations and fit temperature- and insecticide-dependent performance curves to assess impacts on the overall performance and the thermal optimum, minimum, and maximum. C_LIO_LITemephos exposure profoundly altered the thermal performance of juvenile survival by reducing survival across all temperatures, shrinking the thermal breadth, and shifting the thermal optimum to warmer temperatures. Through this, temephos also altered the thermal performance of population growth primarily by reducing its thermal breadth. C_LIO_LISynthesis and applications: Our findings demonstrate that interactions between temperature and insecticide exposure can fundamentally reshape pest population dynamics, rather than acting as independent stressors. By quantifying this interaction, we showed that temphos is most effective below the pests thermal optimum, suggesting that larvicides may yield the greatest population suppression in cooler regions or during cooler periods of the year. Incorporating such temperature-dependent efficacy into pest management strategies could improve the timing and spatial targeting of control efforts. More broadly, these results highlight the need to integrate anthropogenic stressors with climatic drivers when predicting pest risk and optimizing management under ongoing environmental change. C_LI

The tobacco hornworm moth (Manduca sexta) is evaluated as a model of bacterial virulence and host-pathogen dynamics. Infections of Pseudomonas aeruginosa were established by injection of 5th-instar larvae, and multiple assays of virulence were evaluated. Infected larvae exhibited dose-dependent mortality, reduced growth, melanization, behavioral changes, and altered frass constitution. Even low-dose infections that were not fatal exhibited impaired growth, but individual growth trajectories revealed considerable heterogeneity among worms given the same dose. Twice-daily antibiotic treatment with gentamicin or cefepime improved survival four- to five-fold but did not rescue 100%. Heat-killed cells and filtered culture supernatant alone induced significant morbidity and mortality, suggesting secreted bacterial products are important to pathogenesis. Bacterial burden analysis revealed a shifting bacterial distribution over time, with decreasing hemolymph titers and increasing localization in fat body, gut, and carcass. Hornworms thus offer a more sensitive analysis of bacterial infection dynamics and consequences than do larvae of the more commonly used wax moth.

BackgroundThe World Health Organization (WHO) has raised concerns over increasing Pfhrp2/3 deletions, undermining the sensitivity of Pfhrp2-based rapid diagnostic tests (RDTs). Close monitoring of the population and a change in diagnostic methods are recommended if the prevalence of parasites with Pfhrp2/3 deletions exceeds 5%. In high transmission settings, accurate estimates are hampered by the frequent occurrence of mixed-clone infections (multiplicity of infection; MOI). Objective and MethodsIf parasites with and without deletions are present in an infection, standard molecular assays cannot detect the presence of the former. To accurately estimate frequencies of haplotypes with Pfhrp2/3 deletions in the presence of mixed infections, a novel statistical model that combines genetic/molecular information from Pfhrp2/3 with that from neutral markers is introduced. Maximum-likelihood estimates (MLEs) are obtained for haplotype frequencies characterized by markers at Phrp2/3 loci and loci for neutral markers. The expectation-maximization algorithm is used to derive the MLEs. The adequacy of the method (precision and accuracy) is assessed by numerical simulations. ResultsThe method was applied to an active surveillance study conducted in a tribal community in Jagdalpur, India, which enrolled febrile community members (n = 432) between October and November 2021. Four markers each at Pfhrp2 and Pfhrp3 are combined with one marker each at Pfmsp1 (which encodes P. falciparum merozoite surface protein 1) and Pfmsp2. Data from a total of 117 patients who had both P. falciparum infections and genetic information for the molecular markers underwent further analysis with the novel statistical method. ConclusionResults indicate that this novel method has promising statistical properties (asymptotic and in finite samples) and can be readily applied to real-world situations. A stable implementation of the method in R is provided. This novel approach enables accurate estimation of Pfhrp2/3 deletion frequencies in complex P. falciparum infections, addressing a key limitation of current molecular surveillance methods. Author summaryPlasmodium falciparum (Pf) causes the most severe form of human malaria, accounting for over 90% of cases. Rapid diagnostic tests (RDTs) have become a cornerstone of malaria control. These RDTs detect Pf-specific antigens in a blood drop. HRP2/3 emerged as the best antigen for such tests because it is Pf-specific and expressed in abundance. However, some parasites lack the genes that code for HRP2/3 proteins. If parasites in an infection have such gene deletions, RDT results can be false negative. The WHO considers the containment of such deletions a public health priority and recommends monitoring their prevalence. The detection of HRP deletions is challenging if parasites with and without deletions co-occur in infections because standard molecular assays cannot detect deletions in this situation. To overcome this challenge, we introduce a novel statistical method to estimate the frequency distribution of parasite variants with deletions. The method combines information from neutral molecular markers and from HRP-related markers to correct for unobservable information. Here we provide a derivation of the statistical model, a stable implementation, and test its statistical properties with synthetic and real data, thereby showing that our method is well-suited for the underlying problem.

In wildlife forensic practice, species identification and estimation of the Minimum Number of Individuals (MNI) for highly processed specimens have long relied on weight-based conversion methods, which may result in underestimation of the number of individuals involved in a case. Focusing on confiscated casque products of the helmeted hornbill (Rhinoplax vigil), this study combines macroscopic morphological examination with mitochondrial DNA barcoding (16S rRNA, COI, and Cytb) to explore a more robust approach for individual quantification. The results demonstrate that the conventional "weight-based" approach overlooks critical biological information contained in anatomical structures and cannot accurately reflect the actual number of individuals involved. Based on this, we propose an anatomy-based criterion centered on the principle of structural uniqueness: specimens retaining biologically unique beak or casque structures should be directly assigned to a single individual, whereas weight-based estimation should only be applied when original anatomical features are entirely absent. In addition, considering material loss during processing, we propose approximately 85 g as a reference threshold for estimating the number of individuals in heavily processed solid casque products. This approach improves the scientific rigor and accuracy of forensic identification and provides reliable technical support for the conviction, sentencing, and law enforcement of wildlife trafficking cases involving helmeted hornbill and other endangered species.

The liver fluke, Fasciola hepatica, is a major parasitic threat to ruminant health and productivity worldwide, with important implications for food security, animal welfare, and zoonotic risk. This study developed and validated a multiplex deep amplicon sequencing assay targeting the mitochondrial NADH dehydrogenase 1 (mt-ND1) and cytochrome c oxidase subunit 1 (mt-COX1) loci for high-throughput genotyping of F. hepatica. DNA was extracted from eggs sedimented from sheep and cattle faeces (n = 78) received from farms and from adult worm pools (n = 12) isolated at abattoirs from diverse regions across the UK. Following high-throughput sequencing, bioinformatics analysis was performed to demultiplex Illumina sequence reads and extract amplicon sequence variants (ASVs). A total of 11 ASVs were identified at each locus (mt-ND1: 264-279 bp; mt-COX1: 312-319 bp), with two or three predominant ASVs per locus, along with rare variants. Network and PCA analyses revealed two distinct clusters at the mt-ND1 locus: one primarily associated with sheep and another shared between sheep and cattle. In contrast, mt-COX1 sequence reads formed a single dominant cluster. Population analyses revealed extensive ASV sharing across regions, indicating high gene flow, likely facilitated by livestock movement and parasite adaptation.

Human leptospirosis is a disease of public health importance in Thailand, but the animal species involved in the transmission cycle have not been fully uncovered. This study investigated Leptospira infection in dogs and terrestrial micromammals in rural Nan Province, Thailand, and the pathogen diversity. Sera from 95 seemingly healthy dogs and kidney samples from 399 micromammals were analyzed using real-time PCR for Leptospira detection, followed by conventional PCR and sequencing of infecting Leptospira. We investigated environmental factors associated with Leptospira infection in micromammals, using data collected during trapping. Real-time PCR revealed ongoing infection in 8.4% (8/95) of dogs and 10.0% (40/399) of terrestrial micromammals, with 12 infected species including Bandicota indica, Berylmys berdmorei, Berylmys bowersi, Mus cervicolor, Mus cookii, and Hylomys suillus. In this qPCR-positive micromammals, three pathogenic Leptospira species were identified: L. interrogans, L. weilii, and L. borgpetersenii. This represents the first confirmed detection of L. weilii in rodents in Thailand. Infected micromammals were found in agricultural and forest habitats but not in human settlements. Our study demonstrates potential complex leptospirosis epidemiology in rural Thailand, with multiple species serving as pathogenic Leptospira reservoirs across diverse habitats, and some shared pathogen diversity with human leptospirosis cases in Thailand. Free-roaming dogs may serve as bridge hosts, transmitting zoonotic Leptospira from micromammals to humans by visiting both animal habitats and human settlements. These findings emphasize the need for integrated One Health surveillance approaches to control leptospirosis in rural communities.

Gastrointestinal disorder caused by the ingestion of (oo)cysts of Cryptosporidium and Giardia is one of the major health problems in developing countries. Fruits and vegetables that are usually consumed unpeeled, poorly washed and or cooked and are the major modes of transmission. Frequent large-scale screening of the food samples is necessary to prevent outbreaks but screening of vegetables for such microbes is limited in Nepal. In this study, we used a smartphone microscopy system to study prevalence and quantification of (oo)cysts of Cryptosporidium and Giardia in 651 vegetable samples collected from nine major vegetable collection sites across Nepal. The overall prevalence rate of vegetable samples was 37.5% with at least with one of the parasites. We found that 23.2% samples were contaminated with Giardia and 33.3% samples were contaminated with Cryptosporidium. Among eight vegetable types, the prevalence rate was lowest in carrot (20%) and highest in spinach (48%). The prevalence rate of vegetable samples at different sites ranged from 13% in Dhading to 61% in Dhangadi. The contamination rate was 28% for winter, 43% for summer and 33% for monsoon seasons in samples collected from Kathmandu. These vegetables should be considered as a potential source of parasitic contamination in people. These vegetables can cause infection if consumed poorly washed and or cooked, posing a potential source of parasitic contamination in people.