Medical and Veterinary Entomology

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

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.

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

Aedes albopictus is a major arboviral vector whose global expansion, driven by human activities and climate change, poses a growing public health concern for a number of neglected tropical diseases in both tropical and temperate regions. As a poikilotherm, its biology and population dynamics are strongly influenced by temperature, thereby shaping disease transmission. To thwart and control its geographic expansion, effective vector control strategies are increasingly critical. Densoviruses (DVs), such as AalDV2, are being explored as mosquito viral biocontrol agents due to their restricted host range and ability to disseminate through oviposition sites. However, the influence of environmental parameters on the interactions between Ae. albopictus and AalDV2 remains poorly understood. This makes their performance under realistic, fluctuating thermal regimes difficult to estimate. In this study, we investigated the combined effects of temperature and AalDV2 exposure on Ae. albopictus survival and development across its full life cycle. Mosquitoes were reared under fluctuating temperature regimes (26-28 {degrees}C and 32-34 {degrees}C, 12:12 day[ndash]night cycles) and exposed to AalDV2 or a control treatment. Chronic exposure to 32-34 {degrees}C significantly reduced overall survival, decreasing median lifespan by approximately 10 days (HR=2.21, p=0.0018), with a deleterious effect increasing over time. It extended aquatic lifespan and increased pupal mortality. It also reduced adult lifespan in both sexes with a stronger effect in females. AalDV2 exposure had no significant effect on overall survival, stage-specific mortality, or adult lifespan. However, a significant interaction between viral exposure and thermal stress was detected on aquatic lifespan: AalDV2-exposed females showed further extended larval and pupal development specifically under the 32-34 {degrees}C regime, without any effect on survival. These results indicate that the biocontrol potential of AalDV2 cannot be assessed independently of thermal context: while lethal effects were absent under both fluctuating regimes, the prolongation of aquatic development by the virus under thermal stress may have indirect consequences for mosquito population dynamics that warrant further investigation.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

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.

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

Entomological surveillance is essential for the control of lymphatic filariasis (LF). This xenomonitoring study, conducted in 2022-2023, evaluated the effectiveness of three mosquito trapping methods to identify the most suitable tool for surveillance following the discontinuation of mass drug administration. The surveys took place in two Malian villages: Konfra (seasonal watercourse) and Siradjouba (permanent watercourse). Three techniques were compared: Pyrethrum spray catch (PSC) conducted at the end of each visit inside 30 randomly selected houses, and different collection points were chosen based on the characteristics of the study area to place the Ifakara Type C tent (Ifakara) and the gravid trap (GT). Collections were conducted simultaneously at both sites. All Anopheles gambiae and Culex spp. collected in 2022-2023 were morphologically identified and then analyzed in the laboratory for their infectious status. Data was processed using SPSS v25; Fishers exact test was used to compare proportions, with a significance threshold of p < 0.05. A total of 4,732 mosquitoes were captured (95.05% in 2022). In 2022, the Anopheles were predominant in Konfra (91.38%), while Culex predominated in Siradjouba (63.41%). In 2023, Anopheles was found only in Konfra (3 specimens), while Culex was predominant in Siradjouba (64.07%). In 2022, PSC collected 90.57% of Anopheles, Ifakara 9.43%, and GT 0%. For Culex, GT collected 53.67%, PSC 36.62%, and Ifakara 9.71%. In 2023, the few Anopheles (3) came from PSC; Culex were mainly collected by the Ifakara tent (43.29%). For FL xenomonitoring, the combination of PSC and the GT appear to be effective collection methods.

Sleep is a ubiquitous, yet highly variable, behavior across species. The duration and timing of sleep are influenced by ecological demands and dietary context. In the mosquito Aedes aegypti, a blood-feeding insect with specialized nutritional requirements, the relationship between feeding and sleep remains poorly understood. Here, we investigated how blood-derived dietary protein influences sleep regulation. Using postural analysis, videography, and arousal-threshold assays, we established that immobility bouts of greater than 10 minutes reliably define sleep in Ae. aegypti. Mosquitoes lacking the circadian clock gene cycle still maintained daily sleep rhythms but exhibited reduced sleep duration and heightened overall activity. Infrared activity monitoring revealed that blood-fed females showed a marked increase in sleep beginning immediately after feeding and persisting for several days, accompanied by reduced locomotor activity. Notably, this sleep elevation lasted well beyond the cessation of previously reported host-seeking phases, raising the possibility of distinct phases of opportunistic versus targeted host pursuit. To determine the dietary basis of this effect, we tested mosquitoes fed a bovine serum albumin (BSA)-based diet. BSA feeding alone was sufficient to mimic the sleep-promoting and activity-reducing effects of blood, suggesting dietary protein is a major nutritional regulator. Moreover, RNAi-mediated knockdown of the leucokinin receptor (Lkr), which has previously been associated with fluid homeostasis and feeding behavior, resulted in enhanced sleep and reduced activity, implicating mosquito LK signaling in the modulation of postprandial sleep. Together, these findings demonstrate that blood-derived proteins drive sustained increases in sleep and reductions in locomotor activity in Ae. aegypti. This work positions Ae. aegypti as a model for dissecting nutrient-specific regulation of sleep and highlights potential adaptive functions of protein-induced quiescence, such as energy conservation and predator avoidance. More broadly, it provides insight into how specialized diets shape the neural and behavioral architecture of sleep.

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Background: Dirofilaria repens is a zoonotic parasite expanding unnoticed across Europe due to climate change. We hypothesised that in this process, the Carpathian Basin has a facilitating effect. Methods: Using 426 georeferenced European cases, the probability of infection occurrence was determined in relation to climatic factors, surface water availability, regional social deprivation, and stray dog population density. To analyse the potential impacts of ecological and social factors (deprivation and stray dog population density), the MaxEnt algorithm, and spatial Empirical Bayes smoothing and Bivariate Local Indicators of Spatial Association (BiLISA) index calculation were employed, respectively. Results: MaxEnt analysis revealed that the mean warmest month temperature (22.8 - 25.1 oC), winter mean minimum temperature (> -2.1 oC), and summer precipitation (28.6 - 231 mm) have the strongest impact on the probability of the parasite's occurrence in Europe. Social factors have significance in the eastern Balkans and the Carpathian Basin, but not in Western Europe. The Carpathian Basin appears to be a hotspot, similar to Mediterranean coastal areas. Furthermore, the Danube Valley acts as an ecological corridor for subtropical vector-borne parasites. Conclusions: Our findings confirm that summer warmth is the primary ecological driver of the parasite's range expansion, which is facilitated by the Carpathian Basin due to climatic and socioeconomic conditions.

The global invasion of the Asian tiger mosquito Aedes albopictus has led to an increase in arboviral disease, including within Mesoamerica. Understanding vector invasion routes is important for public health because it directs biosecurity and identifies sources of adaptive allele spread. Panama is an important hub of global trade with opportunities for Aedes introduction through both maritime and overland routes but dispersal into the Isthmus has not yet been investigated. We therefore sought to investigate the population structure and invasion history of Ae. albopictus into Panama, targeting both its mitogenome and associated Wolbachia. Historical demographic analysis with Bayesian phylogeographic diffusion models and estimates of divergence revealed that Panamanian Ae. albopictus and its associated Wolbachia have a convergent evolutionary history resulting from multiple introductions. Both could be traced to Asian-derived lineages introduced via the Americas, with invasion primarily through the maritime trade of the Panama Canal rather than overland dispersal from neighboring Costa Rica. An investigation of the relative density of Wolbachia in Panama revealed that both the strains wAlbB and wAlbA were at a notably lower density compared to other worldwide locations. This finding has implications for arbovirus transmission and raises important questions about how Wolbachia density is impacted by the environment and impacts on population control. Overall, the Panama Canal is a key route for vector introductions into Mesoamerica.

Objectives identified as critical for effective management of brown marmorated stink bugs, Halyomorpha halys Stal (BMSB), were addressed in the overwintering life stage. Research was conducted on a 675-acre farm in western Maryland, U.S.A. Developing and testing were done during September - October of each of the years 2013 - 2020. Traps and related materials were developed. Four invented traps plus the development and use of "weed beater" polyethylene plastic in combination with the other traps were used as attractions to BMSB during the overwintering testing periods. Overwintering BMSB showed very little interest other than to find a place to hide that was dry, dark, easy to get into and safe over the winter months (Nielson et al. 2016). Placement of traps show effect of cardinal directions due to heat and light were north = east > west = south of numbers of BMSB. These data were affected by the vegetation, type of buildings such as silos, cabins, houses, etc., around BMSB prior to the en masse flight. Silos were used as attractants from long range during the en masse flight. Five trap types were invented and tested for use against overwintering BMSB found various places such as buildings of any type, pathways in the woods and other habitats present. Trap #1 was made of plastic out of special plant pots, turned upside down with a plastic lid and entry holes top and bottom that allowed the BMSB to reach the hexcel inside and stay there. Trap #2 was made of white, rectangular coroplast and pressed into 0.5 x 0.5 x 7.32 m (5" x 5" x 24") box also holding hexcel. Traps #1 and #2 are useful for attachment to most anything with BMSB such as the eaves in houses, other buildings, vegetation, crevasses, etc. They can be placed as horizontal or vertical with other ways that enhance efficacy. Both #1 and #2 are cheap and easy to make and use. Trap #3 is made of a cardboard tube, as a fake or "faux" tree (dead and/or dying) in vegetation 20 cm (6-8") diameter and 1.22 m (3 or 4) long. The tubes are placed on tomato stakes, have hexcel and holes made as do the others and are placed vertically in the ground. The tubes are covered with small widths of white, black and or burlap around them. Lids of plastic are on top and the trap appears as a dead and/or dying tree. The fourth trap is a "mechanism" or resembling some kind of "Haven" if you will, made of "weed beater" plastic materials 1.98 m (66") wide in brown or black. The materials are placed on trees by wrapping them around 3-4 trees near 4.6 m (10-15 long). Traps 1 and 2 can then be placed on the outside of the plastic walls and the #3 fake trees can be added around the outside areas. Around 60,000 BMSB were collected and counted in these experiments by the author, and several different factors were raised and addressed to better understand the BMSB behavior during the overwintering stage. The results provide several new tools as well as understanding of approaches to address BMSB management better.

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.

Background: The development of safe and effective plant-based repellents is crucial to control malaria transmission, particularly given the spread of insecticide resistance in major vectors like Anopheles stephensi. Essential oils (EOs) are promising candidates, yet their high volatility and hydrophobicity limit their efficacy. This study aimed to design and evaluate nanoliposomal gels containing Syzygium aromaticum (clove) or Melaleuca alternifolia (tea tree) EOs to enhance their repellent durability against An. stephensi. Methods: The chemical profiles of the EOs were determined via Gas Chromatography-Mass Spectrometry (GC-MS). Nanoliposomes bearing 3% of each EO were made ready with the ethanol injection method, and incorporated into a carboxymethyl cellulose (CMC) gel. Formulations were characterized for particle size, zeta potential, viscosity, and chemical interactions (FTIR). Repellent efficacy was evaluated using the arm-in-cage method, recording the complete protection time (CPT) for nanoliposomal gels (LipoGel 3%), in comparison with nonformulated EOs and the gold-standard repellent, DEET (40%). Results: GC-MS analysis identified eugenol (79.51%) and terpinen-4-ol (73.53%) as the major constituents of clove and tea tree EOs, respectively. Nanoliposomes exhibited sizes of 82.3 nm (clove) and 102 nm (tea tree), with narrow size distributions. The clove LipoGel demonstrated a significantly enhanced CPT (341 min), which was statistically comparable to 40% DEET (351 min, P>0.05). In contrast, the nonformulated EOs resulted in only 45 min of protection, highlighting the critical role of the nanocarrier system. Conclusion: The nanoliposomal gel formulation, particularly containing clove EO, represents a potent and safe botanical alternative to conventional synthetic repellents. This approach offers a promising strategy for integrated vector management, warranting further field-based investigations.

Wildlife hosts play important roles in the ecology and transmission of vector-borne parasites, yet information on host associations remains scarce in many biodiverse tropical regions. Within a One Health framework, characterizing parasite diversity in wildlife can improve understanding of ecosystem health and disease emergence. Road-killed animals provide a non-invasive opportunity to investigate host-parasite interactions while minimizing disturbance to natural habitats. We screened 127 liver and intestinal tissue samples obtained from 76 road-killed vertebrates collected near protected areas in two Ecuadorian biodiversity hotspots, the Tropical Andes and Choco-Darien, for trypanosomatids and other vector-borne microorganisms. Molecular analyses targeted the 18S rRNA and cytochrome b genes of trypanosomatids and included additional screening for Trypanosoma cruzi, Trypanosoma rangeli, Rickettsia spp., and piroplasmids. Twenty-nine samples were positive for kinetoplastids. We detected diverse trypanosomatids representing the genera Leishmania, Porcisia, Trypanosoma, Phytomonas, Blastocrithidia, and Obscuromonas, as well as free-living kinetoplastids of the order Neobodonida. The most frequently detected species was Leishmania amazonensis, identified in 17 samples from at least 13 species of birds, reptiles, and caecilians, predominantly in liver tissue, suggesting previously unrecognized host associations. We also identified a putatively novel species of Porcisia and three potentially undescribed avian trypanosomes belonging to the subgenus Ornithotrypanum. No evidence of T. cruzi, T. rangeli, Rickettsia spp., or piroplasmids was found. Our findings identify birds, reptiles, and caecilians as potential reservoir hosts of L. amazonensis. In addition, we substantially expanded current knowledge of kinetoplastid diversity in Ecuadorian wildlife. This study demonstrates the value of road-killed animals as a practical, non-invasive resource for wildlife pathogen surveillance and highlights the importance of integrating biodiversity research into One Health approaches to better understand parasite transmission dynamics in rapidly changing tropical ecosystems. Author summaryMany parasites that affect humans circulate naturally in wildlife, but identifying their animal hosts is often difficult in remote, biodiverse regions. We used road-killed animals as a non-invasive source of biological material to investigate parasites in wildlife from two biodiversity hotspots in Ecuador. By analyzing tissues from birds, reptiles, amphibians, and mammals, we found a remarkable diversity of kinetoplastid flagellates, a group that includes the agents of Chagas disease and leishmaniasis. Although we did not detect human-infective trypanosomes, we repeatedly identified Leishmania amazonensis (a species causing human disease) in birds, reptiles, and caecilians. These vertebrate groups have not previously been recognized as potential hosts of this parasite. We also discovered several undescribed trypanosomatid species, emphasizing how little is known about parasite diversity in tropical wildlife. Our results show that road-killed animals can provide valuable information on host-parasite interactions without disturbing living populations. Such surveillance contributes to One Health efforts by improving our understanding of how environmental change, wildlife, and human health are interconnected.

Rift Valley fever (RVF) is a mosquito-borne disease that can cause severe illness and death in both humans and livestock. Since 2016, Uganda has experienced recurrent but localized RVF outbreaks concentrated in the countrys southwestern region. The ecological drivers of this emergence remain unclear, as outbreaks have occurred throughout the year and show little association with meteorological patterns. We evaluated whether crop cultivation, particularly banana cultivation, is associated with RVF outbreak occurrence after controlling for likely confounders. We conducted a longitudinal study of human-inhabited 5 x 5 km grid cells across southwestern Uganda from 2016-2024. Annual Sentinel-2 satellite imagery composites were used to classify land cover into banana, coffee, ground crops, and non-crop categories, and the proportion of each land type was calculated for every grid-cell year. Because land cover proportions are compositional, isometric log-ratio transformations were used to estimate the independent effects of each land type. Confounding was addressed through propensity weighting, and crop substitution effects were estimated using g-computation. Banana land cover was the only land type consistently associated with increased RVF outbreak likelihood. In grid-cell years with low baseline banana cover, a 10-percentage point substitution from other land classes into banana was associated with a 1.64-fold increase in the odds of an RVF outbreak (95% CI: 1.17-2.29). In a simplified banana-only model, each 10-percentage point increase in banana cover was associated with a 1.21-fold increase in outbreak odds (95% CI: 1.02-1.43). Holding banana cover constant, substitutions among coffee, ground crop, and non-crop land showed weak or null associations. These findings suggest that banana cultivation may be an important ecological feature influencing RVF transmission dynamics and outbreak risk in southwestern Uganda. Author SummaryRift Valley fever (RVF) is a mosquito-borne disease that affects both humans and livestock and has caused repeated outbreaks in southwestern Uganda since 2016. While rainfall and flooding are often linked to RVF outbreaks elsewhere, Ugandas recent outbreaks have occurred across seasons and are not well explained by weather patterns alone. We investigated whether agricultural land use could help explain where outbreaks occur. Using satellite imagery from 2016-2024, we measured the amount of banana cultivation, coffee cultivation, ground crops, and non-crop land across southwestern Uganda and evaluated their association with RVF outbreak occurrence. We found that areas with greater banana cultivation were consistently more likely to experience RVF outbreaks, even after accounting for environmental and demographic factors. In contrast, coffee, ground crops, and non-crop land showed little evidence of an independent association with outbreak risk. These findings suggest that banana cultivation may create ecological conditions that favor RVF transmission. Rather than indicating that bananas themselves cause disease, the results point to banana-growing landscapes as potential environments where interactions among mosquitoes, livestock, and humans may increase transmission opportunities. Understanding these local ecological drivers could help improve surveillance, risk assessment, and prevention strategies for RVF in Uganda and other endemic regions.

Ticks transmit pathogens across wildlife, domestic animals, and humans, and are therefore considered important vectors under the One Health framework. In India, the burden of tick-borne diseases, such as Kyasanur Forest Disease and Crimean-Congo Hemorrhagic Fever, remains poorly quantified due to fragmented surveillance and limited spatial data. Here, we present TickMapKB, a curated spatial knowledgebase documenting 72 tick species across more than 600 georeferenced locations in India. The majority of the species belonged to the genera Haemaphysalis, Rhipicephalus, and Hyalomma. Additionally, the resource integrates host associations, pathogen and disease information, morphological identification keys, acaricide resistance profiles, and protein annotations into a single, interactive platform accessible at https://cb.imsc.res.in/tickmapkb/. Specifically, the resource captures 53 pathogens, over 3000 protein annotations, and morphological keys from 26 published resources. TickMapKB thus provides researchers, clinicians, and other stakeholders with integrated spatial, biological, and resistance information to support surveillance planning, resistance management, and tick-borne disease risk assessment.

In many leishmaniasis foci, reservoirs that maintain infection remain unknown. Here, we developed a field-applicable toolkit based on the analysis of individual blood fed sand flies (IBF) to identify reservoirs. Sand flies were given a Leishmania donovani-infected first blood meal (iBM1) by feeding artificially on a membrane or naturally on a clinically ill animal followed by two subsequent uninfected blood meals (BMS+). Bulk-RNAseq was used to identify two target parasite genes, sherp and a novel hypothetical gene (HPB), which exhibited a significantly higher expression in BMS+ compared to iBM1 sand flies. DNA and RNA were co-extracted from IBF. DNA was used to detect Leishmania infection and the blood meal source; RNA was used to assess expression of target genes by qRT-PCR. Linear discriminant analysis (LDA) of target gene expression classified sand fly specimens based on their iBM1 or BMS+ status. Co-extraction yielded a mean of >800ng per IBF for DNA and RNA. We detected [&ge;]1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. LDA identified iBM1 parasites with a predictive accuracy of [~]87% and [~]82%, in membrane or naturally fed sand flies, respectively. This toolkit provides an innovative approach to identification of leishmaniasis reservoirs informing targeted control strategies.