Parasitology

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

A hosts diet can alter the course of parasite infection. This is especially true of trophic parasites, which a host acquires through feeding. While a large body of work attests to the role of diet in the spread of disease within-hosts, diet can also impact host density and encounter rate with parasites, both of which are expected to modify disease dynamics. When parasites are acquired through feeding, epidemics may be larger and more severe on high-quality diets if these diets support a higher density of hosts that feed more and thus ingest more parasites. Alternately, epidemics may be more severe on low-quality diets if malnourishment decreases hosts ability to resist disease. To differentiate these hypothesized effects of diet on disease, we quantified individual infections and epidemic dynamics for the natural microsporidian parasite Nematocida ironsii infecting its nematode host Caenorhabditis elegans. We measured feeding rate, parasite transmission, and host fitness across three bacterial diets that vary in quality and elicit distinct feeding behaviors in C. elegans. We found that low-quality diets reduced feeding rate, which corresponded to reduced acquisition of parasite spores. However, these diet-mediated differences in parasite acquisition did not directly map onto fitness consequences: hosts eating the poor-quality diet had similar reductions in fitness to those on higher quality diets. During epidemics, a combination of increased parasite acquisition and higher population growth rates resulted in higher parasite abundance for hosts on high-quality diets. Our work underscores the importance of considering both individual- and population-level impacts acting in concert to determine how diet affects the spread of infectious disease.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors. Author summaryRhodnius prolixus is a blood-feeding insect and a major vector of Chagas disease, a parasitosis endemic in Latin America and affecting millions of people worldwide. In the absence of effective drugs and vaccines, the control of the insect population represents a promising strategy to reduce the diffusion of the disease. Yet, genetic and functional studies in Rhodnius are extremely challenging due to its feeding habit and life cycle. To overcome these limitations, researchers have previously developed two stable cell lines derived from Rhodnius embryos. In this study, we provide the first characterization of the genes expressed in these cell lines. We found that, while the two cell lines share many expressed genes, each of them also has distinct gene expression patterns pointing to two different cell types with specialized functions. These differences likely affect the way they respond to stress and regulate biological processes. Our findings provide an important resource for researchers studying Rhodnius prolixus and other insect vectors, helping advance our understanding of the genetic and molecular mechanisms that control the insect development and mediate the interactions between insect vectors and the parasites they transmit

The tropical house cricket, Gryllodes sigillatus, is a mass-produced insect that is used as a protein source for pets and livestock. However, intensive mass-rearing conditions, coupled with high genetic relatedness, create an ideal environment for the spread of pathogenic microbes that severely impact production. Cricket iridovirus (CrIV) is a pathogen that impedes cricket growth and causes significant losses for cricket farmers. Interestingly, recent studies have shown that CrIV is often present asymptomatically, yet the molecular basis of the emergence of disease symptoms remains unknown. To address this, we sampled healthy and diseased crickets and examined differences in cricket and CrIV gene expression via RNAseq. Using differential gene expression analysis and functional enrichment analysis, we found significant differences in host and viral gene expression between healthy and diseased crickets, including genes involved in immunity. Interestingly, while we observed high CrIV gene expression across the entire CrIV genome in sick populations, healthy asymptomatic populations showed elevated expression at a single viral locus. Our results shed light not only on the cricket immune response to CrIV infection but also identify a viral gene that is highly expressed during covert infections, suggesting its potential role in suppressing the hosts immune response. These findings enhance our understanding of how CrIV interacts with our cricket host, providing essential insights for developing targeted strategies to manage CrIV outbreaks in cricket mass-rearing facilities.

Helminths are widespread parasites that can modulate host immunity, potentially increasing susceptibility to viral infections. However, evidence for these effects varies across systems and environments, and links between laboratory and wild populations remain unclear. We developed a tractable system using wood mice, Heligmosomoides spp. nematodes, and wood mouse herpes virus (WMHV) to bridge this gap. Combining laboratory and field experiments with population modelling, we examined how helminth infection, anthelmintic treatment and diet affect viral dynamics. Across lab and field data, helminth infection consistently increased WMHV risk, with stronger effects at higher worm burdens. Field results showed that anthelmintic treatment reduced viral infection, and laboratory experiments showed that improved nutrition mitigates helminth-induced increases in viral susceptibility. Our population-level modelling suggested that helminth burden-dependent facilitation can generate nonlinear effects on viral spread, dependent on helminth virulence. Our findings highlight the potential importance of helminths as facilitators of viral infections, and suggest that anthelmintic treatment may provide indirect benefits for viral control. We also show the value of integrating lab and field approaches on the same (or closely related) species, in particular the potential offered by the wood mouse - Heligmosomoides - WMHV system, to understand the drivers and consequences of host-helminth-viral interactions.

Ectotherm insects growth and development are dictated by temperature and humidity. Conducive habitats and the availability of resources set ideal conditions for insect population growth. Mosquitoes require water, favorable temperature, and blood meal to survive. In this research, we picked a rapidly growing megacity, Ahmedabad, in western India, to explore and establish potential linkages between disease spread and meteorological conditions. Ahmedabad, with a population of over 8 million, is experiencing changes in rain and humidity patterns, pushing the city towards changing vector-borne disease dynamics. We examined dengue cases over ten years, 2012-22, and explored their connections with two prominent climatic variables, temperature and relative humidity. Our findings indicate that stable temperature (25-27.5 {degrees}C) and humidity (> 60%) interaction is a ruling factor in spikes in dengue cases in the city. While stable temperature ranges triggers the dengue cases, RH drives the explosive phases and sustainability of such episodes. Statistically significant increasing trends in temperatures, narrowing down of the day-night temperature ranges, and increasing night temperatures provide more stable temperature regimes in a warming world thereby likely to extend the dengue season beyond the usual monsoon season. Plain Language SummaryDengue incidences have been found to be associated with mosquito population outbreaks. Every year, thousands of lives are lost due to this deadly virus spread by mosquitoes. Particularly in the Indian subcontinent, a large proportion of these cases is associated with the monsoon season and rain patterns. In recent years, there have been abrupt spikes in dengue cases across Indian cities, particularly in western India. To understand this complex interaction of viral proliferation and local environmental conditions, the last ten years of dengue case patterns have been scanned in parallel to the climate data. Our findings suggest that stable temperature windows and humidity levels above certain thresholds trigger a rise in dengue cases. While stable temperature ranges trigger dengue cases, humidity drives such episodes explosive phases and sustainability. Our work pinpoints specific temperature-humidity combinations and suggests that local municipal corporations use them as warning indicators to initiate preventive measures.

AimHome range size is a fundamental aspect of animal spatial ecology, and understanding the factors that shape it is important for conservation purposes. Several hypotheses, based on energy needs or competition, assume that home range size negatively correlates with population density. However, this pattern has been little investigated on a global scale, and it remains unclear whether it would stand at both intra- and interspecific levels. To fill this gap, we conducted a global exploration of this relationship at the level of an animal family. Location: Global. Time period: Contemporary. Major taxa studied: Wild Felidae. MethodsIndividual home range size records (n = 1022) and population density estimates (n = 1061) were retrieved from the literature for 23 felid species across the world. We first investigated the interspecific relationship by modelling the median home range size of a species as a function of its median population density. To study the intraspecific relationship, we spatially merged data points based on their spatial or temporal proximity. We then applied a mixed-effects linear model using species as a random factor. ResultsWe found that home range size was negatively associated with population density, at both interspecific (-1.323 {+/-} 0.180, p < 0.001) and intraspecific levels (-0.569 {+/-} 0.201 to - 0.537 {+/-} 0.201 depending on the merging approach, p < 0.01). Landscape features were also predictors of home range size, without confounding the effect of population density. Main conclusionsSeveral processes likely govern the relationship between home range size and population density: differences in body mass between species may drive the interspecific relationship, whereas the intraspecific pattern is probably explained by conspecific competition. Although more research is needed to quantify their relative contribution, our study highlights a worldwide ecological pattern that exists at multiple biological levels in the wild.

Guinea worm disease eradication efforts may benefit from environmental surveillance methods capable of detecting infected copepod intermediate hosts in aquatic habitats. We developed a three-dimensional, spatially explicit agent-based model to examine how ecological processes influence detection probability for a hypothetical water sampling method. The results show that surveillance sensitivity is shaped by the combined effects of larval diffusion, copepod density, and pond size, with interactions among these factors producing nonlinear relationships. Detection, in our model, was concentrated within a relatively restricted period after larvae matured to the infective stage and before dispersal and mortality reduced presence, indicating a limited spatiotemporal window for effective sampling. Surveillance performance peaked under intermediate dispersal regimes that generated sufficient spatial overlap between larvae and intermediate hosts, while both limited dispersal and excessive diffusion reduced detection by constraining encounters or diluting larval concentrations. Increasing habitat size reduced detection by diluting larval concentrations, but the magnitude of this effect depended on copepod density and dispersal dynamics, producing nonlinear and threshold responses rather than simple scaling with pond volume. Spatial and temporal patterns of detection shifted as larvae dispersed, with the most favorable detection periods occurring when both larval abundance and intermediate host encounters were elevated. These findings indicate that surveillance can be guided by local ecological conditions. When the timing of larval introduction is uncertain, effective surveillance requires repeated sampling over time to capture transient windows of detectability and the sampling will be less effective in very stagnant and highly mixed waterbodies. Overall, this study demonstrates how mechanistic modeling can support the design and interpretation of environmental surveillance strategies for Guinea worm eradication programs. Author summaryGuinea worm disease is close to eradication but confirming that transmission has fully stopped remains difficult because detecting infectious larvae in water is challenging. Transmission depends on freshwater copepods that become infected after ingesting Guinea worm larvae. These copepods are short-lived and unevenly distributed within ponds, and infected individuals may die before larvae reach the infective stage. As a result, environmental detection is inherently uncertain. We developed a three-dimensional agent-based model to simulate larval dispersal, copepod infection, and water sampling in a pond environment. The model shows that detection is constrained to a brief period when mature larvae and copepods overlap in space and time, and that this window depends strongly on local ecological conditions such as larval dispersal, copepod density, and pond size. Because infected copepods can be present outside these narrow detection windows, negative water samples do not necessarily indicate absence of transmission, highlighting the need for repeated, spatially targeted surveillance during the final stages of eradication.

Nature-Based Solutions are increasingly promoted to address current urban challenges. While their potential effects on vector-borne disease risks have been documented, data on Aedes albopictus, a known arbovirus vector, remain limited in France. A previous study showed that urban vegetation moderately increases the abundance of adult mosquitoes of this species, but the monitoring period lasted only six months. Using ovitraps, we monitored Ae. albopictus egg density dynamics over multiple years (2022 to 2024) and analysed its environmental predictors in various urban environments. We included lagged meteorological variables, land cover metrics, and the cumulated egg densities recorded in the previous weeks as environmental predictors. Both parametric (GLMM) and non-parametric (Random Forest) models were fitted to weekly egg counts per trap. Our findings highlight that (i) egg density dynamics were related to how vegetation classes structured the landscape, (ii) growing degree days and cumulated number of eggs recorded in specific lagged time windows were the main contributors to egg density, and (iii) the non-parametric and parametric models performed similarly in terms of prediction accuracy.

Tickborne diseases are a significant burden in many parts of the world. In the upper Midwestern United States, Lyme disease is the most common tickborne disease. It is carried by Ixodes scapularis. This vector can also transmit the pathogens causing anaplasmosis, babesiosis, ehrlichiosis, and several more tickborne diseases in this region. There is also concern for other tick species, such as Amblyomma americanum, that are expanding their ranges northward. We launched a citizen science passive tick surveillance program in 2024 to investigate tick species ranges in the upper Midwest, as well as the pathogens carried by I. scapularis. We received over 12,000 ticks in the first two years of this program, primarily from Wisconsin. While we received submissions of adult A. americanum outside of their endemic range, we did not see evidence of establishment in our study area. We measured pathogen prevalence in adult female I. scapularis (n=707) and observed 51% positivity for Borrelia burgdorferi, 9% for Babesia microti, 9% for Anaplasma phagocytophilum, and 3% for Ehrlichia muris eauclairensis. Multiple pathogens were identified in 14% of tested specimens, and significant associations were observed between B. burgdorferi and B. microti, and B. burgdorferi and E. muris eauclairensis. Pathogen prevalences varied across time and geography. Our results can begin to inform risk assessment for tickborne diseases in our region. A non-technical version of this document with interactive maps is available here: https://storymaps.arcgis.com/stories/8008c9d710b5400599f3c6cf88b2c546 Our online data dashboard is available here: redcap.link/TICS

The basic reproduction number R0 is central to malaria epidemiology, yet it is typically treated as a static quantity derived under memoryless assumptions for mosquito demography. In natural systems, however, mosquito populations are shaped by delayed processes such as larval development and density-dependent feedback, introducing biological memory into vector dynamics. We develop a minimal delay-based framework that incorporates this memory into the Ross-Macdonald model by describing adult mosquito abundance with a retarded differential equation. This formulation induces a time-dependent transmission potential R0(t). Using complex analysis and the argument principle, we derive an explicit stability threshold [Formula], which separates stable from oscillatory transmission regimes. Near this threshold, delayed feedback produces slow relaxation times and sustained transient oscillations, implying that transmission potential may vary intrinsically even in the absence of external forcing. To account for ecological variability, we extend this deterministic condition into a probabilistic framework and define the stability probability as [Formula]. Numerical simulations and global sensitivity analysis show that recruitment and developmental delays are the primary drivers of instability, while adult mortality has a weaker stabilizing effect. These results indicate that malaria interventions may influence not only the magnitude of malaria transmission but also its dynamical stability. By linking delay dynamics, transmission theory, and uncertainty quantification, this framework provides a basis for stability-aware modeling and interpretation of malaria transmission under ecological variability. Author summaryMalaria transmission is often summarized by a single number, R0, treated as a fixed indicator of whether transmission will increase or decline. This assumes mosquito populations respond instantly to environmental conditions. In reality, mosquitoes develop through stages where larval conditions, such as crowding, nutrition, or temperature, affect adult populations only after a delay. This creates biological memory: todays mosquitoes reflect past environments. We show that this memory can fundamentally reshape transmission dynamics. When developmental delays are included, transmission potential is no longer constant but can fluctuate over time, even in stable environments. These fluctuations can persist or amplify depending on the balance between mosquito growth, mortality, and delay. As a result, variability in mosquito abundance or malaria transmission may arise from intrinsic dynamics rather than external drivers alone. Under ecological variability, stability becomes probabilistic, allowing estimation of how likely transmission is to remain stable. Interventions that reduce larval productivity or increase adult mortality may therefore both lower transmission and make it more predictable, improving interpretation and control strategies.

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Telomere length (TL) is increasingly used in ecology as a biomarker of individual quality and environmental stress, yet research on non-model species with complex life histories remains limited. Because TL varies among tissues and across ages in a species-specific manner, identifying non-lethal tissues that reliably reflect whole-organism telomere dynamics is essential for longitudinal telomere studies in the field. This study aimed to evaluate tissue-specific TL in Japanese eel (Anguilla japonica), an endangered catadromous fish. We first mapped the chromosomal distribution of telomeric sequences using fluorescent in situ hybridization (FISH), the first application of this method in this species. We then tested whether muscle and caudal fin, which can be sampled easily and non-lethally, can serve as suitable proxy tissues for TL measurements in wild individuals. Relative telomere length (RTL) was quantified by qPCR in blood, brain, caudal fin, gonads, heart, liver, and muscle. FISH analysis confirmed telomeric repeats at all chromosomal ends, with only weak interstitial signals on three chromosomal pairs unlikely to affect qPCR-based estimates. A generalized additive mixed model and Wilcoxons signed-rank tests revealed significant inter-tissue differences: RTL was shortest in the brain and muscle and longest in liver, blood and caudal fin. Muscle and caudal fin RTL were significantly correlated with RTL in many other tissues, supporting their use as proxy tissues for longitudinal TL monitoring, including responses to environmental variation. Both total length and age were tested as explanatory variables for RTL, and the model including total length showed a better fit than the age-based model. Non-linear relationships between RTL and total length observed in several tissues suggest physiological shifts associated with growth and sexual differentiation. Overall, these findings advance understanding of telomere dynamics in eels and establish muscle and caudal fin as suitable tissues for repeated, non-lethal TL assessment in ecological and conservation contexts.

There is disagreement on whether secondary endosymbionts are found in the major cereal pest aphid, Rhopalosiphum padi. Some papers report a diversity of secondary bacterial endosymbionts while others have failed to find evidence of these bacteria in this species. Here we revisit this issue by summarizing the relevant literature and through additional sampling of the species in Australia, China and Denmark using a combination of molecular approaches. We find a general absence of secondary endosymbionts beyond the obligate endosymbiont Hamiltonella defensa in R. padi. While the inconsistency in survey results may reflect rapid changes in endosymbiont turnover in populations and/or the impact of ecological factors such as host plant type on endosymbiont diversity, we are concerned that technical issues may be at least partly responsible for inconsistencies in the literature. This leads us to emphasize the importance of multiple sources of evidence required to establish and characterize endosymbiont infections, including PCR and qPCR assays, DNA Sanger sequencing and 16SrRNA gene metabarcoding. We note that several major aphid pests show a low incidence of secondary endosymbionts which raises issues about the importance of these endosymbionts in aphids that constitute pests, even though endosymbionts can in some cases increase host fitness and therefore pest impact.

Sex ratio is a key demographic parameter shaping population dynamics and evolutionary trajectories. In biocontrol agents, demographic bottlenecks during species introduction to a new habitat and subsequent mass rearing can elevate inbreeding, potentially biasing sex ratios through sex-specific mortality associated with inbreeding depression. Moreover, reproductive endosymbionts such as Wolbachia are known to manipulate host reproduction and further skew sex ratios. However, the relative contributions of these processes to sex-ratio variation remain poorly resolved. In this study, we evaluated the effects of cross-generational full-sibling inbreeding and Wolbachia infection on sex ratio and key life-history traits in the biocontrol beetle Zygogramma bicolorata using controlled laboratory crosses across three generations. Inbreeding did not significantly alter offspring sex ratio, which remained close to parity across generations, while pupal mortality increased in later generations, consistent with delayed expression of inbreeding depression. Adult body weight remained largely unaffected by inbreeding. Wolbachia infection was detected in a subset of females and was associated with a modest but significant increase in female-biased offspring production, although the effect was variable across lineages. Strain typing identified a single supergroup A Wolbachia, consistent with previous descriptions of the wBic strain from this species. These findings indicate that sex-ratio variation in introduced populations of Z. bicolorata is not driven by inbreeding alone but instead emerges from the interaction between demographic processes and symbiont-mediated effects, providing crucial insights for optimizing biocontrol programs where sex-ratio stability is essential for population establishment and persistence. SignificanceSex ratio is a key determinant of population growth and stability - the essential parameters determining success of biocontrol programs. Yet, the mechanisms shaping sex-ratio variation remain poorly resolved. Using controlled crosses in Zygogramma bicolorata, we show that short-term inbreeding does not directly alter sex allocation, despite inducing delayed fitness costs through increased pupal mortality. In contrast, Wolbachia infection contributes to female-biased offspring production, although with variable outcome across lineages. These findings demonstrate that sex-ratio variation in Z. bicolorata arises from the interaction of demographic processes and symbiont effects, rather than a single mechanism, with important implications for predicting the establishment, persistence, and efficacy of mass-reared biocontrol populations.

BackgroundControl of Aedes aegypti, the primary vector of dengue and other Aedes-borne viruses, is challenged by insecticide resistance, limited efficacy of existing tools and the large and widespread epidemics. Targeted Indoor Residual Spraying (TIRS), a modification of traditional indoor residual spraying focused on Ae. aegypti resting sites, has demonstrated promising results, yet its indirect community-wide effects remain underexplored. Methodology/Principal FindingsWe conducted an entomological cluster-randomized controlled trial in Iquitos, Peru, to evaluate the direct and indirect entomological impacts of TIRS using pirimiphos-methyl. Thirty clusters were randomized to receive either TIRS (15 clusters, 898 structures) or standard Ministry of Health vector control activities (15 clusters, 1,018 structures). Aedes aegypti indoor densities were assessed in the 45 days pre-intervention and at four time points up to 255 days post-intervention using Prokopack aspiration. Generalized linear mixed models with a negative binomial link were used to estimate incidence rate ratios (IRRs) and calculate efficacy (1-IRR) for houses that received TIRS (direct effect) and untreated houses in TIRS clusters (indirect effect). Direct efficacy reached 96% at 15 days post-spraying and remained significant (40%) at 255 days post-spraying. Indirect efficacy reached 69% at 15 days and declined to 7% by 255 days post-spraying. Despite only 57% household-level TIRS coverage, both direct and indirect impacts on Ae. aegypti were significant during early post-intervention surveys, and after 8 months in TIRS clusters. Conclusions/SignificanceTIRS provided substantial and sustained reductions in indoor Ae. aegypti density, including measurable indirect effects in untreated homes within intervention clusters. These findings demonstrate the entomological value of TIRS even at moderate coverage levels and highlight its potential for both preventive and reactive vector control programs and should be considered for implementation by Ministries of Health in dengue-endemic urban settings as well as by the U.S. military when deployed to tropical or subtropical locations.

Giraffes, unlike other large mammals, typically pose minimal risk to humans, their animals, and crops, so they are traditionally not involved in human-wildlife conflict. Tree crops, however, are expanding across Africa, resulting in crop raiding by giraffes and retaliatory snaring, poaching, and killing of giraffes in response. The dynamics of giraffe crop raiding, however, are poorly understood, making effective interventions difficult to implement. To better understand key factors for humans and giraffes that mediate crop raiding, we used a multi-method approach to estimate giraffe abundance and activity, understand farmers perceptions and decisions, and test countermeasures around Garissa Giraffe Sanctuary in eastern Kenya. We hypothesized that 1) giraffe farm invasion would occur in dry seasons, 2) farms growing mangoes would be more likely to be invaded, 3) reducing invasion with only physical barriers would be less effective than adding behavior-based countermeasures, 4) perceptions would match giraffe activity and 5) countermeasure adoption would be driven by cost. We found that invasion and crop raiding primarily occur during the dry season and are associated with mangoes. Farmers are using many countermeasures. Effective countermeasures target giraffe behavior combined with physical barriers. Countermeasures are most effective when negative associations with humans are reinforced. Floodlights and speakers that play predator calls both reduce invasion, but only if used consistently. Overall, farmers perceptions matched giraffe dynamics. Availability was the most important factor in farmers willingness to try a countermeasure. Our results suggest that conflict can be reduced and there is interest from farmers in doing so, but use of countermeasures should be consistently applied and supported by making necessary equipment and instructions available.

ObjectiveRecently, alternatives to animal testing, such as new approach methodologies, are being developed in the orthopedic research field; animal models still provide valuable insights into the pathogenesis of knee osteoarthritis (OA). However, commonly used models develop OA much more rapidly and severely than those observed in human patients. We aimed to develop a novel murine model that closely mimics the slow progression of human OA with posterior Cruciate ligament (PCL) rupture. Design12-week-old C57BL/6 mice were induced to PCL-rupture (PCL-R) by manually applying an external tibial posterior translation force. We analyzed joint kinematics, histological observations, and bone structure to confirm the absence of concurrent injury on day 0. Then, joint stability and the pathophysiological progression of knee OA were analyzed at 8, 16, and 34 weeks post-PCL-R. The destabilized medial meniscus (DMM) model was also analyzed to compare the OA progression. ResultsNon-invasive PCL-R intervention induced the complete rupture in the central region of PCL without concurrent injury. The PCL-R group showed larger posterior tibial deviation than the INTACT (P=0.008). Regarding the range of motion in the PCL-R group, there was no limitation in range of motion on day 0, but extension limitations occurred at weeks 16 and 34 weeks. Histologically, articular cartilage degeneration in PCL-R was milder than DMM. In the subchondral bone, micro-CT reconstruction images indicated that, compared with the INTACT group, the DMM group observed progressive subchondral bone formation from 16 weeks post-surgery. In contrast, the PCLR group maintained the subchondral bone structure even at 34 weeks. ConclusionsPCL-R model induced mild abnormal mechanical stress depending on posterior instability, and cartilage degeneration occurred more slowly in this model than in DMM models.

IntroductionHealthy and diseased placentae alike often display some degree of pathology. However, quantitative techniques to characterize common pathologies and their relationship to local maternal hemodynamics in healthy primate placentae are currently limited. MethodsPlacentae from seven rhesus macaques were imaged by MRI at three time points across mid-to late-gestation, to quantify placental blood volume, flow, and perfusion from maternal spiral arteries across pregnancy. Near term, we collected placental cotyledons, digitized hematoxylin/eosin-stained slides, then segmented and annotated sub-tissues and major pathologies (intervillous gaps, fibrin deposition, villous agglutination, inflammatory agglutination, and stromal mineralization) within each cotyledon. Individual pathologies were assessed in relation to each other and MRI perfusion metrics, in a cotyledon-specific manner. Parallel analyses were performed to investigate both basic (Spearman correlation) and animal variance-negated (dimensionality-reduction) relationships. ResultsCotyledons with increased stromal mineralization demonstrated low blood perfusion across pregnancy, alongside significant compensatory changes. Mineralization was further associated with decreased fetal weight, across all sub-tissues. Dimensionality reduction revealed maternal vascular malperfusion-associated pathologies as the largest contributor to dataset variance. Additionally, pathologies commonly associated with healthy placental function demonstrated low cotyledon blood flow and volume at all timepoints, with no evidence of compensatory changes across gestation. ConclusionsComprehensive digital annotation revealed several relationships connecting pathology and maternal blood perfusion in the healthy primate pregnancy, at the smallest functional unit of the placenta. This methodological framework embeds pathologist-refined morphological expertise into a quantitative, spatially resolved format that can ground, rather than be replaced by, unsupervised computational approaches to placental analysis.