Anopheles stephensi is rapidly expanding across Africa, posing new challenges for malaria control. Its biting time patterns, however, remain poorly characterized, raising uncertainty about the effectiveness of bed nets against this invasive vector. To address this gap, we investigated diel biting activity, feeding propensity, and flight behavior using complementary behavioral assays on females reared from wild-caught larvae in Hawassa City, Ethiopia. Flight monitoring assays revealed that An. stephensi exhibited pronounced activity at dusk, beginning between 18:00 and 19:00 with scotophase onset, and little to no activity during photophase. Blood-feeding propensity, defined as the proportion of mosquitoes taking a blood meal when offered, peaked during early scotophase (18:00-22:00) at 33.3-51.7% but was markedly reduced during daylight hours (0-16.7%). Human landing catches in large-cage enclosures confirmed this early-evening activity: 83.5% of total landings occurred between 18:00 and 22:00, with a sharp peak at 18:00-19:00, corresponding to a mean biting rate of 17.8 bites per person per hour. These findings demonstrate that invasive An. stephensi primarily seek hosts and bite during the early evening, when people are often unprotected by bed nets. This behavior reduces the protective impact of conventional net-based interventions and underscores the need for African National Malaria Control Programs to deploy complementary measures such as spatial repellents and larval source management to mitigate early-evening transmission. Moreover, this study highlights the utility of integrated behavioral assays for estimating biting time, offering approaches that can be extended to other vector species across Africa.

Vector-borne diseases pose a persistent and increasing challenge to human, animal, and agricultural systems globally. Mathematical modeling frameworks incorporating vector trait responses are powerful tools for assessing risk and predicting vector-borne disease impacts. Developing these frameworks and the reliability of their predictions hinge on the availability of experimentally derived vector trait data for model parameterization and inference of the biological mechanisms underpinning transmission. Trait experiments have generated data for many known and potential vector species, but the terminology used across studies is inconsistent, and accompanying publications may share data with insufficient detail for reuse or synthesis. The lack of data standardization can lead to information loss and prohibit analytical comprehensiveness. Here, we present MIReVTD, a Minimum Information standard for Reporting Vector Trait Data. Our reporting checklist balances completeness and labor intensity to make these important experimental data easier to find and reuse, without onerous effort for scientists generating the data. To illustrate the standard, we provide an example reproducing results from an Aedes aegypti mosquito study.

The dynamics of vector-borne disease transmission depend on the abundances of vectors. The dominant malaria vector species complex of Anopheles gambiae sensu lato is a target of vector control strategies designed to reduce and eliminate malaria transmission. The three most widely distributed dominant malaria vectors within the species complex are An. arabiensis, An. coluzzii and An. gambiae sensu stricto. Previous studies across the extent of the species complex range have been restricted to annual predictions of species occurrence or relative abundance. This study incorporated relative abundance data at the species level and abundance data at the species complex level to estimate and predict daily equilibrium vector abundances of each species. Statistical models with interpretable parameters were used to gain insight into how factors such as meteorological conditions, insecticide-treated net use and human density affect the spatial and temporal predictions. The statistical models were used to predict daily equilibrium vector abundance, which is an important factor in indices of malaria transmission such as the basic reproduction number and vectorial capacity. Meteorological factors, such as precipitation and relative humidity, and human factors, such as population density and insecticide-treated nets, were important predictors of these three malaria vector species in Africa. Insecticide-treated net use was negatively associated with per capita vector abundance of the An. gambiae species complex and interacted with year to suggest a negative effect on the ratio of An. gambiae s.s. to An. arabiensis at the end of the prediction period that extended from 2002 to 2020. A predicted increasing interannual trend for An. coluzzii was potentially caused by changing species identification methods. The predicted equilibrium abundances of the three species showed potentially high levels of geographical overlap, niche overlap and evidence for stable coexistence despite substantial interspecific competition. Improving collection of longitudinal species abundance data across the spatial range of the An. gambiae species complex will facilitate future explorations of causal hypotheses that relate vector abundance to control measures, malaria interventions and meteorological conditions.

The Asian malaria vector Anopheles stephensi is invading Africa, requiring it to adapt to novel climates and ecosystems. In part, this may be facilitated by An. stephensi ’s poorly understood seasonal behavioural plasticity in flight timing, leading to earlier biting activity in cold Asian winters and later biting times in the warm summer. Changes in behavioural timing could be directly imposed by seasonal variation in ambient light and temperature levels or result from altered entrainment of intrinsically expressed circadian rhythms by these factors. We demonstrate that An. stephensi entrained flight activity timing is phase-locked to dawn and is not affected by constant ambient temperature, which cannot explain earlier biting activity in colder winters with later dawn. Instead, we show that where night temperatures are the colder part of daily temperature cycle; the entrained phase-angle between dawn and flight activity is altered, hereby increasingly colder, winter-like nights progressively advance flight activity onset. We propose that seasonal timing plasticity optimizes behaviour to warmer daytime in winter, and colder nights in summer, providing protection against both heat-desiccation and cold immobility. The adaptive advantage of this plasticity could be relevant to the successful invasion and survival of An. stephensi in African climates, and changing climate worldwide. This article is part of the Theo Murphy meeting issue, ‘Circadian rhythms in infection and immunity’.

BACKGROUND:Transmission of vector-borne pathogens follows daily rhythms, occurring at the time of day that vectors forage for blood. Control measures, such as insecticide-treated bed nets (ITNs), have been particularly successful for reducing malaria transmission because they exploit the nocturnal biting behaviour of the Anopheles spp. that vector malaria. However, shifts in biting behaviour to earlier or later hours when people are unprotected can undermine the efficacy of ITNs. Despite the implications for malaria transmission, the mechanisms underlying these shifts remain poorly understood. Because food availability mediates activity and foraging rhythms, and ITNs block access to human blood as a food source, it was hypothesized that nutritional deprivation could cause mosquitoes to shift their biting behaviour to earlier or later in the diel cycle. METHODS:Female Anopheles gambiae sensu lato (s.l.) mosquitoes were provided with a blood meal on day 3 post-emergence, and access to one of three feeding treatments that varied in nutritional resources: (i) 0.5% sucrose, (ii) 10% sucrose, or (iii) 10% sucrose plus an additional blood meal on day 6. Mosquitoes were released into a semi-field system on day 10 with human-mimic traps to investigate how food availability impacted the time of day that mosquitoes host seek. RESULTS:Nutritional resources determine both the likelihood and time of day that host-seeking occurs. Specifically, low-resourced mosquitoes were 2-3-fold more likely to host seek overall, and 5-10-fold more likely to host seek at an earlier time of day than well-resourced mosquitoes (fed 10% sucrose with and without an additional blood meal), which predominantly sought a host in the second half of the night time. CONCLUSIONS:This study reveals that mosquito nutritional condition drives plasticity in biting time of day, suggesting it is an underappreciated contributor to residual malaria transmission. Furthermore, the findings suggest that targeting mosquito nutrition (e.g. sugar-baited traps) could influence mosquito behaviour in ways that affect the success of ITNs. More broadly, understanding the drivers of biting time of day variation is crucial for the future success of vector control tools and controlling malaria transmission.

Background Approximately twenty-one years of historical mosquito abundance and species surveillance data, collected by the University of Notre Dame and the St. Joseph County (IN) Health Department, from 1976 to 1997 are made available following a data rescue effort. St. Joseph County is a county in Indiana, located on the Michigan-Indiana border, 35 miles from Lake Michigan. New information The collected data will allow for trends in species to be followed over a wide time range and facilitate further research regarding mosquito-borne diseases, species distribution, phenology and ecological changes overtime.

Africa and the United States are both large, heterogeneous geographies with a diverse range of ecologies, climates and mosquito species diversity which contribute to disease transmission and nuisance biting. In the United States, mosquito control is nationally, and regionally coordinated and in so much as the Centers for Disease Control (CDC) provides guidance, the Environmental Protection Agency (EPA) provides pesticide registration, and the states provide legal authority and oversight, the implementation is usually decentralized to the state, county, or city level. Mosquito control operations are organized, in most instances, into fully independent mosquito abatement districts, public works departments, local health departments. In some cases, municipalities engage independent private contractors to undertake mosquito control within their jurisdictions. In sub-Saharan Africa (SSA), where most vector-borne disease endemic countries lie, mosquito control is organized centrally at the national level. In this model, the disease control programmes (national malaria control programmes or national malaria elimination programmes (NMCP/NMEP)) are embedded within the central governments' ministries of health (MoHs) and drive vector control policy development and implementation. Because of the high disease burden and limited resources, the primary endpoint of mosquito control in these settings is reduction of mosquito borne diseases, primarily, malaria. In the United States, however, the endpoint is mosquito control, therefore, significant (or even greater) emphasis is laid on nuisance mosquitoes as much as disease vectors. The authors detail experiences and learnings gathered by the delegation of African vector control professionals that participated in a formal exchange programme initiated by the Pan-African Mosquito Control Association (PAMCA), the University of Notre Dame, and members of the American Mosquito Control Association (AMCA), in the United States between the year 2021 and 2022. The authors highlight the key components of mosquito control operations in the United States and compare them to mosquito control programmes in SSA countries endemic for vector-borne diseases, deriving important lessons that could be useful for vector control in SSA.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

A growing body of information on vector-borne diseases has arisen as increasing research focus has been directed towards the need for anticipating risk, optimizing surveillance, and understanding the fundamental biology of vector-borne diseases to direct control and mitigation efforts. The scope and scale of this information, in the form of data, comprising database efforts, data storage, and serving approaches, means that it is distributed across many formats and data types. Data ranges from collections records to molecular characterization, geospatial data to interactions of vectors and traits, infection experiments to field trials. New initiatives arise, often spanning the effort traditionally siloed in specific research disciplines, and other efforts wane, perhaps in response to funding declines, different research directions, or lack of sustained interest. Thusly, the world of vector data - the Vector Data Ecosystem - can become unclear in scope, and the flows of data through these various efforts can become stymied by obsolescence, or simply by gaps in access and interoperability. As increasing attention is paid to creating FAIR (Findable Accessible Interoperable, and Reusable) data, simply characterizing what is 'out there', and how these existing data aggregation and collection efforts interact, or interoperate with each other, is a useful exercise. This study presents a snapshot of current vector data efforts, reporting on level of accessibility, and commenting on interoperability using an illustration to track a specimen through the data ecosystem to understand where it occurs for the database efforts anticipated to describe it (or parts of its extended specimen data).

VectorBase (VectorBase.org) is part of the VEuPathDB Bioinformatics Resource Center, providing free online access to multi-omics and population biology data, focusing on arthropod vectors and invertebrates of importance to human health. VectorBase includes genomics and functional genomics data from bed bugs, biting midges, body lice, kissing bugs, mites, mosquitoes, sand flies, ticks, tsetse flies, stable flies, house flies, fruit flies, and a snail intermediate host. Tools include the Search Strategy system and MapVEu, enabling users to interrogate and visualize diverse 'omics and population-level data using a graphical interface (no programming experience required). Users can also analyze their own private data, such as transcriptomic sequences, exploring their results in the context of other publicly-available information in the database. Help Desk: help@vectorbase.org.

Abstract Mosquitoes and other blood feeding arthropods are vectors of pathogens causing serious human diseases, such as Plasmodium spp. (malaria), Wuchereria bancrofti (lymphatic filariasis), Borrelia burgdorferi (Lyme disease), and viruses causing dengue, Zika, West Nile, chikungunya, and yellow fever. Among the most effective strategies for the prevention of vector-borne diseases are those aimed at reducing human–vector interactions, such as insecticide applications and insecticide-treated bed nets (ITNs). In some areas where ITNs are widely used, behavioral adaptations have resulted in mosquitoes shifting their time of blood feeding to earlier or later in the night when the bed nets are not being employed. Little is known about the genetic basis of these behavioral shifts. We conducted quantitative trait locus (QTL) analysis using two strains of Culex pipiens sensu lato with contrasting blood feeding behaviors, wherein the lab adapted Shasta strain blood feeds at any time of the day or night, while the newly established Trinidad strain feeds only at night. We identified a single locus on chromosome 2 associated with the observed variation in feeding times. None of the core clock genes period, timeless, clock, cycle, PAR-domain protein 1, vrille, discs overgrown, cryptochrome 1, or cryptochrome 2 were located within the QTL region. We then monitored locomotor behavior to determine if they differed in their flight activity. The highly nocturnal Trinidad strain showed little daytime activity while the day-feeding Shasta strain was active during the day, suggesting blood feeding behavior and flight activity are physiologically linked.

Aedes aegypti mosquito is a major vector of arboviral disease. Here, we report that the biting behavior of normally daytime active anthropophilic Ae. aegypti mosquitoes on human hosts is abnormally increased at night following exposure to artificial light at night (ALAN). Biting was examined using a human host assay where caged mosquitoes were exposed to a human arm and blood-feeding measured. Mosquitoes were tested during the daytime, nighttime, or challenged with ALAN. As predicted from the Ae. aegypti diel/circadian biting cycle, maximal biting occurred during daytime and lowest level occurred at night. Biting in the ALAN group was increased compared with time-matched nighttime controls. These data reveal that exposure to ALAN increases nocturnal blood-feeding behavior. This finding highlights the concern that globally increasing levels of light pollution could be impacting arboviral disease transmission, such as dengue fever and Zika, and has implications for application of countermeasures for mosquito vector control.

Arthropods play a dominant role in natural and human-modified terrestrial ecosystem dynamics. Spatially-explicit arthropod population time-series data are crucial for statistical or mathematical models of these dynamics and assessment of their veterinary, medical, agricultural, and ecological impacts. Such data have been collected world-wide for over a century, but remain scattered and largely inaccessible. In particular, with the ever-present and growing threat of arthropod pests and vectors of infectious diseases, there are numerous historical and ongoing surveillance efforts, but the data are not reported in consistent formats and typically lack sufficient metadata to make reuse and re-analysis possible. Here, we present the first-ever minimum information standard for arthropod abundance, Minimum Information for Reusable Arthropod Abundance Data (MIReAD). Developed with broad stakeholder collaboration, it balances sufficiency for reuse with the practicality of preparing the data for submission. It is designed to optimize data (re)usability from the "FAIR," (Findable, Accessible, Interoperable, and Reusable) principles of public data archiving (PDA). This standard will facilitate data unification across research initiatives and communities dedicated to surveillance for detection and control of vector-borne diseases and pests.

Background: Biological rhythms allow organisms to compartmentalise and coordinate behaviours, physiologies, and cellular processes with the predictable daily rhythms of their environment. There is increasing recognition that the biological rhythms of mosquitoes that vector parasites are important for global health. For example, whether perturbations in blood foraging rhythms as a consequence of vector control measures can undermine disease control. To address this, we explore the impacts of altered timing of blood-feeding on mosquito life history traits and malaria transmission. Methods: We present three experiments in which Anopheles stephensi mosquitoes were fed in the morning or evening on blood that had different qualities, including: (i) chemical-induced or (ii) Plasmodium chabaudi infectioninduced anaemia; (iii) Plasmodium berghei infection but no anaemia; or (iv) stemming from hosts at different times of day. We then compared whether time-of-day variation in blood meal characteristics influences mosquito fitness proxies relating to survival and reproduction, and malaria transmission proxies. Results: Mosquito lifespan is not influenced by the time-of-day they received a blood meal, but several reproductive metrics are affected, depending on other blood characteristics. Overall, our data suggest that receiving a blood meal in the morning makes mosquitoes more likely to lay eggs, lay slightly sooner and have a larger clutch size. In keeping with previous work, P. berghei infection reduces mosquito lifespan and the likelihood of laying eggs, but time-of-day of blood-feeding does not impact upon these metrics nor on transmission of this parasite. Conclusion: The time-of-day of blood-feeding does not appear to have major consequences for mosquito fitness or transmission of asynchronous malaria species. If our results from a laboratory colony of mosquitoes living in benign conditions hold for wild mosquitoes, it suggests that mosquitoes have sufficient flexibility in their physiology to cope with changes in biting time induced by evading insecticide-treated bed nets. Future work should consider the impact of multiple feeding cycles and the abiotic stresses imposed by the need to forage for blood during times of day when hosts are not protected by bed nets.

The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour ("circadian") manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are 'missing' from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of 'missing' peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly.

Infection can dramatically alter behavioural and physiological traits as hosts become sick and subsequently return to health. Such “sickness behaviours” include disrupted circadian rhythms in both locomotor activity and body temperature. Host sickness behaviours vary in pathogen species-specific manners but the influence of pathogen intraspecific variation is rarely studied. We examine how infection with the murine malaria parasite, Plasmodium chabaudi , shapes sickness in terms of parasite genotype-specific effects on host circadian rhythms. We reveal that circadian rhythms in host locomotor activity patterns and body temperature become differentially disrupted and in parasite genotype-specific manners. Locomotor activity and body temperature in combination provide more sensitive measures of health than commonly used virulence metrics for malaria (e.g. anaemia). Moreover, patterns of host disruption cannot be explained simply by variation in replication rate across parasite genotypes or the severity of anaemia each parasite genotype causes. It is well known that disruption to circadian rhythms is associated with non-infectious diseases, including cancer, type 2 diabetes, and obesity. Our results reveal that disruption of host circadian rhythms is a genetically variable virulence trait of pathogens with implications for host health and disease tolerance.

Members of the Culex pipiens complex differ in physiological traits that facilitate their survival in diverse environments. Assortative mating within the complex occurs in some regions where autogenous (the ability to lay a batch of eggs without a blood meal) and anautogenous populations are sympatric, and differences in mating behaviors may be involved. For example, anautogenous populations mate in flight/swarms, while autogenous populations often mate at rest. Here, we characterized flight activity of males and found that anautogenous strain males were crepuscular, while autogenous strain males were crepuscular and nocturnal, with earlier activity onset times. We conducted quantitative trait locus (QTL) mapping to explore the genetic basis of circadian chronotype (crepuscular vs. crepuscular and nocturnal) and time of activity onset. One major-effect QTL was identified for chronotype, while 3 QTLs were identified for activity onset. The highest logarithm of the odds (LOD) score for the chronotype QTL coincides with a chromosome 3 marker that contains a 15-nucleotide indel within the coding region of the canonical clock gene, cryptochrome 2. Sequencing of this locus in 7 different strains showed that the C-terminus of CRY2 in the autogenous forms contain deletions not found in the anautogenous forms. Consequently, we monitored activity in constant darkness and found males from the anautogenous strain exhibited free running periods of ~24 h while those from the autogenous strain were ~22 h. This study provides novel insights into the genetic basis of flight behaviors that likely reflect adaptation to their distinct ecological niches.

Despite the major impact of mosquitoes on human health, knowledge gaps exist regarding their natural population dynamics. Even the most basic information-such as spatiotemporal abundance-is mostly unavailable. In the USA, municipalities have created agencies for mosquito control and monitoring, yet no national open-access repository for mosquito surveillance data exists. Vectors, and the pathogens they transmit, know no jurisdictions. We identify >1,000 mosquito control agencies and identify those which make their population abundance surveillance data publicly available. We directly survey Floridian mosquito districts to estimate, from one state alone, the potential amount of hidden data. We generate a large, standardized data set from publicly available online data and demonstrate that spatiotemporal population abundance can be reconstructed and analyzed across data generators. We propose that the ensemble of US mosquito control agencies can, and should, be used to develop a national-and potentially international-open-access repository of mosquito surveillance data, generating the data capital needed to gain a mechanistic understanding of vector population dynamics, and identify existing digital infrastructure that could be leveraged for digitizing and collating extant and future surveillance data for such a repository.

Daily rhythms in behaviour, physiology and molecular processes are expected to enable organisms to appropriately schedule activities according to consequences of the daily rotation of the Earth. For parasites, this includes capitalizing on periodicity in transmission opportunities and for hosts/vectors, this may select for rhythms in immune defence. We examine rhythms in the density and infectivity of transmission forms (gametocytes) of rodent malaria parasites in the host's blood, parasite development inside mosquito vectors and potential for onwards transmission. Furthermore, we simultaneously test whether mosquitoes exhibit rhythms in susceptibility. We reveal that at night, gametocytes are twice as infective, despite being less numerous in the blood. Enhanced infectiousness at night interacts with mosquito rhythms to increase sporozoite burdens fourfold when mosquitoes feed during their rest phase. Thus, changes in mosquito biting time (owing to bed nets) may render gametocytes less infective, but this is compensated for by the greater mosquito susceptibility.