Biology subjects

Hol, F. J.

Publications and source records attributed to Hol, F. J..

8 recordsLinked to original sources

bioRxiv2026

Deep behavioral phenotyping of pathogen infected mosquitoes reveals species-specific behavior changes enhancing transmission

Mosquito-borne pathogens are often proposed to alter mosquito behavior to enhance transmission, yet the prevalence, magnitude, and consequences of such effects remain unclear. Using custom high- throughput behavioral assays and deep-learning analysis, we quantified the blood-feeding behavior of more than 5,000 mosquitoes, and long-term activity patterns of more than 1,000 mosquitoes across six clinically relevant mosquito-pathogen combinations. Infection effects were highly species-specific. Anopheles stephensi infected with an African Plasmodium falciparum strain showed elevated flight activity throughout infection, an increased tendency to blood-feed upon host contact, and prolonged feeding times. In contrast, infection with an Asian P. falciparum strain or P. vivax had little impact on An. stephensi behavior. We detected no behavioral changes associated with P. falciparum infection in Anopheles gambiae s.s. or Anopheles coluzzii. Interestingly, dengue virus infection in Aedes aegypti increased landing, probing, and engorgement rates. Together, these results demonstrate that infection-induced behavioral change is not a general phenomenon among mosquito-borne pathogens, yet differs markedly per mosquito and pathogen species. A transmission model parameterized with our data showed that even modest behavioral shifts can substantially increase transmission, underscoring the potential epidemiological importance of subtle phenotypic shifts.

animal behavior and cognition↗

bioRxiv2026

Quantifying sporozoite inoculum dynamics with the SpitGrid reveals temporal decay and behavioral determinants of Plasmodium transmission

The number of Plasmodium sporozoites expelled by an infected mosquito during a bite, the inoculum size, is a critical but rarely measured factor in transmission. Its temporal dynamics, individual variation, and behavioral underpinnings remain poorly understood. To address this, we developed the SpitGrid, a modular platform that supports natural probing and feeding on artificial substrates, while enabling behavioral characterization combined with non-lethal, sensitive measurement of inoculum size for individual mosquitoes in high-throughput. Using the SpitGrid, we determined inoculum size of Plasmodium falciparum-infected Anopheles stephensi mosquitoes over time and identified a robust temporal structure. Inoculum size peaked shortly after the extrinsic incubation period, and declined approximately log-linearly post peak. Salivary gland parasite load decreased only moderately and was positively associated with inoculum size during the early phase of sporozoite expelling, yet explained only part of the variation observed in inoculum size. Fine-grained behavioral classification confirmed that probing alone can deliver substantial inocula, and revealed that probing more often increased inoculum size while the duration of probing, or if this led to feeding, had little effect. Application of the SpitGrid to Plasmodium vivax-infected mosquitoes similarly showed a temporally structured expelling profile, with generally smaller inocula and a higher proportion of non-expelling mosquitoes. Our observations reveal that Plasmodium infectivity in mosquitoes is a highly dynamics process, challenging the assumption that per-bite transmission potential remains constant over time. We establish the SpitGrid as a flexible tool to gain a nuanced understanding of the determinants of transmission, refine epidemiological models, and evaluate the efficacy of vector- based interventions under biologically relevant conditions. HighlightsWe develop the SpitGrid, a flexible, accessible, low-cost and animal free platform to measure sporozoite expelling We identify a peak in P. falciparum and P. vivax sporozoite expelling by infected Anopheles stephensi mosquitoes early after completion of the extrinsic incubation period, followed by a gradual decline We confirm a correlation between salivary gland load and inoculum size that weakens over time, indicating that a smaller fraction of the remaining salivary gland load is being expelled by older mosquitoes We confirm that mosquito probing alone can deliver substantial sporozoite inoculum independently of a successful bloodmeal By dissecting the behavioral parameters that determine inoculum size, we observe that the initiation of probing is a major determinant of inoculum size with relatively minor roles for probing time and whether biting results in engorgement Compared to P. falciparum, we observe comparatively smaller inocula, in P. vivax-infected mosquitoes a higher rate of non-expellers and similar albeit more moderate temporal structure with an earlier onset of expelling, illustrating cross-species utility of the SpitGrid

microbiology↗

bioRxiv2025

Expelling of P. falciparum sporozoites by Anopheles stephensi mosquitoes during repeated feeding

It is currently unclear whether repeated mosquito feeding attempts achieve pathogen transmission. We measured Plasmodium falciparum sporozoite expelling in Anopheles stephensi mosquitoes during two interrupted feeding attempts. The number of expelled sporozoites was positively correlated with salivary gland sporozoite load during the first feeding effort ({rho}= 0.36, p= 0.0181) but less so during subsequent feeding ({rho}= 0.06, p= 0.6811). The median number of expelled sporozoites was of similar magnitude during the first and second feeding event, with a strong correlation between them ({rho}= 0.56, p= 0.0002). We conclude that repeated feeding results in repeated sporozoite inoculation at similar intensity.

microbiology↗

bioRxiv2025

Blood, sweat, and beers: investigating mosquito biting preferences amidst noise and intoxication in a cross-sectional cohort study at a large music festival

IntroductionWe live in a world split between mosquito magnets and those lucky enough to remain (nearly) untouched. The reasons why some people attract more mosquito bites than others remain largely mysterious. In the Mosquito Magnet Trial, we investigated differences in mosquito attraction amongst festivalgoers with varying levels of hygiene and intoxication. MethodsOur study was conducted in a slightly steamy pop-up laboratory inside four connected shipping containers at Lowlands Festival in The Netherlands (August 18-20, 2023). Participants completed an anonymous questionnaire on hygiene, diet, and festival-related behaviour (including alcohol uptake and shared sleeping arrangements). Mosquito attraction was measured using a custom designed setup: a transparent cage with perforations where female Anopheles mosquitoes were offered a choice between a sugar-feeder and the participants arm. Mosquitoes could only smell, not bite, the participants arm. Attraction was quantified through video imaging, measuring arm landings relative to total landings. Mosquito attraction was correlated with questionnaire responses and skin microbiota profiles collected from forearm skin swabs. ResultsAmongst the 465 included participants, mosquitoes showed a clear fondness for those who drank beer over those who abstained from the liquid gold (Fold Change 1.35, 95% CI 1.12-1.63, PFDR<0.001). Attraction was also contagious: participants that successfully lured a fellow human into their tent the previous night also proved more enticing to mosquitoes (FC 1.34, 95% CI 1.14-1.58, PFDR=0.002). Meanwhile, skipping the morning showering routine and using sunscreen reduced mosquito attraction (FC 0.52, 95% 0.38-0.70, PFDR<0.001). Streptococci were more abundant on the skin of highly attractive individuals (Puncorrected=0.017), and the overall abundance of malodour associated bacteria was high - possibly reflecting the containers smell. ConclusionThe Mosquito Magnet Trial, to our knowledge the largest study of its kind, was conducted in a loosely controlled setting with a selection bias towards science loving festivalgoers. That said, using our custom designed experimental set-up, we found that mosquitoes are drawn to those who avoid sunscreen, drink beer, and share their bed. They simply have a taste for the hedonists among us.

animal behavior and cognition↗

bioRxiv2025

BuzzWatch: uncovering multi-scale temporal patterns in mosquito behavior through continuous long-term monitoring

Understanding the temporal dynamics of mosquito behavior is essential for developing effective interventions against pathogen transmission. However, limited knowledge exists about the environmental, physiological, and genetic factors influencing mosquito activity patterns. This knowledge gap is partly due to a lack of tools to accurately quantify the behavior of free-flying mosquitoes over extended periods. Here, we introduce BuzzWatch, an open-source, low-cost platform designed to continuously monitor mosquito flight behavior over several weeks with high temporal resolution. BuzzWatch records videos of mosquitoes freely flying in a transparent cage and automates the extraction, analysis, and visualization of behavioral data, including flight trajectories and population-level flight and sugar-feeding statistics. Using BuzzWatch, we quantified the daily rhythms of 10 Aedes aegypti populations from various geographic origins. Globally invasive Ae. aegypti showed increased sugar feeding and flight activity during midday compared to native African populations. Our platform further revealed subtle, long-lasting effects of blood feeding on activity patterns and a complex response to extended daylight periods. By integrating a host-seeking module in BuzzWatch to deliver CO2 and heat pulses, we observed a twofold increase in Ae. aegyptis response to host-associated cues during the daytime compared to nighttime. Combined, these results demonstrate BuzzWatchs potential to investigate responses to host cues over seconds, natural variability in daily rhythms over hours, and phenotypic plasticity over days. BuzzWatch offers a novel perspective on mosquito behavior over multiple timescales, paving the way for advanced ecological and epidemiological studies that can inform targeted and effective vector control strategies. Short SummaryThis study introduces a new experimental platform to monitor multiple features of mosquito behavior (flight activity, sugar feeding, short-range host-seeking) with high temporal resolution over several weeks. HighlightsO_LIContinuous month-long tracking of mosquito flight activity in a laboratory setting. C_LIO_LIMultiscale analysis of flight behavior, from seconds to weeks. C_LIO_LIMapping of natural variation in daily rhythm of Aedes aegypti populations. C_LIO_LIQuantifying the long-lasting effect of physiological (blood-meal digestion) and environmental (photoperiod increase) perturbations. C_LIO_LIAutomatically monitoring response to short-range host-seeking cues at specific times of the day. C_LI

animal behavior and cognition↗

bioRxiv2020

Vectorchip: Microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Mosquito bites transmit a number of human pathogens resulting in potentially fatal diseases including malaria, dengue, chikungunya, West Nile encephalitis, and Zika. Although female mosquitoes transmit pathogens via salivary droplets deposited during blood feeding on a host, very little is known about the genomic content of these nanoliter scale droplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform for molecular interrogation of individual mosquito bites in a high-throughput fashion. An ultra-thin PDMS membrane coupled to a microfluidic chip acts as a biting interface, through which freely-behaving mosquitoes deposit saliva droplets by biting into isolated arrayed micro-wells enabling molecular interrogation of individual bites. By modulating membrane thickness, the device enables on-chip comparison of biting capacity and provides a mechanical filter allowing selection of a specific mosquito species. Utilizing Vectorchip, we show on-chip simultaneous detection of mosquito DNA as well as viral RNA from Zika infected Aedes aegypti mosquitoes - demonstrating multiplexed high-throughput screening of vectors and pathogens. Focus-forming assays performed on-chip quantify number of infectious viral particles transmitted during mosquito bites, enabling assessment of active virus transmission. The platform presents a promising approach for single-bite-resolution laboratory and field characterization of vector pathogen communities, to reveal the intricate dynamics of pathogen transmission, and could serve as powerful early warning artificial "sentinel" for mosquito-borne diseases.

bioengineering↗

bioRxiv2020

The Taste of Blood in Mosquitoes

Blood-feeding mosquitoes survive by feeding on nectar for metabolic energy, but to develop eggs, females require a blood meal. Aedes aegypti females must accurately discriminate between blood and nectar because detection of each meal promotes one of two mutually exclusive feeding programs characterized by distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. We investigated the role of the syringe-like blood-feeding appendage, the stylet, and discovered that sexually dimorphic stylet neurons are the first to taste blood. Using pan-neuronal GCaMP calcium imaging, we found that blood is detected by four functionally distinct classes of stylet neurons, each tuned to specific blood components associated with diverse taste qualities. Furthermore, the stylet is specialized to detect blood over nectar. Stylet neurons are insensitive to nectar-specific sugars and responses to glucose, the sugar found in both blood and nectar, depend on the presence of additional blood components. The distinction between blood and nectar is therefore encoded in specialized neurons at the very first level of sensory detection in mosquitoes. This innate ability to recognize blood is the basis of vector-borne disease transmission to millions of people world-wide.

neuroscience↗

bioRxiv2020

BiteOscope: an open platform to study mosquito blood-feeding behavior

Female mosquitoes need a blood meal to reproduce, and in obtaining this essential nutrient they transmit deadly pathogens. Although crucial for the spread of mosquito-borne diseases, our understanding of skin exploration, probing, and engorgement, is limited due to a lack of quantitative tools. Indeed, studies often expose human subjects to assess biting behavior. Here, we present the biteOscope, a device that attracts mosquitoes to a host mimic which they bite to obtain an artificial blood meal. The host mimic is transparent, allowing high-resolution imaging of the feeding mosquito. Using machine learning we extract detailed behavioral statistics describing the locomotion, pose, biting, and feeding dynamics of Aedes aegypti, Aedes albopictus, Anopheles stephensi, and Anopheles coluzzii. In addition to characterizing behavioral patterns, we discover that the common insect repellent DEET repels Anopheles coluzzii upon contact with their legs. The biteOscope provides a new perspective on mosquito blood feeding, enabling high-throughput quantitative characterization of the effects physiological and environmental factors have on this lethal behavior.

animal behavior and cognition↗