Ecology

Altogether, we found that, while traffic volume declined by  > 7% during the pandemic year (with a maximum monthly decline of nearly 40%), the absolute number of annual WVCs was largely unchanged. This resulted in significant increases of  > 8% in collision rates between vehicles and wildlife during the pandemic year, peaking at a  > 27% nationwide increase in April 2020. Other studies from the first several months of the pandemic documented similar transient declines in the number of WVCs when the pandemic began which then reversed in many jurisdictions as the pandemic progressed and traffic rebounded26,27. We observed a similar pattern over the first five months of the pandemic at the national scale (Fig. 2): WVCs initially declined during the pandemic in step with declines in traffic volume, but then started to increase to baseline levels at a faster rate than traffic, possibly due to behavioral lags by wildlife following traffic-mediated increases in wildlife road use. Though based on coarse-scale data, our research aligns with assertions from studies during27 and prior to the pandemic3,15,16,28,29 that the relationship between traffic volume and WVCs is non-linear.We postulate that the observed non-linear relationship between traffic volume and WVCs is the result of greater use of roads and roadsides by certain wildlife species, namely large mammals (Table S1), in response to decreasing traffic volume, as prior research has suggested3,14,15,16. This explanation is consistent with accounts of various wildlife species making increased use of human spaces during the pandemic17,20,21: with less cars on the roads, wildlife might be less deterred from roads by the noise and light pollution that accompany high traffic volumes9,10,11,20 and perceive roads as less risky, thereby increasing their willingness to attempt road crossings3,8,15,16. Beyond incidentally crossing roads while moving about the landscape8,9, wildlife might be attracted to roads for travel, mates, or other resources8,10,11. Many animals are shown to utilize roads to move efficiently across the landscape11,12, and roads and the surrounding areas are comparatively open, such that wildlife might select roads and roadsides for enhanced visibility to find mates, detect predators, or locate prey10,13. Roadsides also can provide foraging opportunities and essential nutrients for wildlife via abundant, high-quality early successional vegetation and high salt concentrations10,11. As such, decreased road traffic during the pandemic might have caused certain wildlife species to tolerate the risks associated with roads in order to access the benefits of roads and roadsides.An alternative explanation for the observed increases in collision rates is that human driving behavior, rather than animal behavior, changed during the pandemic. With fewer cars on the road, people might drive faster35, rendering it more difficult for both humans and wildlife to avoid collisions3. Preliminary studies from throughout the United States have indeed suggested changes to human driving behavior during the pandemic, with several jurisdictions reporting increased vehicle speeds35,36. Despite reported increases in vehicle speeds, however, the total number of vehicle collisions (the sum of both wildlife and non-wildlife collisions) mirrored trends in traffic volume and declined considerably during the pandemic37,38. Thus, because changes to human behavior appear to have had a minimal effect on vehicle collisions overall, it is unlikely that the observed changes in collision rates are due to increased vehicle speeds alone. Still, we cannot discount the possibility that changes to human driving behavior contributed to the patterns documented here, and future work should more explicitly test the relative effects of changes in traffic volume on both human driving behavior and wildlife space-use, as well as the resultant impacts on WVCs.A greater understanding of human driving behavior would also help explain our findings regarding changes in traffic patterns during the pandemic. Nationwide, the severity of COVID-19 restrictions accounted for a large amount of the variation in changes in monthly traffic volume (R2 = 0.968), but the severity of restrictions was less influential on changes in yearly traffic across states (Tables S3 and S4). Restrictions implemented throughout the pandemic were largely enacted for the purpose of minimizing travel, and other research has demonstrated that these restrictions were effective at reducing human mobility18,21. Our state-level findings, however, imply that it was not only the restrictions themselves that reduced travel, but possibly also the associated anxiety regarding the risk of contracting the SARS-CoV-2 virus, as has been suggested in other studies21,22,23,24; although we observed the greatest declines in traffic volume early in the pandemic (Fig. 2A) when restrictions were most stringent (Fig. S2)21, there was widespread anxiety about the risks posed by SARS-CoV-2 during this time22,23, which likely motivated people to stay home independent of restrictions24. Indeed, anxiety and risk perception might explain the relationship between traffic volume and the other covariates in our top models (Table S4). Declines in traffic were greatest in the most densely populated states (Fig. 4A) and in states that had the highest and the lowest disease burdens (Fig. 4B). The risk of SARS-CoV-2 transmission is greater in more densely populated states due to the close proximity of and frequent interactions amongst people21. As such, people may have altered their road use more in densely populated states as compared to sparsely populated ones due to differing perceptions of disease transmission risk23—though differences in infrastructure in relation to population density likely contributed to this pattern as well39. Similarly, declines in traffic volume in states with larger outbreaks of SARS-CoV-2 might have been driven by increases in the perceived risk of contracting the virus21,23. Alternatively, traffic reductions in states with low disease burdens might reflect increased compliance with stay-at-home orders, and therefore less opportunity for disease spread40,41; essentially, reductions in traffic volume might be the cause of locally low disease burdens therein, rather than a consequence. Altogether, we posit that the observed heterogeneity in traffic volume between states is, at least in part, attributed to differences in the perceived risk posed by the SARS-CoV-2 virus.Regardless of the mechanisms underlying changes in traffic volume and WVCs, our observation that the annual number of WVCs was largely unchanged despite substantive declines in traffic volume has implications for mitigating WVCs going forward. Most directly, the lack of a directional change in WVCs suggests that road traffic levels in the United States are currently such that even large decreases in traffic volume would have minimal long-term effects on the absolute number of WVCs. As such, decreasing collisions by reducing traffic volume would require even larger and longer-lasting changes in traffic than those observed during the pandemic. Since such massive and sustained reductions in traffic are unlikely4,5,6, WVCs in the United States essentially represent a fixed cost as of now, both for human society and wildlife populations. As such, these transient decreases in traffic likely provided minimal reprieve to large mammals from collision-induced mortality, in contrast to speculation that changes in human mobility during the COVID-19 pandemic had substantial positive effects for wildlife populations by freeing wildlife from the pervasive direct and indirect effects of humans17,18,19,20,26,27,42.Indeed, it is possible that short-term decreases in traffic volume might ultimately be harmful to those wildlife species that increased their road use. Although the increases in collision rates we observed at the beginning of the pandemic were rapid and corresponded to nationwide declines in traffic volume (see also26,27), collision rates remained elevated even as traffic approached baseline levels in July (Fig. 2B). If wildlife responses to changes in traffic are asymmetric (i.e., increases in wildlife road use following declines in traffic occur more rapidly than decreases in wildlife road use in response to increased traffic), then short-term declines in traffic volume might lead to net increases in the number WVCs over longer timeframes, ultimately proving detrimental to certain wildlife populations1,3. Future work should evaluate the long-term effects of the pandemic on wildlife populations, specifically with regards to collision-induced mortality17,20,26,27,42.Although the COVID-19 pandemic provided an opportunity to examine the short-term effects of transient decreases in traffic volume on WVCs, the longer-term effects of expanding human populations, greater road densities, and altogether higher traffic volumes on WVCs are less clear. Similar to the increases in wildlife road use in response to decreases in traffic volume theorized here, steady increases in traffic might reduce wildlife road use long-term3,14,15,16; since road traffic is indeed increasing through time4,5,6, we might therefore see declines in WVCs as roads become more effective at repelling wildlife1,3,14. Although these reductions in vehicle-induced wildlife mortality are welcome, this would see roads increasingly serve as barriers to animal movement and gene flow43, further fragmenting already disconnected wildlife populations8. Thus, policy makers and urban planners should invest in infrastructure such as overpasses, underpasses, and fencing that enables wildlife to cross high-traffic roads safely or directs wildlife towards low-risk areas8,9. Even substantive short-term declines in road traffic are not sufficient to mitigate wildlife-vehicle conflict on their own. More

Gene Expression and Therapy Group, King’s College London, Faculty of Life Sciences & Medicine, Department of Medical and Molecular Genetics, Guy’s Hospital, London, UKRobin Mesnage & Michael N. AntoniouCentre for Ecology, Evolution & Behaviour, Department of Biological Sciences, School of Life Sciences and the Environment, Royal Holloway University of London, Egham, UKEdward A. Straw, Mark J. F. Brown & Ellouise LeadbeaterHeartland Health Research Alliance, Port Orchard, WA, USACharles BenbrookANSES, Sophia Antipolis Laboratory, Unit of Honey Bee Pathology, Sophia Antipolis, FranceMarie-Pierre ChauzatAgricultural Economics and Policy Group, ETH Zürich, Zürich, SwitzerlandRobert FingerSchool of Life Sciences, University of Sussex, Brighton, UKDave GoulsonBC3 — Basque Centre for Climate Change, Scientific Campus of the University of Basque Country, Leioa, SpainAna López-BallesterosCentre D’Études Biologiques de Chizé, UMR 7372, CNRS & La Rochelle Université, Villiers-en-bois, FranceNiklas MöhringInstitute of Bee Health, Vetsuisse Faculty, University of Bern, Bern, SwitzerlandPeter NeumannSchool of Agriculture and Food Science, University College Dublin, Dublin, IrelandEdward A. Straw, Dara Stanley & Linzi J. ThompsonDepartment of Botany, School of Natural Sciences, Trinity College Dublin, Dublin, IrelandJane C. Stout & Elena ZiogaDepartment of Ecoscience, Aarhus University, Aarhus, DenmarkChristopher J. ToppingSchool of Chemical Sciences, Glasnevin Campus, Dublin City University, Dublin, IrelandBlánaid WhiteInstitute of Zoology, University of Natural Resources and Life Sciences, Vienna, Vienna, AustriaJohann G. ZallerCorrespondence to
Robin Mesnage or Edward A. Straw. More

Device design and fabricationVectorchips with integrated PDMS elastomer membranes were fabricated using a combination of laser patterning and soft lithography. We selected PDMS as the material of choice due to its low cost, biocompatibility, optical transparency, and chemical stability. PDMS-based devices can be fabricated using reproducible and scalable fabrication techniques26. Such devices have routinely been used for molecular analysis of small sample volumes using microfluidics27, and are compatible with molecular assays and cell cultures21,22. We used laser patterning to create open-ended arrayed compartments (3−4 mm deep) to capture saliva droplets from mosquito bites in PDMS micro-wells (Fig. 1a–d). In order to obtain ultra-thin PDMS films that the mosquito mouth parts can pierce, we utilized spin-coating of uncured PDMS over a flat silicon surface. We obtained membrane-integrated devices after plasma-induced PDMS-PDMS bonding of the laser-patterned array with the PDMS film (Fig. 1e). Detailed steps describing chip fabrication are included in the methods section below. The versatile fabrication process can provide user-defined variation in the size and density of the individual compartments. We were able to fabricate chips with hole diameters ranging from 150 μm to 1 cm (Supplementary Fig. 1) showing the scales of operation for the laser-ablation process while maintaining membrane stability. Additionally, the membrane can be easily integrated with fluidic networks for direct interfacing with mosquito bites, enabling assays involving on-chip fluid exchange (Fig. 1f). The microfluidic compartments on the chips can hold feeding media such as blood or sugar water (Fig. 1g), collect saliva during biting events, and act as isolated reaction chambers for molecular assays.Fig. 1: Fabrication of Vectorchip for collection of mosquito saliva and molecular assays.a Schematic showing the fabrication process of Vectorchip. Laser patterning was used to obtain through-holes in blocks of PDMS. A thin sacrificial layer of photoresist (Microposit™ S1813 G2, DOW Inc., USA) was spread onto a silicon wafer followed by spin-coating a thin film of PDMS on the wafer surface. The laser-patterned body of the chip was then bonded to the thin film of PDMS using plasma-activated PDMS-PDMS bonding. Membrane-bonded chips were obtained by placing the wafer in an acetone bath, where the sacrificial layer of resist was dissolved. b A PDMS block with laser-generated through-holes (diameter 250 μm). c A PDMS chip bonded to a 1.6 μm thin PDMS membrane on a 4-inch silcon wafer. d A Vectorchip with around 3000 wells (dia: 250 μm) is shown. e Chip with well diameter 1.75 mm and a visible PDMS membrane (thickness 1.6 μm). f Microfluidic channel-integrated chips with a PDMS membrane on top. g These wells can be loaded with feeding solution, reaction reagents, and can store mosquito saliva after biting assays. Wells loaded with feeding media (10% sucrose laced with blue food color) shown in the image. Scale bars represent 1 mm in (b), 5 mm in (e), and 2 mm in (f, g).Full size imageMosquito-chip interactionsNext, we examined the ability of mosquitoes to pierce through the PDMS membranes. Mosquitoes were attracted to the chips using heat as a guiding cue although several other baiting methods could be used. We placed a camera above the chip and observed stylet insertion through 1.6 μm thick PDMS membranes (Fig. 2a, Supplementary Movies 1 and 2). Abdominal engorgement in mosquitoes was observed after biting, indicating that mosquitoes can successfully feed through these membranes. Four species of mosquitoes were tested (Aedes aegypti, Aedes albopictus, Culex tarsalis, Culex quinquefasciatus) and all demonstrated successful probing and feeding through 1.6 μm thick PDMS membranes.Fig. 2: Mosquito biting on Vectorchip.a Stylet entry through a PDMS membrane for an Aedes aegypti female. b Mosquito feeding success as a function of membrane thickness for two mosquito species—Aedes aegypti and Culex tarsalis. We demonstrate that by changing the membrane thickness we can turn biting on or off. Biting off is defined as when no mosquito in the cage was able to feed from the chip in a duration of 30 min. The feeding assays were repeated at least three times (n = 3) at every given membrane thickness to confirm the ability of mosquitoes to feed through the membrane. c A Culex tarsalis female mosquito biting through a 20 μm thick PDMS membrane. d Bending of the proboscis observed for an Aedes aegypti female, indicating failure in biting through the 20 μm membrane. e Fluorescent salivary droplets expectorated by an Aedes aegypti mosquito while probing. These mosquitoes were fed on rhodamine-laced sugar water resulting in fluorescent saliva. Three salivary droplets (1, 2, and 3) are encircled. f Timelapse images show a magnified view of fluorescent salivary droplet deposition (droplets numbered 1, 2, and 3 as shown in panel (e) over a period of 4 s). Scale bars represent 500 μm in (a) and 2 mm in (c, d).Full size imageWe also examined whether the membrane thicknesses can be engineered to selectively prevent the biting of some species while letting other species feed through the barrier. We loaded the laser-patterned microfluidic compartments with blood and warmed the chips to attract mosquitoes (Supplementary Fig. 2 and Supplementary Movie 3). We considered biting to be off when none of the mosquitoes in the cage demonstrated abdominal engorgement within 30 min since the start of the experiment. Tests were performed with Aedes aegypti (~45 mosquitoes per cage) or Culex tarsalis (~25 mosquitoes per cage) while varying the thickness of the PDMS membrane from 900 nm to 100 μm. We observed significant differences in the biting ability of the two species, where complete inhibition of feeding by Aedes aegypti was observed with membrane thickness at or greater than 15 μm (Supplementary Movies 4 and 5). In contrast, complete inhibition of feeding by Culex tarsalis was only observed for membranes that were 100 μm thick (Fig. 2b). This remarkable difference in the biting capacity of these two species has not been reported earlier. While some studies have focused on understanding the biting mechanics of a single mosquito species (Aedes aegypti or Aedes albopictus)28,29, we are not aware of any study which quantifies the differences in biting strength between different species and the evolutionary or biomechanical differences involved in this variation. Evolutionary pressure on mosquitoes biting different hosts could generate great variability of biting strength in mosquito species, which may be needed to probe different skin thickness. This intriguing biomechanical phenomenon can be advantageous for deploying devices where membranes of desired thickness can act as species-selective filters, providing a means to better track highly relevant species and pathogens in the field.In order to extract molecular information from mosquito bites, we exploited the release of saliva during probing and biting events. The volume of saliva expectorated by mosquitoes in oil has been reported previously to be around 0.03 nL/min for Culex pipiens30, and 6.8 nL in approximately an hour for Aedes aegypti31 using a forced salivation method. Aedes aegypti have been estimated to salivate around 4.7 nL during blood feeding events32. We demonstrate that feeding rhodamine-labeled sugar water to mosquitoes renders their saliva fluorescent such that the release of saliva droplets can be directly visualized using fluorescence imaging (Fig. 2e and Supplementary Movie 6). Time sequences (Fig. 2f) show magnified images of three locations on a membrane surface where short probing events result in the deposition of fluorescent salivary droplets. We estimate that the volume of salivary droplets expectorated during this process is approximately 0.66 nL (Supplementary Fig. 3). This calculated mean volume is considerably lower compared to the volume associated with blood-feeding events reported earlier32; however, this discrepancy likely reflects differences between probing events such as observed in Fig. 2e, f and full feeding events as reported by Devine et al.32. The deposited salivary droplets harbor several biomarkers in the form of mosquito salivary proteins33, active pathogens, mosquito cells, and/or nucleic acids that can be used for their identification14,20,34.Distribution of bites on chipAn important consideration for molecular surveillance of mosquitoes is the granularity at which the population is sampled. Methods can range from pooling strategies with low coverage where samples are pooled together at the population scale, to the individual collection of saliva from mosquitoes while recording species identity. Vectorchip potentially provides an adjustable method between these two strategies by increasing or decreasing the density of bite wells presented to a population of mosquitoes.We first analyzed the distribution of bites on Vectorchip by tracking mosquito activity during biting assays. In order to perform sucrose biting assays for molecular diagnostics, we filled the wells with 10% sucrose, and covered the open side of the chip with a glass slide. A resistor that acts as a heat source was placed atop the glass slide and the chip-resistor assembly was placed on top of the mesh ceiling of a mosquito cage. A part of the chip (50 percent of the total chip area) was protected by paper tape such that mosquitoes could not bite into that section of the device. The area of the chip protected by tape served as negative control for the downstream molecular assays. A camera (Raspberry Pi 3 camera module V2.1) was placed on the bottom of the cage to track and record individual mosquito activity on chip (Supplementary Fig. 4). Image recognition and mosquito tracking algorithms have been described by us previously25, where we used computer vision to analyze image sequences and robustly track the presence, movement, and biting behavior of individual mosquitoes at high resolution. In this study, we have used an imaging setup where the camera is attached to the bottom of the cage (Supplementary Fig. 4a). The obtained image datasets reveal mosquito trajectories on the chip as well as areas with prolonged interactions (Fig. 3a–d, Supplementary Fig. 4, and Supplementary Movie 7). Unique trajectories are identified as movements performed by an individual mosquito while in the field of view (e.g., landing, exploration, biting, and take-off by a single mosquito would constitute a trajectory). The movement of mosquitoes on a chip is shown in Fig. 3e–g and Supplementary Fig. 4. Depending on the duration of mosquito-chip interactions, multiple trajectories do not overlap (Fig. 3e), partially overlap (Fig. 3f), or completely overlap (Fig. 3g). Trajectory plots can indicate the likelihood that a set of wells has been probed by more than one mosquito. These results indicate that by modulating the time of interaction between the mosquito population and the chip, the user can control the degree of probing experienced by the chip. As an example, a chip with no overlap between trajectories should exclusively contain wells probed by single mosquitoes. Tracking mosquito movement on chip was used to locate trajectories and thus regions with unique mosquito presence.Fig. 3: Tracking mosquito activity on chip.a Image recognition software was used to identify and track mosquitoes on the chip. Wells are highlighted in yellow. b Trajectories of mosquito movement can be plotted and indicate the overlap between different movement tracks. c Location plot of mosquitoes on the chip are indicated by red circles. Regions with overlapping red circles are darker in color. d Trajectory and location plot can be used to identify regions with individual mosquito locomotion activity as compared to regions with more crowding. e−g Varying experimental parameters (symbols indicate the duration of experiments and number of mosquitoes) can be used to obtain either individual mosquito plots or population data. Three cases are discussed with e no overlap between multiple probing trajectories, f partial overlap, and g complete overlap. h Representative image for a chip where the presence of fluorescent droplets on wells has been indicated by a red dot. i Zoomed-in fluorescent image of the same chip showing wells with single, or multiple droplets. j Number of bites per well were calculated by counting fluorescent droplets deposited on the wells after biting events. k We defined the number of mosquitoes = Nm, the number of wells = Nw, probing frequency = η, and time = t. The probability of obtaining ‘k’ bites on a well for the parameters (Nm, Nw, η, t) utilized in (h−j) has been shown. Scale bars represent 5 mm in (a−c, e−g), 2 mm in (d), and 1 mm in (i).Full size imageWhile tracking provided macro-scale information about mosquito position and activity, we also utilized the release of fluorescent salivary droplets deposited by biting mosquitoes to resolve mosquito probing events at higher resolution. We quantified the number of fluorescent spots seen in every well of the PDMS chip by using fluorescent scans of the chip before and after biting (Fig. 3h). No spots were observed in the negative control region where access was blocked to mosquitoes, while several fluorescent spots observed on the open areas of the chip indicate the wells which have been probed. The images show the presence of wells with multiple bite marks, as well as single bite marks.We further examined the design parameters used to build Vectorchip that can dictate the resolution of this sampling strategy. The primary determinants of how often a single mosquito will bite a specific well are dependent on multiple factors, including the number of mosquitoes in the cage, the time mosquitoes have access to the chip and biting behavior (e.g., frequency), and the size and number of wells on the chip. Other additional factors like the feeding media may affect the number of biting events performed by a female mosquito. In this study, we focused on sucrose as the feeding media since it is a diet source that does not inhibit polymerase chain reaction (PCR) assays. We considered a simple mathematical model to arrive at a first approximation for the distribution of bites on a chip. In this model, we made a simple assumption that mosquitoes probe the chip at a fixed frequency (η). We also assumed that mosquito bites are random and independent events, since no spatial gradients have been created on the chip. Detailed analysis of this model can be found in Supplementary Section 1.We defined the number of mosquitoes = Nm, the number of wells = Nw, and probing frequency = η, and estimated the probability (P) that a well receives k bites in given time t as (Supplementary Note 1):$$P[k]=({N}_{m}eta t)!/(k!times ({N}_{m}eta t-k)!)times 1/{N}_{w}^{k}times {(1-1/{N}_{w})}^{{N}_{m}eta t-k}$$
(1)
This was approximated by the form (Supplementary Note 1):$$P[k]approx {{{{{{mathrm{e}}}}}}}^{-{N}_{m}eta t/{N}_{w}}times {({N}_{m}eta t/{N}_{w})}^{k}/k!$$
(2)
and represents a Poisson distribution of the form,$$P[k]={{{{{{mathrm{e}}}}}}}^{-lambda }times {(lambda )}^{k}/k!$$
(3)
with the expected rate of events (i.e., bites per well), λ = Nmηt/Nw. Probability distributions for the expected number of bites on a well were plotted while varying the number of mosquitoes, wells, and probing frequencies (Supplementary Fig. 5). The probing frequency of Aedes aegypti mosquitoes on a parafilm membrane has been estimated previously to be in the range of 0–1 bites per min35. Supplementary Fig. 5 shows a diverse phase space where a single bite per well is the most dominant outcome, i.e., λ was less than or equal to 1, when up to 50 mosquitoes interact with a 150-well Vectorchip for 15 min—in line with our experiments. In the context of a larger number of mosquitoes interacting with the chip, increasing the number of wells can ensure that the majority of wells will not get bitten more than once. We quantified bites on wells as indicated from fluorescent droplets (Supplementary Table 1), and compared it to the analytical model (Fig. 3j, k). The fitting parameter was η = 0.1 bites per minute, which is close to the range of probing frequencies reported recently35. With water as the feeding media and parafilm as the probing membrane, Jove et al. report that the majority of tested mosquitoes showed a probing frequency below 0.5 bites per minute35. While we observe and model slightly lower probing frequencies, this difference is possible due to the change in the membrane material from parafilm to PDMS, and other differences in the setup. The methods discussed above provide multiple ways to understand mosquito−chip interactions as well as help us define parameters to obtain data at single-bite resolution.PCR diagnostics on-chipHaving established that mosquitoes can bite through the membrane and deposit salivary droplets in microwells while biting on a Vectorchip, we analyzed these bites for their DNA and RNA content. Running PCR-based nucleic acid amplification directly on individual wells establishes a low-cost assay to detect bite content in a high-throughput manner. We utilized devices with a PDMS membrane thickness of 1.6 μm and well diameter of 1.75 mm to perform molecular analysis of salivary samples; providing 145 independent reactions per chip (within a chip area of 5 cm × 2.5 cm).Detection of mosquito mitochondrial DNA (mtDNA) and RNA from viruses in saliva was performed using on-chip PCR with end-point fluorescence readout. A detailed protocol for performing PCR in Vectorchip is provided in the Methods section and Supporting Information file (Supplementary Fig. 6). We tested the efficiency of PCR amplification in Vectorchip by manually loading a known concentration of DNA and RNA into the reaction wells (Fig. 4a–c). We performed the PCR for 42 cycles and detect DNA amplification from approximately 5 DNA copies in 4 μL of reaction mix (~1 copy/μL) (Fig. 4b). Simultaneously detection of Zika virus RNA using reverse transcription-PCR (RT-PCR), was successfully demonstrated from as low as 15 RNA copies in 4 μL of reaction mix (Fig. 4c). PCR reactions were also performed in 96-well plates to test if amplification accuracy and sensitivity were similar as compared to Vectorchips. Both qPCR amplification curves and end point fluorescence information was obtained from the well plates. The 96-well plate reactions were performed in DI water and also in the presence of dried sucrose (10%) to ensure that the amplification response provided a good comparison to the Vectorchip-based reactions using manually spiked concentrations, and to test if the presence of sucrose would inhibit the reactions (see Supplementary Fig. 7a, b).Fig. 4: On-chip PCR for detection of mosquito and pathogen.The symbols indicate manually spiked assays, uninfected mosquito assays, and Zika infected mosquito assays from top to bottom. a–c A spiked assay with successful amplification of Aedes aegypti DNA and Zika RNA on chip. a Indicates wells filled with PCR mix, with ROX dye providing the background fluorescence. Fluorescent wells indicate amplification of b mosquito DNA, and c Zika RNA on chip. Assays were performed with uninfected mosquitoes where d tracking patterns were collected and demonstrate extensive translocation activity on the chip. e PCR shows detection of mosquito DNA on the chip directly from biting. f Percentage of wells available for biting on Vectorchip that display a positive PCR outcome. The PCR assay was repeated 6 times on different chips (n = 6). The false positive rate (amplification in wells that were covered by tape) was close to zero (0.23%). Assays performed with Zika infected mosquitoes indicate the presence of both g Aedes aegypti DNA and h Zika virus RNA after bites on chip. i Percentage of wells available for biting that show a positive signature for Zika RNA, mosquito DNA or both. The PCR assay was repeated 3 times on different chips (n = 3). Scale bars represent 5 mm in (a−e) and 3 mm in (g, h). Source data are provided as a Source Data file.Full size imageVectorchip provides an opportunity for tracking both pathogens in salivary bites, as well as identifying host species by detecting DNA from the host biting the well. Mosquito DNA has previously been reported in salivary droplets deposited by feeding Culex mosquitoes20. Utilizing uninfected Aedes aegypti mosquitoes, we performed PCR assays on-chip to detect the presence of mosquito DNA in deposited saliva droplets. Figure 4d, e shows the tracking data and PCR detection of mtDNA for a chip, which was placed on a cage with 75 uninfected Aedes aegypti females for 45 min. Areas highlighted in green were protected by paper tape such that mosquitoes could not probe them and serve as negative controls. The tracking data obtained from the chip indicate significant activity of the mosquitoes on-chip as represented by several overlapping trajectories. PCR data confirms that mosquito DNA indeed can be detected in probed wells. Multiple experiments indicate that a subset (3−12%, n = 6) of the wells where mosquitoes are active test positive for the presence of mosquito DNA (Fig. 4f). The rate of false positives obtained from wells not accessible to mosquitoes was very close to zero (0.23%, n = 6). In order to further validate the rate of detection of mosquito DNA on Vectorchips, we performed a biting assay using a 96 well plate loaded with agarose and covered with a parafilm membrane. Agarose gel was used as feeding media during this test, to minimize the risk of cross-contamination between wells during the peeling off of the parafilm membrane (details in “Methods” section). We recorded the tracking and DNA amplification response from uninfected mosquito bites on the well plate (Supplementary Fig. 7c, d). Approximately 15% of the wells where mosquitoes were active tested positive for mosquito DNA, which is close to the fractions observed on Vectorchips. Since mosquito DNA can only be detected in a subset of wells showing mosquito activity, this suggests that the presence of detectable levels of mosquito DNA in saliva is a noisy physiological process, likely dependent on multiple variables such as duration of feeding and time of last bite. It is furthermore important to note that our tracking algorithm only detects the presence of mosquitoes, but does not provide information regarding if a well was bitten or not.Next, we utilized the device to perform assays on infected mosquitoes, for the detection of both mosquito species and deposited pathogens. Figure 4h shows results obtained when Aedes aegypti infected with Zika virus (ZIKV)36 interacted with the chip for 20 min. We subsequently performed RT-PCR on the chip and observed that Aedes aegypti mtDNA and ZIKV RNA could be detected in 24/118 and 37/118 wells, respectively. We summarize the detection rates of mosquito DNA and ZIKV RNA for three assays performed with ZIKV-infected mosquitoes in Fig. 4i. Interestingly, not all wells positive for ZIKV RNA showed positive for mosquito DNA and vice versa, highlighting the stochastic genetic composition of bite-derived nanoliter saliva droplets. A higher fraction of wells (mean ~ 22%, n = 3) showed positive for ZIKV RNA as compared to mosquito mtDNA (mean ~ 17%, n = 3), indicating a higher prevalence of viral genomic material in mosquito saliva as compared to mosquito mtDNA. Detection of mosquito DNA and virus RNA in these assays demonstrates the capacity of this tool to identify mosquito and pathogen species directly from mosquito bites on-chip.Detection of infectious viral particles directly from bitesWhile PCR-based end-point assays are an important strategy for multiplexed detection of vectors and pathogens in various settings, they cannot determine the presence or concentration of infectious virus particles in bites. Detection of infectious pathogen load in mosquito bites is important to understand the vector competence (including potential and efficiency of pathogen transmission through bites) of mosquito-pathogen systems and its dependency on factors such as local climate37, mosquito species38, physiology39, and presence of endosymbionts (e.g., Wolbachia)40. Typical methods to measure vector competence rely on manual “forced salivation” of individual mosquitoes12, where the viral loads may differ from biting events and mosquitoes are sacrificed preventing time-course measurements. Sugar feeding on filter papers has also been utilized recently, and provides promising results towards longitudinal sampling of viral transmission by individual mosquitoes20. As compared to filter paper-based approaches, the Vectorchip uses an integrated PDMS membrane which can enable purely bite-based collection and avoid possible contamination from e.g., excretion events. Furthermore, our method improves the throughput of the assay as mosquitoes do not need to be individually housed and sampled. Finally, filter paper assays are reliant on sampling nucleic acids, and cannot perform live cell culture assays to detect infectious pathogens.We tested the potential of Vectorchip to perform focus forming assays (FFA) and detect infectious viral particles directly as a result of mosquito bites on chip (Fig. 5). Vectorchip wells were filled with cell culture media (Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The efficiency of FFA on-chip was tested using manual spiking of viral particles into Vectorchip (Supplementary Fig. 8). In order to perform biting assays, Aedes aegypti mosquitoes were infected with Mayaro virus, an emerging zoonotic pathogen that causes a dengue-like disease41. The mosquitoes were able to bite through the membrane into the wells and transfer live Mayaro viral particles directly into the cell culture medium (Fig. 5a). These viral particles then infect a monolayer of vero cells cultured on the PDMS membrane. These infections can be identified via fluorescent antibodies specific to viral envelope proteins (Fig. 5c). Each fluorescent patch (focus) is attributed to an infection resulting from a single viral particle. Foci counted on two sample chips can be seen in Fig. 5d. No foci were visible from negative control samples. The distribution of viral foci on the chips indicates the heterogeneity of infectious particle dose in mosquito bites. Figure 5 shows that the Vectorchip supports the growth of a cell monolayer on the chip and can enable the direct collection of mosquito saliva through bites in cell culture media. Antibody assays can be used to directly quantify infectious viral particles in the wells. These results demonstrate the suitability of using the Vectorchip to probe the transmission of infectious viral particles.Fig. 5: Quantifying active viral particles using focus-forming assays.Focus forming assays on Vectorchip can directly quantify the number of active viral particles in mosquito bites, without the need for isolation of individual mosquitoes and manual salivary extraction. a Image shows Aedes aegypti mosquitoes infected with Mayaro virus biting on Vectorchips filled with DMEM cell culture media. b A monolayer of vero cells was cultured on Vectorchip membrane. The formation of vero cell monolayers on membranes was verified on 5 independent chips (n = 5). c FFA performed on a Vectorchip. Symbols indicate wells in the top row which were bitten by mosquitoes with no infection (negative control), and rows of wells bitten by mosquitoes infected with Mayaro virus. The green channel shows fluorescence from an antibody against a viral envelope protein. Every green island represents infected cells likely to have resulted from an active viral particle. No active viral particles were seen in the control wells. FFA on Vectorchips was verified on 3 chips (n = 3) for both uninfected and Mayaro infected mosquitoes. d FFA formed on two chips and viral foci were counted using the antibody fluorescence. Scale bars represent 2 mm in (a), 30 μm in (b), and 100 μm in (c).Full size imageFinally, we examined if the feeding media provided in Vectorchip can enable blood meal-mimic biting behavior through the addition of phagostimulants such as ATP, while maintaining accurate molecular analysis (Supplementary Note 2). We observed that the addition of 100 μM ATP, (which is a phagostimulant and used by mosquito sensory neurons to identify blood35), to the feeding media did not impact nucleic acid amplification reactions (Supplementary Fig. 9a) (n = 3). Interestingly, mosquitoes feeding on DMEM supplemented with 10% FBS and ATP (1 mM) led to a similar or higher number of abdominal engorgements in 45 min as compared to blood meals (n = 3) (Supplementary Fig. 9b). This is reasonable as DMEM (supplemented with FBS and ATP) contains a variety of the components used by mosquito sensory neurons to identify blood at relevant concentrations (Supplementary Note 2)35). In summary, our data indicate that Vectorchip can provide physiologically relevant mosquito feeding behavior, enabling accurate assessment of vector and pathogen identity through nucleic acid amplification reactions for field surveillance, as well as on-chip assessment of transmission of infectious viral particles in bites through focus forming assays. More

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Type-I errorIn the case of two identical unimodal von Mises distributions, seven tests did not maintain Type-I error near the nominal 5% level, at least when sample sizes were small. These tests were the Kuiper two-sample test, the non equal concentration parameters approach ANOVA, the P-test, the Watson’s large-sample nonparametric test, the Watson–Williams test and the Rao dispersion test (Fig. 2). The Type-I error results were similar for the unimodal wrapped skew-normal distribution, except that the Wallraff test and Fisher’s method also showed Type-I error inflation (Fig. S1). No other methods showed evidence of failure to control Type-I error rate across different testing situations (Figs. S2–S5), except for the Log-likelihood ratio ANOVA in the case of two identical asymmetrical bimodal distributions (Fig. S3). In summary, only eight out of 18 tests reliably controlled the Type-I error rate near the nominal 5% level across all the situations investigated. These included five tests for identical distribution, the Watson’s U2 test, the Large-sample Mardia–Watson–Wheeler test, the Watson-Wheeler test, the embedding approach ANOVA, the MANOVA approach, the Rao polar test for differences in mean direction, and two tests for differences in concentration, the Levene’s test and the concentration test. We focus only on these tests in our explorations of statistical power.Figure 2Type-I error of all tests using von Mises distributions for different sample sizes: 10 and 10 (A), 20 and 20 (B), 50 and 50 (C), 20 and 30 (D) and 10 and 50 (E). Concentration (κ, kappa) increases for both distributions from 0 to 8. Tests are grouped according to their null hypotheses.Full size imagePower to detect differences in concentrationThe most powerful test to detect concentration differences between two von Mises distributions was the MANOVA approach, which offered superior power especially at lower sample sizes (Fig. 3). The Watson’s U2 test was also very powerful, followed by the Watson–Wheeler and the Large-sample Mardia–Watson–Wheeler tests with only marginally lower power. The embedding approach ANOVA had lower power, but, notably, was still more powerful than the Concentration test and Levene’s test, both specifically designed to detect differences in concentration. As expected, the Rao polar test was not sensitive to differences in concentration. The general results for two unimodal wrapped skew-normal distributions were comparable to the results for unimodal von Mises distributions, with the only exception of superior performance of Levene’s test in situations with highly asymmetric samples sizes (Fig. S6).Figure 3Power of all included tests when comparing von Mises distributions of differing concentrations using different sample sizes: 10 and 10 (A), 20 and 20 (B), 50 and 50 (C), 20 and 30 (D) and 10 and 50 (E). The first distribution is fixed at κ = 0, the second increases from 0 towards 8.Full size imageWhen comparing axial von Mises distributions, only the Watson’s U2 test offered acceptable power (Fig. S7). For the symmetrical trimodal distributions, overall power was very low, and again, only the Watson’s U2 providing some power (Fig. S8). The asymmetrical bimodal (Fig. S9) situation showed acceptable power of the MANOVA approach and Watson’s U2, however, for the asymmetrical trimodal distribution power was low with the Watson’s U2 providing the best results (Fig. S10).Power to detect differences in the mean/medianThe power to detect angular differences between two von Mises distributions was highest for the MANOVA approach at small sample sizes (n = 10), followed by the Watson’s U2, Watson-Wheeler test and the Large-sample Mardia-Watson-Wheeler test (Fig. 4). Notably, the Levene’s test also showed acceptable power levels, clearly failing to detect specifically concentration differences (to which it was less sensitive, see Fig. 4). The concentration test was not sensitive to the differences in mean direction. Special cases were the embedding approach ANOVA and the Rao polar test. The ANOVA approach showed, with the exception of very unequal sample sizes (n = 10/50), a unimodal response, with increasing power levels from 0° to 90° difference, but then rapidly decreasing power towards 180° difference. The Rao polar test showed an even stranger pattern, with, at higher sample sizes, very good power when the difference was either around 45° or 135°, but with power levels dropping to 0.05 in between these two peaks (at 90°). The results were similar for the wrapped skew-normal distribution, with the exceptions that the Rao polar test showed strongly reduced power and switched from a bimodal to a unimodal power curve with a peak around 60°, and the Levene’s test completely lost its power (Fig. S11).Figure 4Power of all included tests when comparing von Mises distributions (kappa for both = 2) of differing directions using different sample sizes: 10 and 10 (A), 20 and 20 (B), 50 and 50 (C), 20 and 30 (D) and 10 and 50 (E). The first distribution is fixed at 0°, the second increases from 0° towards 180.Full size imageFor axial distributions, only the Watson’s U2 test offered acceptable power levels, although large sample sizes (~ n = 100) were required for the power to reach over 50% (Fig. S12). All other tests failed to detect the difference in mean direction between two axial distributions. For symmetric trimodal distributions none of the tests used was sensitive to differences in mean direction (Fig. S13).When comparing asymmetrical bimodal distributions, the general trends were similar to the unimodal case. However, over all sample sizes the MANOVA approach offered the best power. The Watson–Wheeler test was considerably less powerful in this situation, as were the Watson’s U2 test and the Large-sample Mardia–Watson–Wheeler test (Fig. S14). The Levene’s test showed a unimodal-shaped power curve. The asymmetrical trimodal situation was, again, similar to the asymmetrical bimodal situation (Fig. S15), with the exception of the Levene’s test, which showed steady power increase with angular difference (instead of the hump-shaped curve).Power to detect differences in distribution typeWhen comparing a unimodal and an axial bimodal distribution, which increased similarly in concentration, we found that the MANOVA approach again offered the best power in particular at low samples sizes, followed by the Watson’s U2 test, the Large-sample Mardia–Watson–Wheeler test and Watson–Wheeler test (Fig. 5). While the embedding approach ANOVA and the Levene’s test had varying but usable power levels, the concentration test was only sensitive to such differences at low concentration values. The Rao polar test was not sensitive to such differences.Figure 5Power of all included tests when comparing von Mises distributions of differing number of modes (unimodal and axially bimodal) using different sample sizes: 10 and 20 (A), 20 and 40 (B), 50 and 100 (C), 20 and 60 (D) and 10 and 100 (E). The concentration (κ) of both increases from 0 to 8.Full size imageThe picture was only marginally different when comparing a von Mises with a wrapped skew-normal distribution (Fig. S16). For low sample sizes (n = 10) the MANOVA approach offered great power, followed by the embedding approach ANOVA. The latter offered good power throughout the range of sample sizes tested, followed by the Watson’s U2 test, the Large-sample Mardia–Watson–Wheeler test and Levene’s test. Also, the Rao polar test showed lower, but acceptable sensitivity to distribution type. The concentration test only showed very low power, that (as expected) increased with increasing concentrations of the respective distributions.We summarize the results obtained in the power analysis in Table 2. In all situations, either the Watson’s U2 test or the MANOVA approach offered the best power.Table 2 Ranking of tests based on the power comparisons for the main scenarios encountered in potential data sets (using different distributions: unimodal, axial, asymmetrical bimodal, symmetrical trimodal, asymmetrical trimodal), in cases were only one test performed acceptable the others ranks were left blank (see Table 1 for abbreviations).Full size tableReal data examplesTesting the performance of the robust tests on real data sets revealed, predominantly, the expected test behavior. In the example of homing pigeons where a difference in concentration was expected, all tests, with the exception of the Rao polar test and, notably, the concentration test, showed a significant difference between the distributions (Fig. 6A). Therefore, we can conclude, in accordance with the respective publication19, that sectioning of the olfactory nerve disrupted the homing behavior of pigeons.Figure 6Results from example data. Shown are results of pigeon (A), ant (B) and bat orientations (C). Control groups are on the left panels and experimental groups on the right. The tests are abbreviated according to Table 1, significant test results are indicated in red with asterisk and non-significant in blue. For each circular plot directional data is shown as dots on the circle (each dot is one individual), the arrows represent the mean direction and the dashed line the 95% confidence interval.Full size imageIn the ant example, where no difference between the groups was expected, there was no significant difference between the distributions detected by most of the tests (Fig. 6B). Only the concentration test showed a significant difference. Based on the other tests we would conclude that there was no biological meaningful difference between the two distributions. Therefore, ants appear to be able to transfer visual information from one eye to the other.In the bat example, where a difference in mean direction was expected, the Watson’s U2, the Mardia–Watson–Wheeler, Watson-Wheeler test and the MANOVA approach showed a significant difference (Fig. 6C). Notably, the Rao polar, Levene’s, and concentration tests and the embedding approach ANOVA failed to show a significant difference. At least for the Rao polar test, one would have expected a significant difference, as the two distributions are clearly 180° apart. This outcome concurs with our simulation results where the Rao polar test failed to distinguish distributions on the same and orthogonal axes (Fig. 4). As the results of the tests where quite mixed this example highlights the need for choosing a test with appropriate power to detect the expected differences. Based on the results of the most powerful tests, we conclude that the bats showed a mirrored orientation, as expected in the experimental design. More