Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Host associations of Culex panocossa (Diptera: Culicidae) in southern Florida and its implications for arbovirus transmission.

Culex panocossa, Dyar and Knab, an important enzootic vector of Venezuelan equine encephalitis virus subtype ID in Central and South America, was found to have invaded and become established in southern Florida in 2016. No information is currently available regarding the ecology of this invasive mosquito in the United States. Here, we use PCR-based blood meal analysis to investigate vertebrate host associations of Cx. panocossa from Florida to provide information necessary for determining the potential importance of this mosquito for arbovirus transmission in the United States. Culex panocossa fed mainly upon birds (49.5%) but took a substantial fraction of blood meals from mammals (33.3%) and reptiles (17.1%). By feeding upon amplifying hosts of Everglades virus (hispid cotton rat) and eastern equine encephalitis virus (wading birds) and humans, Cx. panocossa could act as a bridge vector for these pathogenic Alphaviruses in Florida, potentially resulting in increased human disease.

Culex erraticus (Diptera: Culicidae) utilizes gopher tortoise burrows for overwintering in North Central Florida.

Mosquito-borne diseases represent a significant threat to human and animal health in the United States. Several viruses, including West Nile, Saint Louis encephalitis, and Eastern equine encephalitis are endemic. In humans, the disease is typically detected during the summer months, but not during the winter months. The ability of these viruses to reemerge year after year is still not fully understood, but typically involves persistence in a reservoir host or vector during periods of low transmission. Mosquito species are known to overwinter at different life stages (adults, larvae, or eggs) in manufactured or natural sites. Gopher tortoise burrows are known to serve as refuge for many vertebrate and invertebrate species in pine savannas. In this study, we surveyed the interior of gopher tortoise burrows for overwintering mosquitoes. We identified 4 species (Anopheles crucians s.l., Culex erraticus, Mansonia dyari, and Uranotaenia sapphirina). Cx. erraticus was the most abundant, and its presence and abundance increased in winter months, implying that this species utilized gopher tortoise burrows for overwintering. Bloodfed Cx. erraticus and An. crucians s.l. females were detected. While An. crucians s.l. fed exclusively on the white-tailed deer, Cx. erraticus had a more diverse host range but fed primarily on the gopher tortoise. Tortoises and other long-lived reptiles like the American alligator have been shown to sustain high viremia following West Nile virus (WNV) and Eastern equine encephalitis virus (EEEV) infection and therefore could play a role in the maintenance of these viruses. In addition, Cx. erraticus is naturally infected with WNV and is a known bridge vector for EEEV. As such, these overwintering sites may play a role in perpetuating over-winter arboviral activity in Florida.

Changes in mosquito species and blood meal composition associated with adulticide applications.

Although adulticide application is a pillar in the integrated management of nuisance and vector mosquitoes, non-target effects of adulticide applications within ecosystems are a substantial concern. However, the impacts of adulticide applications on non-target organisms are not necessarily detrimental, and in some cases, may provide benefits to certain organisms or wildlife. Here, we hypothesized that adulticide applications have beneficial non-target impacts on vertebrate wildlife through reduced biting pressure. To test this, we collected mosquitoes from ultra-low volume Permanone-treated (intervention) and untreated (reference) areas and assessed mosquito abundance and diversity, and abundance of blood-engorged female mosquitoes. We performed DNA barcoding analysis on mosquito blood meals to identify host species. Our results demonstrated a significant reduction in mosquito abundance by 58.9% in the intervention areas, taking into account the reduction in reference areas. Consequently, this decline led to a 64.5% reduction in the abundance of blood-engorged females. We also found a temporal dynamic of mosquito composition driven by mosquito control actions in which different mosquito species became dominant at treated sites while composition at reference areas remained similar during the same period. The present study suggests that the beneficial effects of mosquito control treatments for humans extend to other vertebrates, which represents an unstudied and rarely recognized non-target impact.

Phylogenomics reveals the history of host use in mosquitoes.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188-250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Integrated pest management strategies targeting the Florida kissing bug, Triatoma sanguisuga: Preventing this vector of Chagas disease from invading your home.

Triatomines (Hemiptera: Reduviidae: Triatominae), commonly called "kissing bugs", are blood-sucking pests and vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). Eleven species of kissing bugs occur throughout the southern half of the USA, four of which are well known to invade human dwellings. Certain kissing bugs in the USA are known to transmit T. cruzi to humans and other animals and their bites can also lead to serious allergic reactions, including anaphylaxis. In Florida, the kissing bug Triatoma sanguisuga frequently invades homes, bites residents, and has been found infected with T. cruzi, placing humans and companion animals at risk for CD. This review outlines integrated pest management (IPM) strategies for minimizing human exposure to T. sanguisuga and CD. A comprehensive IPM plan for kissing bugs includes detailed inspections, removal of vertebrate host nesting areas, and kissing bug harborage, home improvements to exclude kissing bugs from entering structures, pest removal, and judicious use of pesticides. This approach can limit or eliminate kissing bug entry into residential structures, thereby preventing kissing bug bites, and CD infections in humans and companion animals.

Everglades virus: an underrecognized disease-causing subtype of Venezuelan equine encephalitis virus endemic to Florida, USA.

Everglades virus (EVEV) is subtype II of the Venezuelan equine encephalitis virus (VEEV) complex (Togaviridae: Alphavirus), endemic to Florida, USA. EVEV belongs to a clade that includes both enzootic and epizootic/epidemic VEEV subtypes. Like other enzootic VEEV subtypes, muroid rodents are important vertebrate hosts for EVEV and certain mosquitoes are important vectors. The hispid cotton rat Sigmodon hispidus and cotton mouse Peromyscus gossypinus are important EVEV hosts, based on natural infection (virus isolation and high seropositivity), host competence (experimental infections), and frequency of contact with the vector. The mosquito Culex (Melanoconion) cecedei is the only confirmed vector of EVEV based upon high natural infection rates, efficient vector competence, and frequent feeding upon muroid rodents. Human disease attributed to EVEV is considered rare. However, cases of meningitis and encephalitis are recorded from multiple sites, separated by 250 km or more. Phylogenetic analyses indicate that EVEV is evolving, possibly due to changes in the mammal community. Mutations in the EVEV genome are of concern, given that epidemic strains of VEEV (subtypes IAB and IC) are derived from enzootic subtype ID, the closest genetic relative of EVEV. Should epizootic mutations arise in EVEV, the abundance of Aedes taeniorhynchus and other epizootic VEEV vectors in southern Florida provides a conducive environment for widespread transmission. Other factors that will likely influence the distribution and frequency of EVEV transmission include the establishment of Culex panocossa in Florida, Everglades restoration, mammal community decline due to the Burmese python, land use alteration by humans, and climate change.

An Inexpensive System to Investigate the Daily Rhythms of Insects.

Insects' daily rhythms occur in response to their surrounding environment. Recognizing the daily rhythms of pathogen vectors can be helpful in developing effective, safe, and sustainable management strategies to control vector insects and reduce the spread of pathogens. However, studying the daily rhythm of insects often requires costly or labor-intensive trapping, and few tools are available to quantify daily rhythms in the field. We developed a simple collection system to study the flight activity of mosquitoes and biting midges using a contained, programmable, rotating, automatic pet feeder. A diverse assemblage of nuisance and vector species were collected with our system, including mosquitoes of the genera Aedes, Anopheles, Culex, and Deinocerites and biting midges (Ceratopogonidae) such as the coastal pest Culicoides furens. Surprisingly, mosquitoes and biting midges were less active during crepuscular periods (1800-2100h; 0600-0900h) than during dark periods (2100h-2400h; 0300h-0600h). A number of urban and agricultural pest insects were captured, including Coleoptera, Hymenoptera, Isoptera and Lepidoptera. This study shows that relatively inexpensive products can be adapted to study the daily rhythms of flying vectors and nuisance arthropods, with implications for vector-borne disease transmission and control. The collection system could also be used with flight intercept or pitfall traps, permitting study of the circadian activity patterns of a diverse array of arthropods.

Mosquito Blood Meal Analysis.

The host associations of mosquitoes vary by species, with some species being relative generalists, whereas others specialize, to varying extents, on a particular subset of the available host community. These host associations are driving factors in transmission dynamics of mosquito-vectored pathogens. For this reason, characterizing the host associations of mosquito species is critical for understanding the epidemiology of mosquito-vectored pathogens. Diverse methods have been used to associate mosquito species with their hosts. These typically include collecting mosquitoes that bite a restrained host (bait) or collecting wild blood-engorged mosquitoes and matching their blood meal to reference samples (blood meal analysis). Blood meal analysis refers to a collection of molecular techniques for determining the taxonomic identity of the source of a mosquito blood meal using cytological, serological, or DNA-based characteristics of the blood meal. Blood meal analyses that are based on DNA markers have advantages over cytological and serological methods and are effective for determining species-level identities of hosts from a broad range of potential host taxa. Here, we discuss effective techniques for analyzing blood meals.

Extracting DNA from Preserved Mosquito Blood Meals.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

Preservation of Field-Collected Mosquito Blood Meals.

All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.

Amplification and Identification of Vertebrate Host Cytochrome c Oxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals.

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.

Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

Host associations of biting midges (Diptera: Ceratopogonidae: Culicoides) at deer farms in Florida, USA.

Documenting the host use of vector species is important for understanding the transmission dynamics of vector-borne pathogens. Biting midges (Diptera: Ceratopogonidae: Culicoides) are vectors of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) worldwide. However, relative to mosquitoes and many other vector groups, host associations of this group are poorly documented. In this study, we used PCR-based bloodmeal analysis to determine species-level host associations of 3,603 blood-engorged specimens belonging to 18 Culicoides species at 8 deer farms in Florida, USA. We used a binomial mixed model with a Bayesian framework to compare the effect of host composition on the feeding patterns of Culicoides spp. and employed the Morisita-Horn Index to investigate the similarity of host use between farms for Culicoides stellifer and Culicoides insignis. Results show that the estimated probability of Culicoides spp. feeding upon white-tailed deer depends on the availability of cattle or exotic game and demonstrates differences in host-feeding selection among species. Culicoides insignis had high host similarity across farms suggesting that its host-use patterns are somewhat conserved. Culicoides stellifer had lower host similarity across farms suggesting that it is a more opportunistic feeder. White-tailed deer are fed upon by many Culicoides species on deer farms in Florida, and while most Culicoides species feed on white-tailed deer, the ratio of white-tailed deer bloodmeals to other bloodmeals is likely influenced by host availability. Culicoides spp. taking a majority of their bloodmeals from farmed white-tailed deer should be assessed for their vector competence for EHDV and BTV.

Culex (Phenacomyia) lactator (Diptera: Culicidae) in southern Florida, USA: a new subgenus and species country record.

The Culex subgenus Phenacomyia is a small and poorly studied group of three mosquito species native to the American tropics. Here, we report the first detections of established populations of Culex (Phenacomyia) lactator Dyar & Knab in three counties of southern Florida. Culex lactator was first detected in May 2018 in southern Miami-Dade County, and, at this locality, was collected in subsequent years from 2018 to 2022 as both adults and immatures. Larvae and adults were subsequently collected in 2022, ~175 km northwest of the initial locality at nine sites in Collier and Lee Counties. Identification of specimens collected in these counties as Cx. lactator is supported by molecular analysis and morphological characters of the adult female, male genitalia, and larva. The host associations and vector competence of Cx. lactator have not been extensively studied, and the public health implications, if any, of the addition of this species to Florida's mosquito fauna are unclear. These collections represent the first detections of Cx. lactator, or any Phenacomyia species, in the United States, adding to a trend in which detections of established populations of mosquito species from the American tropics in Florida appear to be increasing.

Aedes albopictus host odor preference does not drive observed variation in feeding patterns across field populations.

Laboratory and field-based studies of the invasive mosquito Aedes albopictus demonstrate its competency to transmit over twenty different pathogens linked to a broad range of vertebrate hosts. The vectorial capacity of Ae. albopictus to transmit these pathogens remains unclear, partly due to knowledge gaps regarding its feeding behavior. Blood meal analyses from field-captured specimens have shown vastly different feeding patterns, with a wide range of anthropophagy (human feeding) and host diversity. To address this knowledge gap, we asked whether differences in innate host preference may drive observed variation in Ae. albopictus feeding patterns in nature. Low generation colonies (F2-F4) were established with field-collected mosquitoes from three populations with high reported anthropophagy (Thailand, Cameroon, and Florida, USA) and three populations in the United States with low reported anthropophagy (New York, Maryland, and Virginia). The preference of these Ae. albopictus colonies for human versus non-human animal odor was assessed in a dual-port olfactometer along with control Ae. aegypti colonies already known to show divergent behavior in this assay. All Ae. albopictus colonies were less likely (p < 0.05) to choose the human-baited port than the anthropophilic Ae. aegypti control, instead behaving similarly to zoophilic Ae. aegypti. Our results suggest that variation in reported Ae. albopictus feeding patterns are not driven by differences in innate host preference, but may result from differences in host availability. This work is the first to compare Ae. albopictus and Ae. aegypti host preference directly and provides insight into differential vectorial capacity and human feeding risk.

Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

Pollinator biological traits and ecological interactions mediate the impacts of mosquito-targeting malathion application.

Mosquito adulticides are perceived by the public as detrimental to nontarget arthropods, contributing to declines of native and beneficial insects. However, the actual impact of adulticides on nontarget arthropods in nature needs to incorporate biological and ecological elements. Here, we investigated the effect of biological/behavioral traits (butterfly roosting at different heights, roosting in sites underneath foliage, bumblebee hive usage) and interactions (parasitism, predation) of pollinators (butterflies and bumblebees) that could mediate the impacts of malathion application in manipulative semi-field experiments in Florida, USA. Roosting height from the spray route had a significant negative relationship with mortality of butterflies treated with ULV malathion, with high survival at the highest roosting height (7 m), but butterflies roosting among vegetation did not have higher survival. Bumblebees with hive access had significantly higher survival than bumblebees without hive access. Host plants treated with ULV malathion significantly reduced parasitism of monarch eggs by Trichogramma platneri, but increased predation of monarch caterpillars by Polistes paper wasps. These data provide insight into the realistic impacts of adulticide applications on pollinators in nature which will enable mosquito control districts to better limit nontarget effects of adulticide treatments and may help to address concerns related to potential nontarget effects.

Key to the Adult Females of Species of Culex Subgenus Melanoconion in Florida, USA.

The subgenus Melanoconion of Culex is a diverse and taxonomically challenging group of mosquitoes (Diptera: Culicidae) endemic to the American tropics, with a few species occurring in temperate regions. With the introduction and establishment of Culex (Melanoconion) panocossa in southern Florida, the existing taxonomic resources for identifying species of Melanoconion in Florida need updating. Here we provide an up-to-date photographic key for identifying females of Culex (Melanoconion) spp. known to occur in Florida, USA, and summarize relevant literature on the biology of each species. Given the challenge of identifying members of subgenus Melanoconion, updated resources for identifying females of this medically important group are warranted. The keys, photographs, and summarized biological information in this report should facilitate research, surveillance, and decisions regarding control.

Field Comparison of Removed Substrate Sampling and Emergence Traps for Estimating Culicoides Orbivirus Vectors in Northern Florida.

The larval ecology of Culicoides (Diptera: Ceratopogonidae) influences their spatial distributions and the pathogens they transmit. These features are of special concern for deer farmers in Florida where epizootic hemorrhagic disease virus (EHDV) is a major source of mortality in captive herds. Rarity of larval morphological expertise leads many researchers to study larval ecology by quantifying emergence, either with field emergence traps or removing substrate from the field for observation under laboratory conditions. We investigated the comparability of these methods in Florida seepages where two recently implicated EHDV vectors, Culicoides stellifer Coquillett and Culicoides venustus Hoffman, are common. We compared the abundance and composition of emerging Culicoides collected from emergence traps with removed substrate samples (soil plugs) at three seepages. Soil plugs were sampled adjacent to the emergence trap and from underneath the trap footprint, and then monitored under laboratory conditions for 11-13 wk to compare the methods and to assess the role of incubation period for removed substrate samples. Emergence traps and removed substrate sampling largely agreed on community compositions and trends within different seepages. However, comparatively large numbers of C. stellifer emerged later than expected and well into the incubation period with emergence still occurring after 13 wk (90 d). Removed substrate samples were more similar to emergence traps at shorter incubation times. The importance of time for the capture of Culicoides in removed substrate sampling was more pronounced than we anticipated and is important from both a methodological and biological perspective.