Ixodids suck blood from host animals and transmit pathogens that cause important medical and veterinary diseases. As synthetic conventional acaricide resistance becomes increasingly common, alternative tactics are likely to enhance control efforts. Laboratory bioassays assessed the lethality of CimeXa, a commercial silica gel desiccant dust, and EcoVia, a commercial product containing silica gel + thyme oil, against lone star tick, Amblyomma americanum (L.), larvae and nymphs. Both life stages were completely killed by CimeXa by 24 h, and EcoVia achieved total larval mortality within 1 h when they were initially immersed in the dusts and when they crawled across treated substrate. Larvae were killed faster than nymphs. Temporary immersion of A. americanum larvae and nymphs in aqueous suspensions of the dusts were less effective than exposure to the dusts applied dry. Larval and nymphal mortality associated with crawling on dried aqueous suspensions of the products was also not as strong as when the immature life stages crawled across substrate treated with dry dusts, and EcoVia was not more efficient than CimeXa. CimeXa and EcoVia can likely be used to protect cattle in a prophylactic context because silica gel does not degrade, and EcoVia might be effective at eliminating ticks that are feeding on cattle as well. We suggest that the two dust products be assessed for efficacy against other ixodid species, such as the southern cattle fever tick, Rhipicephalus microplus (Canestrini), which transmits the causal agents of babesiosis to cattle.

Insecticide-impregnated traps and visual targets are used for tsetse (Diptera, Glossinidae) population control. Such devices are made with textiles and deltamethrin is frequently the insecticide of choice. However, persistence of an insecticide on textiles is affected by exposure to weather. Here we examine the effect of weathering on the capacity of four textiles with increasing proportions of polyester (0, 35, 65, and 100%) with cotton and viscose to retain deltamethrin. Textiles tested were those used to make visual targets in a pan-African program to maximize target efficiency for controlling tsetse vectors of African trypanosomiasis. Following impregnation in an aqueous suspension of deltamethrin at 1,000 mg/m², textiles were weathered for 18 mo at Lambwe Valley, Kenya and sampled every 3 mo to make knockdown tests on the tsetse fly Glossina pallidipes Austen. Deltamethrin content of the textiles was established using gas chromatography mass-spectrometry at impregnation and after 9 mo of weathering. Textiles with higher proportions of polyester retained deltamethrin better: respectively, 100% polyester and 65:35 polyester/ viscose textiles retained deltamethrin at 17 and 11 mg/m² 9-mo post-treatment that caused 100% knockdown in G. pallidipes after 1 h, and killed 67 and 47% of flies, respectively, after 24 h. Eighteen-month weathered 100% polyester treated textile still knocked down all tsetse exposed to it within 2 h. The LD₅₀ for deltamethrin on filter paper for G. pallidipes was estimated at 28.8 mg/m², indicating that deltamethrin is more available on polyester to kill tsetse.

Graphical Abstract

Tick-borne diseases are a growing threat to public health in the United States, especially among outdoor workers who experience high occupational exposure to ticks. Long-lasting permethrin-impregnated clothing has demonstrated high initial protection against bites from blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), in laboratory settings, and sustained protection against bites from the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), in field tests. However, long-lasting permethrin impregnation of clothing has not been field tested among outdoor workers who are frequently exposed to blacklegged ticks. We conducted a 2-yr randomized, placebo-controlled, double-blinded trial among 82 outdoor workers in Rhode Island and southern Massachusetts. Participants in the treatment arm wore factory-impregnated permethrin clothing, and the control group wore sham-treated clothing. Outdoor working hours, tick encounters, and bites were recorded weekly to assess protective effectiveness of long-lasting permethrin-impregnated garments. Factory-impregnated clothing significantly reduced tick bites by 65% in the first study year and by 50% in the second year for a 2-yr protective effect of 58%. No significant difference in other tick bite prevention method utilization occurred between treatment and control groups, and no treatment-related adverse outcomes were reported. Factory permethrin impregnation of clothing is safe and effective for the prevention of tick bites among outdoor workers whose primary exposure is to blacklegged ticks in the northeastern United States.

Aedes aegypti (L.) is an important vector of viruses causing dengue, Zika, chikungunya, and yellow fever and as such presents a serious threat to public health in tropical regions. Control programs involving ‘rear and release’ of modified male Ae. aegypti are underway and require effective trapping methods for surveillance of both the released insects and the impacted wild mosquito population. The BG-Sentinel trap (BGS) is widely used in Ae. aegypti surveillance but its level of efficiency, that is, what proportion of the mosquitoes encountering the trap are captured, is unknown. This is especially true for male mosquitoes, the behavior of which is incompletely understood. We tested the efficiency of two versions of the BGS for capturing male Ae. aegypti under semifield conditions with and without CO₂ and a human skin odor mimic lure and with these baits combined. A navy-blue BGS trap emitting CO₂ and a human skin odor mimic captured 18% of the released male Ae. aegypti, with a capture efficiency of 9 % (of the total encounters with the trap). Male Ae. aegypti had multiple encounters with the BGS that did not result in capture; they crossed over the trap entrance without being captured or landed on the sides of the trap. Swarming behavior around the BGS was also recorded, even when only a visual cue was present. Understanding male Ae. aegypti behaviors during an encounter with the BGS can inform improvement of trap design and therefore capture efficiency for surveillance in control programs.

Resistance to insecticides used to control mosquito vectors threatens the ability of mosquito-control organizations to protect public health. Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) are invasive species widely distributed throughout Florida and have been implicated in recent epidemics of Zika, dengue, and chikungunya viruses. Knowledge of the susceptibility status of these mosquito species to pyrethroid and organophosphate active ingredients (AIs) is needed to inform product selection and treatment decisions. The susceptibility of 37 Ae. aegypti and 42 Ae. albopictus populations from Florida was assessed in response to six pyrethroid and three organophosphate AIs using the CDC bottle bioassay method. Of all bioassays completed with a pyrethroid AI, 95% for Ae. aegypti and 30% for Ae. albopictus resulted in a resistant outcome. For organophosphate AIs, ∼31% of assays conducted for both species were classified as resistant. The highest frequency of susceptibility for both species was observed in response to the organophosphate AI, naled. Lambda-cyhalothrin was the only pyrethroid to result in a susceptible status for Ae. aegypti and also had the highest frequency of susceptibility for Ae. albopictus. Resistance was detected to every AI tested for both Ae. aegypti and Ae. albopictus, but there was a pronounced trend of pyrethroid resistance in Florida populations of Ae. aegypti. The results of this work provide evidence for the need to decrease reliance on pyrethroids and to implement different methods of control of Ae. aegypti in Florida.

For the indoor and outdoor pest controls, various types of insecticide formulations are available including aerosols, sprays, electric vaporizers, mosquito coils, and traps. In the present study, the insecticidal activity of aerosols, the most commonly used formulation of household insecticides for mosquito control, against Aedes albopictus (Skuse) was assessed using seven commercial products and some attributes which can affect the efficacy of aerosol were investigated as well. The products had difference in their chemical composition of active ingredients, propellant/liquid phase ratios, solvent types, and nozzle orifice sizes, and these characteristics seem to affect the overall insecticidal activity. In general, solvent type dominantly determined the insecticidal activity, where four products in oil-based solvent system showed greater mortality (97.5% in average) than water-based aerosols (38.3% in average) against the mosquitoes located at the far side of the test chamber. The contribution of solvent type and nozzle orifice size were further examined with the sample aerosols, and the orifice size were determined more influential to the spray distance. Regardless of solvent types, the sample products attached to a bigger actuator (0.96 mm in diameter) showed greater knock-down activity (>98%) than the smaller ones (0.48 mm, 62.5% in average) to the back panel in the chamber. On the other hand, solvent system significantly affected the residual activity, as the oil-based and water-based aerosols showed 2.3- and 4.8-fold decrease in KT₅₀ values, respectively, between 1 and 10 min after the spray.

In recent decades, the occurrence and distribution of arboviral diseases transmitted by Aedes aegypti mosquitoes has increased. In a new control strategy, populations of mosquitoes infected with Wolbachia are being released to replace existing populations and suppress arboviral disease transmission. The success of this strategy can be affected by high temperature exposure, but the impact of low temperatures on Wolbachia-infected Ae. aegypti is unclear, even though low temperatures restrict the abundance and distribution of this species. In this study, we considered low temperature cycles relevant to the spring season that are close to the distribution limits of Ae. aegypti, and tested the effects of these temperature cycles on Ae. aegypti, Wolbachia strains wMel and wAlbB, and Wolbachia phage WO. Low temperatures influenced Ae. aegypti life-history traits, including pupation, adult eclosion, and fertility. The Wolbachia-infected mosquitoes, especially wAlbB, performed better than uninfected mosquitoes. Temperature shift experiments revealed that low temperature effects on life history and Wolbachia density depended on the life stage of exposure. Wolbachia density was suppressed at low temperatures but densities recovered with adult age. In wMel Wolbachia there were no low temperature effects specific to Wolbachia phage WO. The findings suggest that Wolbachia-infected Ae. aegypti are not adversely affected by low temperatures, indicating that the Wolbachia replacement strategy is suitable for areas experiencing cool temperatures seasonally.

With increasing development of resistance to conventional synthetic acaricides in economically and medically important ixodid species, interest in finding alternative control tactics has intensified. Laboratory bioassays were conducted, using the lone star tick, Amblyomma americanum (L.), as a model species, to assess the efficacy of a diatomaceous earth-based product, Deadzone, in comparison with a silica gel-based product, CimeXa. CimeXa is already known to be highly lethal against A. americanum larvae and nymphs. The two dust treatments were 100% effective against larvae and nymphs within 24 h after contact occurred by immersion in dry dusts and after crawling across a surface treated with the dry dusts. Contact by crawling on a dried aqueous film of the dusts, even at a concentration of 10%, was not as effective as exposure to the dusts in dry powder form. As has been demonstrated with CimeXa, it is likely that Deadzone will be capable of providing prophylactic protection of cattle from economically important one-host ixodids, such as the southern cattle fever tick, Rhipicephalus microplus (Canestrini), which vectors the causal agents of babesiosis. Diatomaceous earth can be stored indefinitely, will remain efficacious for as long as sufficient quantities remain on the substrate, it is a natural (organic) substance, and it might be amenable for limited use in environmentally protected habitats.

Rickettsia parkeri, a tick-borne pathogen distributed throughout several countries of the Americas, causes a mild to moderately severe, eschar-associated spotted fever rickettsiosis. Although most U.S. cases of R. parkeri rickettsiosis are reported from southeastern states, some have been reported recently from remote regions of southern Arizona. These cases are linked to R. parkeri-infected ticks of the Amblyomma maculatum (Acari: Ixodidae) group found in several isolated mountain ranges of southern Arizona and New Mexico, referred to as ‘sky islands'. Archival records also document ticks of the A. maculatum group collected from domestic and wild animals in West Texas. We surveyed sites in two sky island chains of Jeff Davis and Brewster counties to document the off-host occurrence of these ticks and identify the presence of R. parkeri in the Trans-Pecos region of Texas. During August 2019, 43 adult A. maculatum group ticks were flagged from vegetation or removed from a road-killed, female mule deer. Of 39 samples evaluated by PCR, eight contained a partial sca0 sequence with complete identity to R. parkeri and two with complete identity to ‘Candidatus Rickettsia andeanae’, a species of undetermined pathogenicity. Four isolates of R. parkeri were obtained using cell culture. Persons at risk for R. parkeri rickettsiosis include those who work or recreate in these mountains, such as hikers, backpackers, research scientists, foresters, and border enforcement personnel. Additional investigations are needed to define the distribution of these medically important arthropods in other parts of the southwestern United States and northern Mexico.

Aedes albopictus (Skuse), an invasive disease vector, poses a nuisance and public health threat to communities in the Northeastern United States. Climate change and ongoing adaptation are leading to range expansion of this mosquito into upstate New York and other northeastern states. Organized mosquito control can suppress populations, but it is time consuming, costly, and difficult as Ae. albopictus oviposits in small, artificial, water-holding containers. Unmanned aerial vehicles (UAVs), with centimeter-resolution imaging capabilities, can aid surveillance efforts. In this work, we show that a convolutional neural network trained on images from a UAV is able to detect Ae. albopictus habitat in suburban communities, and the number of containers successfully imaged by UAV predicted the number of containers positive for mosquito larvae per home. The neural network was able to identify some, but not all, potential habitat, with up to 67% precision and 40% recall, and can classify whole properties as positive or negative for larvae 80% of the time. This combined approach of UAV imaging and neutral network analysis has the potential to dramatically increase capacity for surveillance, increasing the reach and reducing the time necessary for conventional on-the-ground surveillance methods.

Tick-borne disease surveillance in North America has long focused on Lyme disease, though there is currently a significant shift towards comprehensive pathogen surveillance in ticks. Central California has often been overlooked in regular tick-borne pathogen surveillance despite the presence of numerous medically important tick species. The bacterial genus Rickettsia contains tick-borne species that are known pathogens, such as those in the spotted fever group; nonpathogenic endosymbionts; and many species with unknown pathogenic potential. Five common tick species (Ixodes pacificus Cooley and Kohls [Acari: Ixodidae], Dermacentor occidentalis Marx [Acari: Ixodidae], D. variabilis Say, Rhipicephalus sanguineus Latreille [Acari: Ixodidae], and Ornithodoros parkeri Cooley [Acari: Argasidae]) of California were collected by both traditional and modern techniques, and subsequently screened for Rickettsia spp. Many individuals from all five tick species were PCR positive for Rickettsia spp., and a combination of species-specific primers, a restriction fragment length polymorphism assay, and DNA sequencing was used to further characterize the species composition in these ticks. Probable Rickettsia philipii (Rickettsia 364D) was detected in one (1.56%) D. occidentalis collected in Fresno County; R. rhipicephali was detected in 23.4% of D. occidentalis from Fresno Co.; R. bellii was detected in 88.2% of D. variabilis, 7.8% of D. occidentalis, and in one R. rhipicephalus (1.1%) from Fresno Co.; R. monacensis str. Humboldt was detected in three (100%) of I. pacificus collected in both Fresno and Madera Co.; and an uncharacterized Rickettsia was detected in (26.4%) of O. parkeri collected in both Fresno and Madera Co. The findings in this study highlight the need for ongoing surveillance in this region of California.

Eastern equine encephalitis virus (EEEV), an Alphavirus from family Togaviridae, is a highly pathogenic arbovirus affecting the eastern United States, especially Florida. Effects of the Southern Oscillation Index (SOI), precipitation, and cooling degree days on EEEV horse case data in Florida from 2004 to 2018 were modeled using distributed lag nonlinear models (DLNMs). The analysis was conducted at statewide and regional scales. DLNMs were used to model potential delayed effects of the covariates on monthly counts of horse cases. Both models confirmed a seasonal trend in EEEV transmission and found that precipitation, cooling degree days, and the SOI were all predictors of monthly numbers of horse cases. EEEV activity in horses was associated with higher amounts of rainfall during the month of transmission at the statewide scale, as well as the prior 3 mo at the regional scale, fewer cooling degree days during the month of transmission and the preceding 3 mo and high SOI values during the month and the previous 2 mo, and SOI values in the prior 2 to 8 mo. Horse cases were lower during El Niño winters but higher during the following summer, while La Niña winters were associated with higher numbers of cases and fewer during the following summer. At the regional scale, extremely low levels of precipitation were associated with a suppression of EEEV cases for 3 mo. Given the periodicity and potential predictability of El Niño Southern Oscillation (ENSO) cycles, precipitation, and temperature, these results may provide a method for predicting EEEV risk potential in Florida.

West Nile virus (family Flaviviridae, genus Flavivirus) first caused human and veterinary disease, and was isolated from Culex pipiens pipiens L. and Aedes vexans (Meigen) (Diptera: Culicidae) in the United States in 1999. We report that a Connecticut strain of Ae. vexans was competent to transmit West Nile virus both horizontally to suckling mice and vertically to its progeny in the laboratory. Horizontal transmission was first observed on day 6 post-exposure (pe). Daily horizontal transmission rates generally increased with the day post-virus exposure with highest rates of 67–100% recorded on days 28–30 pe. One female vertically transmitted West Nile virus on day 21 pe, but only after it had taken its third bloodmeal. Horizontal and vertical transmission may contribute to West Nile virus infection rates in Ae. vexans in summer, and vertical transmission provides a means of survival of West Nile virus during winter.

Studies related to ants found in hospital environments have aroused interest in their role as mechanical vectors of pathogenic microorganisms. The objective of the current research was to determine the species composition and bacterial contamination of ant species found in a public hospital in the eastern Amazonian region. Ants were captured using bait containing honey and sterilized sardines in 15 locations within the Macapá Emergency Hospital, Amapá. Ants were identified morphologically using specific keys. Bacteria were first inoculed in a Brain Heart Infusion broth and then plated on 5% Agar with blood or MacConkey media. Bacterial species were identified through biochemical procedures. In total, 9,687 ants were collected, with 69.8% from the dry season and 30.2% from the rainy season. Nine species of ants were identified belonging to three subfamilies: the Monomorium pharaonis (Linnaeus 1758) being the most common, comprising 39.2% of the total specimens. Only one ant species was found in each bait, facilitating microbiological analyses. In total, 92 bacteria isolates were identified comprising 12 species. Pseudomonas aeruginosa Schroeter 1872 (Pseudomonadales: Pseudomonadaceae) was pathogenic bacteria, most frequently isolated, comprising 10.9% of the positive samples. The most contaminated ant in the study was M. pharaonis with 38.3%. It was the dominant ant species in this hospital environment. Its wide prevalence, forage day and night of this vector in hospital facilitated bacterial contamination. The presence of bacteria on ants may be associated with the dissemination of pathogens which cause hospital infections, making pest control a necessity in these institutions.

The mosquito Culiseta melanura (Coquillett) is the primary enzootic vector of eastern equine encephalitis virus (EEEV), a zoonotic Alphavirus endemic to eastern North America. In its northern range, Cs. melanura is considered a strict avian biter, transmitting EEEV among susceptible birds in a cycle of enzootic amplification. In its southern range, however, Cs. melanura is more general in host use, feeding heavily upon birds but also reptiles and mammals. The goal of this study was to better understand how host use of Cs. melanura changes throughout the year in Florida, where year-round EEEV transmission is observed. Mosquitoes were sampled in 2018 from nine sites across three central Florida counties. In total, 213 Cs. melanura bloodmeals were identified by PCR consisting of 39 species of birds, reptiles, and mammals. Avian bloodmeals were prominent throughout the year (range = 30–85%), and songbirds were a large portion of identified bloodmeals (37.1%). Reptiles surpassed birds only in spring (April–June), and brown anole (Anolis sagrei Duméril and Bibron, 1837 [Reptilia: Dactyloidae]) was the most commonly detected single host species (22.1% overall). Mammalian bloodmeals were mainly observed in summer, with humans being the most fed on mammal (12.7% overall). This study reveals that in southern foci of EEEV transmission, Cs. melanura host use varies throughout the year with reptiles providing the majority of blood meals in spring (51.3%), and birds are fed on more than other host groups during all other seasons (50.6–70.1%). In addition, feeding on mammals increases during summer months, which may implicate Cs. melanura in epizootic transmission in Florida.

The invasive Asian longhorned tick, Haemaphysalis longicornis Neumann, was first detected in the United States in 2017. It has since been found in 12 states, and there is concern that the tick's parthenogenetic ability and wide variety of host species may allow for broader dissemination. Of the tick-borne diseases endemic to the United States, Rocky Mountain spotted fever (RMSF), a rapidly progressive and potentially fatal disease caused by Rickettsia rickettsii, is the most severe. There is considerable geographical overlap between spotted fever rickettsioses cases, which include RMSF, and the currently known distribution of H. longicornis, providing the potential for this tick to encounter this pathogen. We have evaluated the ability of H. longicornis to acquire and transmit R. rickettsii under laboratory conditions. Haemaphysalis longicornis as larvae and nymphs acquired the pathogen while feeding on infected guinea pigs. The infection persisted through every life stage, all of which were able to transmit R. rickettsii to naïve hosts. The pathogen was also transmitted at a low frequency between generations of H. longicornis through the ova. While H. longicornis was demonstrated to be a competent vector for R. rickettsii under laboratory conditions, the probability of its involvement in the maintenance and transmission of this pathogen in nature, as well as its potential impact on human health, requires further study.

Aedes aegypti L. is considered to have a proclivity for feeding on human blood even when other hosts are available. However, few studies have demonstrated host use by this mosquito in the continental United States, where local transmission of dengue, Zika, and chikungunya viruses has been recently documented. This study investigated the bloodmeal sources of female Ae. aegypti in the subtropical city of Key West and the surrounding county in Florida with the goal of identifying preferred hosts. Blood-engorged Ae. aegypti mosquitoes were collected from BG Sentinel traps used as part of a routine surveillance program in the Florida Keys (Monroe County, Florida). Bloodmeal samples were analyzed using PCR assays, sequencing, and comparison with reference sequences in GenBank. Aedes aegypti females from Key West fed predominantly on humans (79.6%) and did not differ statistically from females collected from the rest of the Florida Keys (69.5%). Culex quinquefasciatus Say (Diptera: Culicidae), considered a host generalist, was collected and analyzed from the same sites for comparative purposes. Females of Cx. quinquefasciatus fed predominantly (70.7%) on birds and nonhuman mammals in the Florida Keys, corroborating the validity of molecular assay breadth and demonstrating that given the same group of available hosts Ae. aegypti selects humans. Our results indicated that Ae. aegypti has a high rate of human-biting in a subtropical area within the United States, supporting its role in recent local transmission of dengue and other viruses.

Nyssomyia whitmani (Antunes & Coutinho) is the sandfly vector of causative agents of American cutaneous leishmaniasis in South and Central America. It is widely spread throughout Brazil, in a close association with domestic animals. Almost nothing is known about male mating aggregations in N. whitmani, let alone the influence of moonlight on this behavior. Thus, the influence of moonlight on male N. whitmani aggregations was investigated by using non-attractant suction traps deployed in chicken cages under full moon and new moon nights. In this 18-night study, 2,160 specimens and seven species were collected. The most frequent species was N. whitmani accounting for 91.48% of the total sample. The abundance of males of N. whitmani was significantly higher in the presence than in the absence of moon. The sex ratio on new moon nights was 1:1 (M:F) and on full moon nights was male-biased (1.7:1). Based on this study, an influence of moonlight on male mating aggregations of N. whitmani was verified. The extent of this influence on increasing sandfly aggregation at hosts, the moonlight influence on the early flight activity of male sandflies and the use of chickens as aggregation sites for males of N. whitmani, are all discussed.

Humans have influenced the epidemiological patterns of American cutaneous leishmaniasis by habitat disturbance, which has led to the emergence of new transmission foci. In these transmission areas, detecting natural infection of sand fly species with Leishmania parasites is of prime importance in epidemiological studies. In this study, we examined the species composition, spatial distribution, seasonality, and natural infection with Leishmania of the sand fly fauna in an emergent leishmaniasis focus located in Colombia. Sand flies were collected from September 2014 to June 2015 using CDC light traps located in indoor, peridomestic, and outdoor habitats within areas with confirmed clinical patients. In total, 13,488 sand flies of 12 species and seven genera were collected. Among these, Pintomyia townsendi (Ortiz) was the most abundant species comprising 76.3% of total flies collected. The sand fly richness and abundance were influenced by habitat and weather conditions. Outdoor areas harbored the greatest diversity of sand flies. Rainfall negatively affected abundance, whereas increased temperature showed a low positive effect. We detected Leishmania amazonensis parasites in Pi. townsendi for the first time.

Dermacentor marginatus Sulkzer is a common tick species found in the Xinjiang Uygur Autonomous Region (XUAR) of China, and is a vector for a variety of pathogens. To determine the potential distribution of this tick species in Xinjiang, a metadata containing 84 D. marginatus presence records combined with four localities from field collection were used for MaxEnt modeling to predict potential distribution of this tick species. Identification of tick samples showed 756 of 988 (76%) were D. marginatus. MaxEnt modeling results indicated that the potential distribution of this tick species was mainly confined to northern XUAR. Highly suitable areas included west side of Altay mountain, west rim of Junggar basin, andYili River valley in the study area. The model showed an AUC value of 0.838 ± 0.063 (SD), based on 10-fold cross-validation. Although tick presence records used for modeling were limited, this is the first regional tick distribution model for D. marginatus in Xinjiang. The model will be helpful in assessing the risk of tick-borne diseases to human and animals in the region.

Graphical Abstract

We added a vector control component to our existing abundance model to simulate intensive vector control in Puerto Rico. Removing 20–30% of gravid females in the model matches observed 60–80% reductions. The model's capacity to reproduce vector control increases its utility for planning and evaluation strategies.

Quantifying arthropod vectors can be a time-consuming process. Here, we describe a technique to count large samples of small arthropods using ImageJ. ImageJ is an open source image processing software, produced by the National Institutes of Health, with a straightforward interface that has proven useful in quantifying small organisms (i.e., cells, pollen, eggs). In 2017, we deployed CDC light traps baited with carbon dioxide among seven sites to capture black flies (Diptera: Simuliidae). Samples of the captured specimens were photographed, and then quantified manually and automatically, using ImageJ. We compared the accuracy of three types of automated counts to manual counts of black flies using an information-theoretic approach. We found that changing the particle size produced counts closest to those obtained by manual counts. Even over a large range of values, from tens to thousands of flies, our automated counts were often identical to and almost always within 5% of the manual counts. When different, automated counts were usually slightly less than manual counts, and thus conservative estimates. This automated technique is simple, repeatable, requires minimal training, and can reduce the time needed to quantify small arthropods such as black flies.

Previous studies suggested the presence of species complex in the so-called Simulium asakoae Takaoka & Davies (Diptera: Simuliidae) in Thailand due to its high morphological variability and genetic divergence. To investigate whether the true S. asakoae is present in Thailand, we performed a detailed morphological identification of S. asakoae and compared its DNA barcodes with the morphospecies S. asakoae from Myanmar and the typical S. asakoae from Malaysia. Phylogenetic analysis revealed the Thai materials analyzed in this study were indeed genetically similar with those from Myanmar and Malaysia, though genetic distances 0–2.27% were observed. We tentatively regard this divergence as intraspecific variation, and the automatic barcode gap discovery analysis further supports them as a single species.