Potential key genes involved in metabolic resistance to malathion in the southern house mosquito, Culex quinquefasciatus, and functional validation of CYP325BC1 and CYP9M12 as candidate genes using RNA interference.

BACKGROUND: Metabolic detoxification is one of the major mechanisms contributing to the development of resistance in mosquitoes, including the southern house mosquito, Culex quinquefasciatus. The three major detoxification supergene families, cytochrome P450s, glutathione S-transferases and general esterases, have been demonstrated to play an important role in metabolic resistance. In this study, we performed differential gene expression analysis based on high-throughput transcriptome sequencing on samples from four experimental groups to give insight into key genes involved in metabolic resistance to malathion in Cx. quinquefasciatus. We conducted a whole transcriptome analysis of field captured wild Cx. quinquefasciatus from Harris County (WI), Texas and a malathion susceptible laboratory-maintained Sebring colony (CO) to investigate metabolic insecticide resistance. Field captured mosquitoes were also phenotypically classified into the malathion resistant and malathion susceptible groups following a mortality response measure conducted using a Centers for Disease Control and Prevention (CDC) bottle assay. The live (MR) and dead (MS) specimens from the bottle assay, along with an unselected WI sample and a CO sample were processed for total RNA extraction and subjected to whole-transcriptome sequencing. RESULTS: We demonstrated that the genes coding for detoxification enzymes, particularly cytochrome P450s, were highly up-regulated in the MR group compared to the MS group with similar up-regulation observed in the WI group compared to the CO group. A total of 1,438 genes were differentially expressed in comparison between MR and MS group, including 614 up-regulated genes and 824 down-regulated genes. Additionally, 1,871 genes were differentially expressed in comparison between WI and CO group, including 1,083 up-regulated genes and 788 down-regulated genes. Further analysis on differentially expressed genes from three major detoxification supergene families in both comparisons resulted in 16 detoxification genes as candidates potentially associated with metabolic resistance to malathion. Knockdown of CYP325BC1 and CYP9M12 using RNA interference on the laboratory-maintained Sebring strain significantly increased the mortality of Cx. quinquefasciatus after exposure to malathion. CONCLUSION: We generated substantial transcriptomic evidence on metabolic detoxification of malathion in Cx. quinquefasciatus. We also validated the functional roles of two candidate P450 genes identified through DGE analysis. Our results are the first to demonstrate that knockdown of CYP325BC1 and CYP9M12 both significantly increased malathion susceptibility in Cx. quinquefasciatus, indicating involvement of these two genes in metabolic resistance to malathion.

Discovery, Development, and Evaluation of a Horn Fly-Isolated (Diptera: Muscidae) Beauveria bassiana (Hypocreales: Cordyciptaceae) Strain From Florida, USA.

The horn fly, Haematobia irritans (L.) is an important cattle pest and traditionally has been managed using insecticides; however, many horn fly populations are insecticide-resistant in United States. Use of alternative control techniques has been limited because of the challenges of managing a fly pest on pastured cattle. After the discovery of a wild horn fly infected with Beauveria bassiana in Florida, the fungus was cultured and evaluated for efficacy against laboratory-reared horn flies. This fungal strain was selected for increased virulence by passage through laboratory-reared horn fly hosts to shorten interval from infection to fly death and subsequent conidia formation, properties important to future use of the fungus as a biological control agent against horn flies. After seven passages through horn fly hosts, fly mortality was not significantly accelerated as evaluated through LT50 values, but conidia were readily produced from these killed flies. Although further development is needed to improve fungal efficacy, this fungal strain holds promise as a biological control agent for inclusion in horn fly integrated pest management programs.

Distribution and host associations of ixodid ticks collected from wildlife in Florida, USA.

A tick survey was conducted to document tick-host associations with Florida (USA) wildlife, and to determine the relative abundance and distribution of ixodid ticks throughout the state. The survey was conducted using collection kits distributed to licensed Florida hunters as well as the examination of archived specimens from ongoing state wildlife research programs. Collected tick samples were obtained from 66% of Florida counties and were collected from nine wildlife hosts, including black bear, bobcat, coyote, deer, gray fox, Florida panther, raccoon, swine, and wild turkey. In total, 4176 ticks were identified, of which 75% were Amblyomma americanum, 14% Ixodes scapularis, 8% A. maculatum, 3% Dermacentor variabilis, and < 1% were I. affinis and I. texanus. americanum, D. variabilis, and I. scapularis had the broadest host range, while A. maculatum, D. variabilis, and I. scapularis had the widest geographic distribution. While the survey data contribute to an understanding of tick-host associations in Florida, they also provide insight into the seasonal and geographic distribution of several important vector species in the southeastern USA.

Aedes albopictus (Diptera: Culicidae) Oviposition Preference as Influenced by Container Size and Buddleja davidii Plants.

Aedes albopictus (Skuse) is a container-breeding mosquito commonly found in residential areas of its range in the United States. Mosquitoes are known to utilize flowering plants for sugar acquisition. Limited information is known about the influences on oviposition site selection, outside of container size. Residential areas are often landscaped with a variety of flowering plants and are known to provide numerous sizes of potential larval developmental sites for container-breeding mosqutioes. Through screened enclosure and field studies, the oviposition preference of Ae. albopictus for containers of three selected sizes (473, 946 and 1,892 ml) and the influence of flowering butterfly bush (Buddleja davidii Franchett cultivar 'Guinevere') plants were examined. Our results document that significantly more eggs were oviposited in the largest containers. Additionally, significantly more eggs were oviposited in containers adjacent to flowering butterfly bushes than in those without a flowering butterfly bush. Finally, our results document that flowering butterfly bushes exerted greater influence over Ae. albopictus oviposition decisions than did container size. Our findings can be applied to several aspects of Ae. albopictus surveillance and control.

Development and Evaluation of an Attractive Self-Marking Ovitrap to Measure Dispersal and Determine Skip Oviposition in Aedes albopictus (Diptera: Culicidae) Field Populations.

Aedes albopictus (Skuse) is a container-breeding species with considerable public health importance. To date, Ae. albopictus oviposition behavior has been assessed in outdoor conditions, but only with laboratory-reared specimens. In outdoor large-cage and field studies, we used an attractive self-marking ovipositional device to assess Ae. albopictus skip oviposition behavior. In field studies, 37 wild Ae. albopictus that visited an attractive self-marking ovisite were subsequently captured at a sticky ovitrap within a 4-d period. Because the average Ae. albopictus gonotrophic period is 4.5-6 d, the wild-caught Ae. albopictus visited at least two oviposition sites within a single gonotrophic period. This provided field-based indirect evidence of skip oviposition. The mean distance traveled (MDT) during the 20-d evaluations ranged from 58 to 78 m. The maximum observed distance traveled was 149 m, which was the outer edge of our trapping ability. As populations of Ae. albopictus increased, the MDT during the 4- and 20-d post-marking period increased significantly. Additional observations of wild-marked and captured Aedes triseriatus (Say) are discussed.

Impact of Topical Application Site On the Efficacy of Permethrin and Malathion To Culex quinquefasciatus.

Although insecticide spray droplets will potentially impinge on many exoskeletal body regions, traditional mosquito topical bioassays focus insecticide application to the mesothoracic pleural or dorsal area. Concentrations of permethrin and malathion found in droplets from ultra-low volume and low-volume sprays were evaluated for efficacy against adult Culex quinquefasciatus using a topical application bioassay. Results document nonuniform insecticide sensitivity across body regions, which has not been previously assessed in mosquitoes. Insecticide contact with appendages, such as the leg and the wing, returned much lower mortality from both insecticides than exposure to the primary body (i.e., head, thorax, and abdomen). No difference was observed in percent mortality 24 h after exposure to different insecticides to the same body region. Sublethal behaviors were also observed and discussed for both insecticides. Our findings provide valuable information for those performing topical bioassays, and may help explain insecticide effectiveness wherever droplets impinge upon the mosquito body during laboratory or field applications.

Evaluation of Four Bed Bug Traps for Surveillance of the Brown Dog Tick (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus (Latrielle), can be a serious residential pest due to its unique ability, among ticks, to complete its lifecycle indoors. A single engorged and fertilized female tick can oviposit around 4,000 eggs, allowing indoor establishment to be rapid and easy to miss in early-stage infestations. Acaricide treatment is currently the primary method of control, but can be costly and can lead to the development of acaricide resistance in tick populations. Traps of various designs can be used to help monitor and manage populations of indoor pests, such as cockroaches and bed bugs, but there are currently no commercially available traps for use with brown dog tick infestations. This study included a comparison of four commercially available bed bug traps (NightWatch [BioSensory Inc., Putnam, CT], Bed Bug Beacon [PackTite, Fort Collins, CO], ClimbUp [Susan McKnight Inc., Memphis, TN], and Verify [FMC Corporation, Philadelphia, PA]) with regard to their efficacy in capturing brown dog ticks, and also compared tick attraction to ClimbUp traps baited with several stimuli including CO2. Significantly more ticks were captured and attracted to the NightWatch and CO2-baited ClimbUp traps than the other two trap models. The results suggest that bed bug traps may be useful in brown dog tick monitoring, and CO2 will likely be an important component of a trapping system employed in the future.

Detection of Permethrin Resistance and Fipronil Tolerance in Rhipicephalus sanguineus (Acari: Ixodidae) in the United States.

Permethrin is a commonly used acaricide for tick control on domestic animals and in residential environments, while fipronil use is restricted to on-animal treatment. Following widespread reports of permethrin and fipronil application failures to control indoor infestations of Rhipicephalus sanguineus (Latreille), the brown dog tick, 31 tick populations were obtained from Florida and Texas for acaricide resistance screening. These field-collected ticks from kennels and residential facilities were challenged with technical grade permethrin and fipronil to create dose response curves that were compared with an acaricide-susceptible strain. Permethrin resistance was successfully screened in nine populations, all of which were resistant or highly resistant. Tick susceptibility to fipronil was conducted on four populations, which were found to be tolerant, with resistance ratios below 10. This is the first documentation of R. sanguineus permethrin resistance and fipronil tolerance in the United States. Potential causes of resistance development and recommendations on future brown dog tick control management plans are discussed.

The Effects of Larval Habitat Quality on Aedes albopictus Skip Oviposition.

Aedes albopictus is an invasive mosquito species that transmits human-disease-causing pathogens. It is a container-inhabiting species that oviposits in resource-limited habitats. To mitigate larval competition, Ae. albopictus females may choose to distribute eggs from a single gonotrophic cycle among multiple containers through skip oviposition. With the use of individual females released in indoor and outdoor caged trials, we evaluated the oviposition choices made by gravid Ae. albopictus offered larval habitats with different qualities. Our results demonstrate that Ae. albopictus performs skip oviposition and that the degree of egg distribution is related to the quality of the larval habitat. In a 4-choice arena, individual Ae. albopictus oviposited in fewer containers when presented with ovisites of high-quality larval habitat (uncrowded conditions) compared with oviposition in low-quality (crowded conditions) larval habitats. Additionally, the females selectively oviposited in high-quality habitats when offered both low- and high-quality habitats, but distributed eggs more evenly among multiple high-quality habitats. Our results have important implications for mosquito management plans that include the use of lethal ovitraps, as well as the role of this behavior in distribution of disease-causing pathogens.

Evaluation of a new spraying machine for barrier treatment and penetration of bifenthrin on vegetation against mosquitoes.

The effectiveness and penetration of a novel, truck-mounted mist sprayer (3WC-30-4P provided by American LongRay) was evaluated with bifenthrin in a large, park-like setting with historic floodwater and woodland mosquito populations. Efficacy evaluations were determined through adult population collections and excised leaf bioassays. Trapping results showed a mean reduction of 77% in mosquito populations in the treated area for 5 sampling events up to 4 wk posttreatment. Leaf bioassays revealed an average mortality of 80% at 2.7 m and 51% at 5.5 m against laboratory-reared Aedes aegypti for 5 posttreatment samples. Leaves collected from the treated areas caused higher mortality at distances closer to the sprayer, though the distance and coverage of bifenthrin application was effective up to 5 m.

Genetics and mechanisms of imidacloprid resistance in house flies.

Imidacloprid is the most widely used neonicotinoid insecticide against house flies, which are major pests at animal production facilities worldwide. However, cases of both physiological and behavior resistance have been reported. Recently, physiological resistance to imidacloprid was found in the United States (California and Florida). However, no studies have been undertaken to characterize this resistance in house flies from the United States. Three imidacloprid selections of a strain originally collected from Florida increased the level of resistance, ultimately resulting in a strain that had 2300-fold resistance in females and 130-fold in males. Imidacloprid resistance was not overcome with piperonyl butoxide (PBO) suggesting that resistance is not due to detoxification by cytochrome P450s. Resistance was mapped to autosomes 3 and 4. There was⩾100-fold cross-resistance to acetamiprid and dinotefuran, but no cross-resistance to spinosad. The resistance in this imidacloprid selected population was unstable and declined over a period of several months. The significance of these results to management of imidacloprid resistance in the field, and potential mechanisms of resistance involved, are discussed.

Unveiling candidate genes for metabolic resistance to malathion in Aedes albopictus through RNA sequencing-based transcriptome profiling.

Aedes albopictus, also known as the Asian tiger mosquito, is indigenous to the tropical forests of Southeast Asia. Ae. albopictus is expanding across the globe at alarming rates, raising concern over the transmission of mosquito-borne diseases, such as dengue, West Nile fever, yellow fever, and chikungunya fever. Since Ae. albopictus was reported in Houston (Harris County, Texas) in 1985, this species has rapidly expanded to at least 32 states across the United States. Public health efforts aimed at controlling Ae. albopictus, including surveillance and adulticide spraying operations, occur regularly in Harris County. Despite rotation of insecticides to mitigate the development of resistance, multiple mosquito species including Culex quinquefasciatus and Aedes aegypti in Harris County show organophosphate and pyrethroid resistance. Aedes albopictus shows relatively low resistance levels as compared to Ae. aegypti, but kdr-mutation and the expression of detoxification genes have been reported in Ae. albopictus populations elsewhere. To identify potential candidate detoxification genes contributing to metabolic resistance, we used RNA sequencing of field-collected malathion-resistant and malathion-susceptible, and laboratory-maintained susceptible colonies of Ae. albopictus by comparing the relative expression of transcripts from three major detoxification superfamilies involved in malathion resistance due to metabolic detoxification. Between these groups, we identified 12 candidate malathion resistance genes and among these, most genes correlated with metabolic detoxification of malathion, including four P450 and one alpha esterase. Our results reveal the metabolic detoxification and potential cuticular-based resistance mechanisms associated with malathion resistance in Ae. albopictus in Harris County, Texas.

Fecundity decline is male derived following transfluthrin exposures in a field strain of Aedes albopictus (Diptera: Culicidae).

Volatile pyrethroids are effective in reducing mosquito populations and repelling vectors away from hosts. However, many gaps in knowledge exist for the sublethal impacts of volatile pyrethroids on mosquitoes. To that end, transfluthrin exposures were conducted on a field strain of Aedes albopictus (Skuse) held as a laboratory colony. Dose-response analysis was conducted on both sexes at either 1-4 days old or 5-10 days old. Resultant concentration data were used to evaluate the LC20 and LC50 values in various mate pairings of treatments and controls in which either the male or female was from a selectively treated group and mated with a counterpart that was treated independently. Blood feeding proportion, delayed mortality after a 24-h recovery period, egg collection totals, and F1 larval survival were determined following transfluthrin treatment in the F0, but outcomes were not significant. In contrast, sterility was predicated on male treatment, with treated females resulting in higher overall egg viability. Treated males in the mating pair resulted in significantly lower egg viability and accelerated larval hatch in the F1. Additionally, the presence of sperm in female spermathecae was significantly diminished in test groups containing treated male mosquitoes. Male sublethal effects may be a critical determinant of a mixed population's reproductive success.

Evidence of Permethrin Resistance and Fipronil Tolerance in Rhipicephalus sanguineus s.l. (Acari: Ixodidae) Populations From Florida and California.

Rhipicephalus sanguineus s.l. (Latreille), is a vector of multiple disease-causing pathogens to humans and dogs. Permethrin and fipronil are two acaricides used to manage R. sanguineus s.l. infestations. Homeowners have reported treatment failures in managing brown dog ticks using permethrin and fipronil based products. Previous studies demonstrated that high permethrin resistance in some R. sanguineus s.l. populations was due to metabolic detoxification and target site insensitivity. In this study, three R. sanguineus s.l. strains, one from a laboratory colony (NC) and two colonies originally collected from Florida (FL) and California (CA), were evaluated for resistance expression against permethrin and fipronil. Metabolic detoxification mechanisms were evaluated in the FL strain using three synergists, while a polymerase chain reaction assay was used to detect a resistance mutation in all strains. The NC strain was susceptible to both permethrin and fipronil, while both the FL and CA strains exhibited high resistance to permethrin and tolerance to fipronil. The synergist tests and PCR results indicated that the FL strain utilized both metabolic resistance and target site insensitivity against permethrin, while the CA strain was documented to have the target-site insensitivity resistant allele. This study confirmed permethrin resistance in both California and Florida populations and its persistence in Florida populations, although its susceptibility can potentially be increased by adding a synergist. Fipronil resistance was not detected suggesting this acaricide may provide suitable tick control.

Using environmental factors to predict Rhipicephalus sanguineus s.l. (Acari: Ixodidae) mortality.

BACKGROUND: Rhipicephalus sanguineus s.l. (Latreille, 1806) can establish indoor populations, which increases the risk of pathogen transmission to humans and companion dogs. Rhipicephalus sanguineus s.l. ticks spend most of their life cycle off the host, which subjects developmental timescale to abiotic factors. Previous studies showed that both temperature and relative humidity (RH) influenced Rhipicephalus sanguineus s.l. survival time across all life stages. However, quantified relationships between environmental factors and Rhipicephalus sanguineus s.l. mortality is not currently available. Here, three Rhipicephalus sanguineus s.l. strains were evaluated for mortality under 20 combinations of five temperatures and four RHs. The data obtained were analyzed to quantify the relationship between environmental factors and Rhipicephalus sanguineus s.l. RESULTS: Mortality probabilities did not show a consistent pattern between the three tick strains. Temperature, RH, and their interaction influenced Rhipicephalus sanguineus s.l. mortality probabilities across all life stages, with mortality probability generally increasing with temperature but decreasing with RH. With 50% and lower RH, larvae cannot survive for more than 1 week. However, mortality probabilities in all strains and stages were more sensitive to temperature than to RH. CONCLUSION: This study identified the predictive relationship between environmental factors and Rhipicephalus sanguineus s.l. survival, which enables estimations of tick survival time under varied residential situations, allows parameterization of population models, and provides guidance for pest control professionals to develop efficient management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effective population size of Culex quinquefasciatus under insecticide-based vector management and following Hurricane Harvey in Harris County, Texas.

Introduction: Culex quinquefasciatus is a mosquito species of significant public health importance due to its ability to transmit multiple pathogens that can cause mosquito-borne diseases, such as West Nile fever and St. Louis encephalitis. In Harris County, Texas, Cx. quinquefasciatus is a common vector species and is subjected to insecticide-based management by the Harris County Public Health Department. However, insecticide resistance in mosquitoes has increased rapidly worldwide and raises concerns about maintaining the effectiveness of vector control approaches. This concern is highly relevant in Texas, with its humid subtropical climate along the Gulf Coast that provides suitable habitat for Cx. quinquefasciatus and other mosquito species that are known disease vectors. Therefore, there is an urgent and ongoing need to monitor the effectiveness of current vector control programs. Methods: In this study, we evaluated the impact of vector control approaches by estimating the effective population size of Cx. quinquefasciatus in Harris County. We applied Approximate Bayesian Computation to microsatellite data to estimate effective population size. We collected Cx. quinquefasciatus samples from two mosquito control operation areas; 415 and 802, during routine vector monitoring in 2016 and 2017. No county mosquito control operations were applied at area 415 in 2016 and 2017, whereas extensive adulticide spraying operations were in effect at area 802 during the summer of 2016. We collected data for eighteen microsatellite markers for 713 and 723 mosquitoes at eight timepoints from 2016 to 2017 in areas 415 and 802, respectively. We also investigated the impact of Hurricane Harvey's landfall in the Houston area in August of 2017 on Cx. quinquefasciatus population fluctuation. Results: We found that the bottleneck scenario was the most probable historical scenario describing the impact of the winter season at area 415 and area 802, with the highest posterior probability of 0.9167 and 0.4966, respectively. We also detected an expansion event following Hurricane Harvey at area 802, showing a 3.03-fold increase in 2017. Discussion: Although we did not detect significant effects of vector control interventions, we found considerable influences of the winter season and a major hurricane on the effective population size of Cx. quinquefasciatus. The fluctuations in effective population size in both areas showed a significant seasonal pattern. Additionally, the significant population expansion following Hurricane Harvey in 2017 supports the necessity for post-hurricane vector-control interventions.

Ticks and Tick-Borne Pathogens from Wild Pigs in Northern and Central Florida.

Invasive wild pigs are distributed across much of the U.S. and are hosts to tick vectors of human disease. Herein, adult ticks were collected from 157 wild pigs in 21 northern and central Florida counties from 2019-2020 during removal efforts by USDA-APHIS Wildlife Services personnel and evaluated for their potential to be used as a method of tick-borne disease surveillance. Collected ticks were identified, screened for pathogens, and the effects of landscape metrics on tick community composition and abundance were investigated. A total of 1415 adult ticks of four species were collected. The diversity of tick species collected from wild pigs was comparable to collections made throughout the state with conventional surveillance methods. All species collected have implications for pathogen transmission to humans and other animals. Ehrlichia, Anaplasma-like, and Rickettsia spp. were detected in ticks collected from wild pigs. These results suggest that tick collection from wild pigs is a suitable means of surveillance for pathogens and vectors. The strongest drivers of variation in tick community composition were the developed open space and mixed forest landcover classes. Fragmented shrub/scrub habitat was associated with increased tick abundance. Similar models may be useful in predicting tick abundance and distribution patterns.

Individual and combined releases of Muscidifurax raptor and M. raptorellus (Hymenoptera: Pteromalidae) as a biological control tactic targeting house flies in dairy calf facilities.

The impact of commercially reared house fly parasitoids released into nine dairy calf coverall facilities were evaluated over 3 yr. Individual and equally mixed ratios of the pteromalid parasitoids Muscidifurax raptor Girault and Saunders and M. raptorellus Kogan and Legner were released at a rate of 500 parasitoids per calf per week for 8 wk. Prerelease, release, and postrelease parasitism was monitored using nearly 100,000 sentinel house fly, Musca domestica L., pupae during the 3 yr study. In general, very few adult parasitoids were recovered during the prerelease period and on the no-release farms during any period. However, considerable numbers of M. raptor and M. raptorellus were recovered from sentinel pupae on respective release farms. As expected, the greatest successful parasitism occurred during release periods, with a drop during postrelease periods. High successful parasitism and uneclosed pupae on M. raptorellus release farms suggests that this parasitoid was aggressive in attacking hosts with progeny production at approximately four wasps per pupa. Solitary releases of M. raptor provided sentinel mortality between 31 and 38%, whereas sentinel mortality on M. raptorellus-release farms was double, at 59-80%. Using mixed releases of the two species, overall fly mortality was slightly lower than that observed on M. raptorellus-only farms. This study documents the advantage of releasing M. raptorellus rather than M. raptor on New York dairy calf facilities, as supported by higher parasitism rates and lower costs (35-75%) for purchase of these gregarious wasps, as 75-80% fewer parasitized pupae are needed to achieve similar adult parasitoid levels.

Variable Effects of Temperature and Relative Humidity on Rhipicephalus sanguineus s.l. (Acari: Ixodidae) Development.

Rhipicephalus sanguineus s.l. (Latreille, 1806) can establish populations in residences and may lead to severe domestic and peridomestic infestations. Detection in the early infestation stage is challenging because of their small body size and the lack of visibility when ticks stay in sheltered refugia. The residents may believe that the infestation has been eliminated when no ticks are observed until ticks reappear when seeking hosts. Thus, it is necessary to improve our understanding of tick phenology to achieve more effective infestation management. In this study, the relationships between environmental conditions and tick development were explored in laboratory and using linear and nonlinear models. Three R. sanguineus s.l. strains, from one colony of the temperate lineage and two of the tropical lineage, were evaluated for the development of all life stages and conversion efficiency index (CEI) under five temperatures and four relative humidities (RHs). The development times differed between the three tick strains across stages and were primarily dependent on temperature. The CEIs had little variance explained by temperature, RH, or strains. Compared with the linear and exponential models with temperature as the only variable, the Brière-1 model was the best approximating model for most of the developmental rates. The developmental temperature thresholds for R. sanguineus s.l. development estimated by the Brière-1 model varied inconsistently across strains and life stages. We developed a more predictive relationship between environmental factors and R. sanguineus s.l. development, which can be utilized to predict tick development using temperature and develop appropriate control strategies.

Host blood meal identification by multiplex polymerase chain reaction for dispersal evidence of stable flies (Diptera:Muscidae) between livestock facilities.

A species-specific multiplex polymerase chain reaction targeting the cytochrome b gene of cattle, horses, humans, and dogs was developed to determine the blood meal sources of stable flies, Stomoxys calcitrans (L.), collected from Florida equine facilities. Of 595 presumptive blood-fed stable flies analyzed, successful host amplification was obtained in 350, for a field host-detection efficiency of 58.8%. The majority of analyzed stable flies had fed on cattle (64.6%), followed by horses (24.3%), humans (9.5%), and dogs (1.6%). A survey of animal-enclosed pastures occurring within 3 km of stable fly collection sites revealed that the nearest cattle were between 0.8 and 1.5 km from the four horse farm sampling sites. Cattle-feeding frequencies were greater on farms where cattle were located at distances of 0.8 km, suggesting that between farm differences in host-feeding frequency is related to the number of and distance from a particular host type. Time course evaluations of previously laboratory-fed stable flies demonstrated that host-detection efficiency with this system was 100, 50, and 0% when flies were evaluated at 16, 24, and 48 h postblood feeding, respectively. The results of this study suggest short-term stable fly dispersal of up to 1.5 km in a 48-h time period. The implications of these findings are discussed.