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Off-host periods are critical for ticks, representing a period when environmental stress, particularly dehydration, can impact tick survival. To prevent dehydration, ticks must be able to detect and move into high humidity areas to allow for water vapor uptake. Ionotropic receptor 93a (Ir93a), which is highly expressed in the front forelegs (location of Haller , s organ), increases expression following dehydration, suggesting a role in humidity detection. Although the Haller , s organ is suggested as the site of humidity detection in ticks, humidity detection has not been extensively examined. Here, we assessed the ability of American dog ticks, Dermacentor variabilis , to sense humidity and how altered humidity detection impacts tick survival by manipulating the Haller , s organ. Permanent (cutting or heat ablation) or temporary blocking (silicone covering) of the Haller , s organ impairs the ability of the American dog tick, Dermacentor variabilis, to rest in areas necessary to maintain hydration. Impaired detection of humidity did not impact tick survival when individuals were held under stable optimal conditions. Still, variable conditions (low and high humidity gradient) reduced survival through dehydration stress and decreased energy reserves, as chronic water vapor uptake is energetically expensive. Field validation of these studies in D. variabilis confirmed that humidity detection is critical to tick survival. Lastly, modeling indicates impaired humidity detection will reduce questing tick populations, specifically in the adult stages. These studies confirm that the Haller , s organ is critical for humidity sensing in ticks. Without this ability, tick survival will be impaired by potential dehydration or more rapid depletion of energy reserves.

Current tick control measures are focused on the use of synthetic acaricides and personal protective measures. However, the emergence of acaricide resistance and the maintenance of tick populations in wildlife has precluded the efficient management of ticks. Thus, host-targeted, non-chemical control measures are needed to reliably reduce ticks parasitizing sylvatic reservoirs. This project aimed to evaluate extracellular vesicles (EVs) from Amblyomma americanum as vaccine candidates for white-tailed deer (Odocoileus virginianus; WTD). Salivary gland (SG) and midgut (MG) EVs were isolated by ultracentrifugation. Three deer were vaccinated with SG and MG EVs and received two boosters at days 28 and 50. Two control deer were injected with adjuvant and PBS only. On day 58, WTD were infested with 100 A. americanum nymphs, 50 females, and 50 males that were allowed to feed to repletion. On-host and off-host mortality, tick engorgement weight, nymph molting, time to oviposition, and egg hatchability were evaluated. Serum samples were recovered every seven days until the last day of tick drop off, and then at one year (Y1) and 1-year and 1-month (Y1M1). Vaccination resulted in seroconversion and significant increases in total IgG levels that remained significantly higher than controls and pre-vaccination levels at Y1 and Y1M1. No negative effects were observed in nymphs, but on-host mortality of female A. americanum was significantly higher in vaccinated animals. No effects were observed on reproductive parameters. These results indicate that proteins within female tick SG and MG vesicles are not good candidates for vaccine design against nymphs; however, the on-host adult mortality suggests that tick EVs harbor protective antigens against A. americanum females., Competing Interests: Declaration of competing interest Dr. Adela Oliva Chavez and Dr. Tammi Johnson have invention disclosures with Texas A&M University and the University of Wisconsin, Madison for the use of tick extracellular vesicle derived proteins for the development of anti-tick vaccines. These disclosures did not affect the performance of these experiments. This article reports the results of research only and mention of a proprietary product does not constitute an endorsement or recommendation by the USDA for its use. USDA is an equal opportunity provider and employer., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Female ticks deposit large egg clusters that range in size from hundreds to thousands. These egg clusters are restricted to a deposition site as they are stationary, usually under leaf litter and other debris. In some habitats, these sites can be exposed to periodic flooding. When the clusters of tick eggs are disturbed, they may float to the surface or remain underneath organic debris entirely submerged underwater. Here, we examined the viability of egg clusters from winter ticks, Dermacentor albipictus, and lone star ticks, Amblyomma americanum, when partially or fully submerged in water and in relation to the developmental stages of the eggs under lab conditions. In general, egg clusters that were older and partially submerged had a higher viability than fully submerged, younger eggs. Of the two species, A. americanum was more resistant to water exposure. These studies highlight that egg clusters for certain tick species can remain viable when exposed to water for at least two weeks. These results also suggest that distribution by flooding of egg clusters could occur for some species and water submersion will differentially impact tick egg survival based on the specific developmental stage of exposure and species., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection., Methods: Nilgai were needle inoculated with ≈10 8 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite., Results: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer., Conclusions: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances., (© 2024. The Author(s).)

Female ticks deposit large egg clusters that range in size from hundreds to thousands. These clusters are restricted to a deposition site, usually under leaf litter and other debris. These sites can be exposed to periodic flooding, where the cluster of tick eggs can float to the surface or remain underneath organic debris entirely underwater. Here, we examined the viability of egg clusters from winter ticks, Dermacentor albipictus , and lone star ticks, Amblyomma americanum , when partially submerged or fully submerged in water in relation to the developmental stages of the eggs. In general, egg clusters that were older and partially submerged had a higher viability than fully submerged, young eggs in water. A. americanum was much more resistant to water exposure between the two species. These studies highlight that egg clusters for specific tick species can remain viable when exposed to water for at least two weeks, where eggs float on the surface. These studies also suggest that water-based distribution of egg clusters could occur for some species, and flooding will differentially impact tick egg survival based on the specific developmental stage of exposure and species.

White-tailed deer Odocoileus virginianus (Zimmermann) (Artiodactyla: Cervidae) are the main host for adult Ixodes scapularis Say (Acari: Ixodidae) (blacklegged tick) and all stages of Amblyomma americanum Linnaeus (Acari: Ixodidae) (lone star tick). However, literature describing the feeding and reproductive parameters of these tick species when feeding on this host is limited. We experimentally infested white-tailed deer with adult pairs of either I. scapularis or A. americanum to improve our understanding of these tick-host relationships. Our study used tick-naïve white-tailed deer and restricted host grooming throughout the infestation. For I. scapularis, the days to repletion (mean ± SE, 6.04 ± 0.07), engorgement weight of replete females (0.20 ± 0.0032 g), duration of oviposition (32 ± 0.45 d), egg mass weight (0.10 ± 0.0027 g), and number of eggs laid per tick (1,803.00 ± 49.00) were recorded. Data from A. americanum were also recorded, including days to repletion (11.00 ± 0.063), engorgement weight of replete females (0.63 ± 0.025 g), duration of oviposition (37.00 ± 1.30 d), egg mass weight (0.34 ± 0.017 g), and number of eggs laid per tick (5,873.00 ± 291.00). These biological parameter data could be used as variables in models (e.g., LYMESIM 2.0) to determine how white-tailed deer influence I. scapularis and A. americanum populations in nature, and to evaluate the protective efficacy of tick-antigen-based antitick vaccines., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

Deer keds, such as Lipoptena cervi Linnaeus (Diptera: Hippoboscidae), are blood-feeding flies from which several human and animal pathogens have been detected, including Borrelia burgdorferi sensu lato Johnson (Spirochaetales: Borreliaceae), the causative agent of Lyme disease. Cervids (Artiodactyla: Cervidae), which are the primary hosts of deer keds, are not natural reservoirs of B. burgdorferi sl, and it has been suggested that deer keds may acquire bacterial pathogens via co-feeding near infected ticks. We screened L. cervi (n = 306) and Ixodes scapularis Say (Ixodida: Ixodidae) (n = 315) collected from 38 white-tailed deer in Pennsylvania for the family Anaplasmataceae, Bartonella spp. (Hyphomicrobiales: Bartonellaceae), Borrelia spp., and Rickettsia spp. (Rickettsiales: Rickettsiaceae). Limited similarity in the bacterial DNA detected between these ectoparasites per host suggested that co-feeding may not be a mechanism by which deer keds acquire these bacteria. The feeding biology and life history of deer keds may impact the observed results, as could the season when specimens were collected. We separately screened L. cervi (n = 410), L. mazamae Róndani (n = 13), L. depressa Say (n = 10), and Neolipoptena ferrisi Bequaert (n = 14) collections from locations within the United States and Canada for the same pathogens. These results highlight the need to further study deer ked-host and deer ked-tick relationships., (© 2022 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

White-tailed deer (Odocoileus virginianus) are a main host for the adult life stages of tick species of medical and veterinary importance. Since white-tailed deer play a vital role in tick ecology, research has been conducted to understand this tick-host relationship. To date, research involving captive white-tailed deer and artificial infestation of these animals with ticks has focused on host suitability, the role of white-tailed deer in tick-borne diseases, and anti-tick vaccine research. The methodology reported for these studies was at times not descriptive and inconsistent regarding how and what region of the white-tailed deer was infested with ticks. Here, we propose a standardized method to artificially infest captive white-tailed deer with ticks for research purposes. The protocol describes a method proven effective to experimentally infest captive white-tailed deer with blacklegged ticks (Ixodes scapularis) to study tick-host interactions. The methods can be reliably transferred for experimental infestation of white-tailed deer by other multi-host and one-host tick species., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions. We recombinantly expressed ScalOBP60, as well as a truncated sequence which lacks the histidine-rich N-terminal region, tScalOBP60. Using fluorescence quenching and electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF), we detected two different types of metal-binding sites. Divalent copper, nickel and zinc bind to the N-terminal histidine-rich region, and divalent copper binds to an internal sequence position. Comparison of the ESI-QTOF spectra of ScalOBP60 and tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of divalent metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact allosterically, possibly through a conformational equilibrium, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP., (Copyright © 2021. Published by Elsevier Ltd.)

Background: White-tailed deer (Odocoileus virginianus) host numerous ectoparasitic species in the eastern USA, most notably various species of ticks and two species of deer keds. Several pathogens transmitted by ticks to humans and other animal hosts have also been found in deer keds. Little is known about the acquisition and potential for transmission of these pathogens by deer keds; however, tick-deer ked co-feeding transmission is one possible scenario. On-host localization of ticks and deer keds on white-tailed deer was evaluated across several geographical regions of the eastern US to define tick-deer ked spatial relationships on host deer, which may impact the vector-borne disease ecology of these ectoparasites., Methods: Ticks and deer keds were collected from hunter-harvested white-tailed deer from six states in the eastern US. Each deer was divided into three body sections, and each section was checked for 4 person-minutes. Differences in ectoparasite counts across body sections and/or states were evaluated using a Bayesian generalized mixed model., Results: A total of 168 white-tailed deer were inspected for ticks and deer keds across the study sites. Ticks (n = 1636) were collected from all surveyed states, with Ixodes scapularis (n = 1427) being the predominant species. Counts of I. scapularis from the head and front sections were greater than from the rear section. Neotropical deer keds (Lipoptena mazamae) from Alabama and Tennessee (n = 247) were more often found on the rear body section. European deer keds from Pennsylvania (all Lipoptena cervi, n = 314) were found on all body sections of deer., Conclusions: The distributions of ticks and deer keds on white-tailed deer were significantly different from each other, providing the first evidence of possible on-host niche partitioning of ticks and two geographically distinct deer ked species (L. cervi in the northeast and L. mazamae in the southeast). These differences in spatial distributions may have implications for acquisition and/or transmission of vector-borne pathogens and therefore warrant further study over a wider geographic range and longer time frame., (© 2022. The Author(s).)

In October 2020, three captive male white-tailed deer, Odocoileus virginianus [Zimmermann] (artiodactyla: Cervidae), were found dead in central Pennsylvania and a fourth was euthanized due to extreme lethargy. The deer presented with high burdens of Dermacentor albipictus (Packard) (Ixoda: Ixodidae) (winter tick). There were no other clinical symptoms and deer were in otherwise good physical condition with no observed alopecia. Winter tick epizootics have been associated with mortalities of moose, Alces alces [Linnaeus] (artiodactyla: cervidae), and more recently elk, Cervus canadensis [Erxleben] (artiodactyla: cervidae), in Pennsylvania, but have not been reported in white-tailed deer. Mild winters are favorable to winter ticks and deer producers and managers should be aware of possible infestations as a result., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies., Results: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways., Conclusions: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Horn flies are one of the most significant economic pests of cattle in the United States and worldwide. Chemical control methods have been routinely utilized to reduce populations of this pest, but the steady development of insecticide resistance has prompted evaluation of alternative control strategies. Behavior modifying compounds from natural products have shown some success in impacting horn fly populations, and a more thorough understanding of the horn fly chemosensory system would enable improvements in the development of species-specific compounds. Using an RNA-seq approach, we assembled a transcriptome representing genes expressed in adult female and male horn fly head appendages (antennae, maxillary palps, and proboscides) and adult fly bodies from which heads were removed. Differential gene expression analysis identified chemosensory gene family members that were enriched in head appendage tissues compared with headless bodies. Candidate members included 43 odorant binding proteins (OBP) and 5 chemosensory binding proteins (CSP), as well as 44 odorant receptors (OR), 27 gustatory receptors (GR), and 34 ionotropic receptors (IR). Sex-biased expression of these genes was not observed. These findings provide a resource to enable future studies targeting horn fly chemosensation as part of an integrated strategy to control this blood-feeding pest.

A survey of ixodid ticks parasitizing white-tailed deer ( Odocoileus virginianus ) and nilgai antelope ( Boselaphus tragocamelus ) was completed during the 2018-2019 public hunt season on the Laguna Atascosa National Wildlife Refuge (Cameron County, Texas) and the East Foundation's El Sauz Ranch in nearby Willacy County (Texas). Anocenter nitens was the predominant tick species identified with 5% of these ticks collected from nilgai. All life stages were encountered in high numbers on white-tailed deer, indicating that deer may be a primary host in this region. Amblyomma maculatum and Amblyomma inornatum were identified from both hosts, while Ixodes scapularis was encountered only on white-tailed deer. This is the first published record of A. inornatum on nilgai. A subset of ticks was used in PCR assays to detect Rickettsia spp., family Anaplasmataceae , Borrelia spp., and Theileria-Babesia spp. Borrelia spp. were not detected in any of the ticks analyzed. Rickettsia parkeri was detected in three A. maculatum adult ticks from deer, Rickettsia sp. endosymbiont sequences were present in all I. scapularis ticks, and Rickettsia amblyommatis was detected in three A. inornatum adult ticks from deer. Sequence analysis of Anaplasmataceae- positive amplicons from A. nitens and A. maculatum had low percent identity to published Anaplasma spp. sequences, suggesting a unique Anaplasma sp. may be circulating in the population. Anaplasma platys was detected from A. nitens larvae and an Ehrlichia sp. Delta strain was present in A. maculatum , both of unknown pathogenicity towards deer. Theileria cervi was detected in all stages of A. nitens ticks, and positive ticks originated from 27 of 31 deer and a single nilgai sampled from throughout the survey site. The primary vector for T. cervi is absent from this region, suggesting T. cervi is possibly maintained by a different tick species., Competing Interests: The authors declare that they have no competing interests.

Acetylcholinesterase (AChE) was previously reported to be present in saliva of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), with proposed potential functions to 1) reduce acetylcholine toxicity during rapid engorgement, 2) modulate host immune responses, and 3) to influence pathogen transmission and establishment in the host. Potential modulation of host immune responses might include participation in salivary-assisted transmission and establishment of pathogens in the host as has been reported for a number of arthropod vector-borne diseases. If the hypothesis that tick salivary AChE may alter host immune responses is correct, we reasoned that similar cholinesterase activities might be present in saliva of additional arthropod vectors. Here, we report the presence of AChE-like activity in the saliva of southern cattle ticks, Rhipicephalus (Boophilus) microplus; the lone star tick, Amblyomma americanum (Linnaeus); Asian tiger mosquitoes, Aedes albopictus (Skuse); sand flies, Phlebotomus papatasi (Scopoli); and biting midges, Culicoides sonorensis Wirth and Jones. Salivary AChE-like activity was not detected for horn flies Haematobia irritans (L.), stable flies Stomoxys calcitrans (L.), and house flies Musca domestica L. Salivary cholinesterase (ChE) activities of arthropod vectors of disease-causing agents exhibited various Michaelis-Menten KM values that were each lower than the KM value of bovine serum AChE. A lower KM value is indicative of higher affinity for substrate and is consistent with a hypothesized role in localized depletion of host tissue acetylcholine potentially modulating host immune responses at the arthropod bite site that may favor ectoparasite blood-feeding and alter host defensive responses against pathogen transmission and establishment., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Sex chromosomes and sex determining genes can evolve fast, with the sex-linked chromosomes often differing between closely related species. Population genetics theory has been developed and tested to explain the rapid evolution of sex chromosomes and sex determination. However, we do not know why the sex chromosomes are divergent in some taxa and conserved in others. Addressing this question requires comparing closely related taxa with conserved and divergent sex chromosomes to identify biological features that could explain these differences. Cytological karyotypes suggest that muscid flies ( e.g. , house fly) and blow flies are such a taxonomic pair. The sex chromosomes appear to differ across muscid species, whereas they are conserved across blow flies. Despite the cytological evidence, we do not know the extent to which muscid sex chromosomes are independently derived along different evolutionary lineages. To address that question, we used genomic and transcriptomic sequence data to identify young sex chromosomes in two closely related muscid species, horn fly ( Haematobia irritans ) and stable fly ( Stomoxys calcitrans ). We provide evidence that the nascent sex chromosomes of horn fly and stable fly were derived independently from each other and from the young sex chromosomes of the closely related house fly ( Musca domestica ). We present three different scenarios that could have given rise to the sex chromosomes of horn fly and stable fly, and we describe how the scenarios could be distinguished. Distinguishing between these scenarios in future work could identify features of muscid genomes that promote sex chromosome divergence., (Copyright © 2020 Meisel et al.)

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC99, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia., (Published by Oxford University Press on behalf of Entomological Society of America 2019.)

Nilgai ( Boselaphus tragocamelus) are nonnative bovines that were originally introduced as game animals to one large, south Texas, US ranch but that are now present throughout southeastern Texas from Baffin Bay to Harlingen and in northern Mexico at least as far west as Durango. Between October 2014 and January 2017, nilgai ( n=517) were examined for the presence of tick ectoparasites, with particular interest in the cattle fever tick, Rhipicephalus ( Boophilus) microplus. These animals were either hunter killed or they were culled as part of federal cooperative harvesting from Cameron and Willacy counties in southeastern Texas. The proportion of fever tick-infested animals differed in a N-to-S pattern, and this was at least partly attributed to differences in habitat. The southern area is a lowland floodplain predominated by halophytes, whereas the northerly area is upland thorn scrub, the latter of which provides a vegetative canopy that is more conducive to tick survival and persistence. A subset of nilgai, all from the Texas-Mexico border area, were screened for livestock pathogens using molecular and serological assays. All nilgai were seronegative for Babesia ( Theileria) equi and Babesia cabalii. Although 11 animals were seropositive for Anaplasma marginale by competitive enzyme-linked immunosorbent assay (cELISA), these were interpreted with caution because of the lack of concordance between cELISA and molecular detection assays. All animals were PCR negative for presence of Babesia spp. DNA, and a single nilgai was seropositive for Babesia bovis and Babesia bigemina by complement fixation. It remains unknown whether cattle Babesia spp. can establish an infection in nilgai.

Musca domestica L. were collected from cattle diagnosed with bovine ringworm to evaluate the potential of the house fly to disseminate Trichophyton verrucosum E. Bodin, a fungal dermatophyte that is the causative agent for ringworm in cattle. Fungal isolates were cultured from 45 individual flies on supplemented Sabouraud dextrose agar, and isolates were identified using morphological and microscopic approaches. Each isolate was identified further by PCR amplification of the ribosomal DNA locus with fungal-specific primers and subsequent amplicon sequencing. Trichophyton verrucosum was not identified using these approaches. However, 35 different fungal species representing 17 genera were cultured from collected flies, including several species that are allergenic and pathogenic to humans and animals. Several species within the fungal orders Hypocreales, Microascales, Onygenales, Saccharomycetales, Xylaniales, and Agaricales were observed for the first time on house flies. The most frequent fungus recovered was Cladosporium cladosporoides Fresen, which is known to be a ubiquitous, airborne allergen to humans., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

Biting arthropods are implicated in the transdermal transmission of Salmonella to bovine peripheral lymph nodes, and such contamination can contribute to increased Salmonella prevalence in processed beef. Since horn flies can acquire Salmonella and then excrete the bacteria in their feces, on-animal fly infestations were conducted in this study to assess whether horn flies have a role in this bacterial transmission. Three Salmonella serotypes were used to assess fly acquisition from and excretion onto cattle. The results indicated that flies can acquire Salmonella from the hide, as assessed by recovery from homogenates of surfacesterilized flies, and that Salmonella persists for at least 5 days in the fly. Fly fecal excreta serves as a bacterial contaminant on the hide, and the overall mean probable estimate of the quantity shed was ≈10(5) most probable number per fly cage area. In 5 days, no transmission of the bacteria to bovine peripheral lymph nodes was evident, prompting an assessment of the effects of prolonged horn fly feeding on transmission. Three groups of animals were infested with flies that had consumed a blood meal containing Salmonella Senftenberg. After 5 days, the study was either terminated or the flies were removed and the cages replenished with unfed flies either once or twice over the course of an 11- or 19-day fly exposure period, respectively. A microlancet-inoculated positive-control animal was included in each group for comparison. The impact of prolonged horn fly feeding was evident, as 8% of lymph nodes cultured were positive from the 5-day exposure, whereas 50 and 42% were positive from 11- and 19-day exposures, respectively. Higher concentrations of Salmonella were recovered from fly-infested animals than from the microlancet-inoculated control, likely a result of repeated inoculations over time by flies versus a single introduction. The data described provide new insights into the transmission dynamics of Salmonella in cattle populations, highlighting a role for biting flies as an important reservoir.

The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller's organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller's organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks.

Wildlife are an important component in the vector-host-pathogen triangle of livestock diseases, as they maintain biological vectors that transmit pathogens and can serve as reservoirs for such infectious pathogens. Babesia bovis is a tick-borne pathogen, vectored by cattle fever ticks, Rhipicephalus spp., that can cause up to 90% mortality in naive adult cattle. While cattle are the primary host for cattle fever ticks, wild and exotic ungulates, including white-tailed deer (WTD), are known to be viable alternative hosts. The presence of cattle fever tick populations resistant to acaricides raises concerns regarding the possibility of these alternative hosts introducing tick-borne babesial parasites into areas free of infection. Understanding the B. bovis reservoir competence of these alternative hosts is critical to mitigating the risk of introduction. In this study, we tested the hypothesis that WTD are susceptible to infection with a B. bovis strain lethal to cattle. Two groups of deer were inoculated intravenously with either B. bovis blood stabilate or a larval extract supernatant containing sporozoites from infected R. microplus larvae. The collective data demonstrated that WTD are neither a transient host nor reservoir of B. bovis. This conclusion is supported by the failure of B. bovis to establish an infection in deer regardless of inoculum. Although specific antibody was detected for a short period in the WTD, the PCR results were consistently negative at multiple time points throughout the experiment and blood from WTD that had been exposed to parasite, transferred into naïve recipient susceptible calves, failed to establish infection. In contrast, naïve steers inoculated intravenously with either B. bovis blood stabilate or the larval extract supernatant containing sporozoites rapidly succumbed to disease. These findings provide evidence that WTD are not an epidemiological component in the maintenance of B. bovis infectivity to livestock.

The Lone Star tick, Amblyomma americanum Linnaeus 1758 (Acari; Ixodidae), causes considerable production losses to the southern U.S. cattle industry due to reduced weight, infertility, secondary infections at bite wound sites, damaged hides, and potentially death, as these ticks tend to infest livestock in large numbers. Increasing environmental concerns, along with the potential for chemical residue in food products, have led to more emphasis on alternative tick control strategies, such as selective breeding practices and anti-tick vaccines. To enable progress toward these goals, a better understanding of bovine host immune mechanisms elicited by ticks is needed. In this study, 7 calves were phenotyped as susceptible, moderately resistant, or highly resistant to adult A. americanum ticks. Tick bite-site biopsies and blood leukocytes were collected at multiple time points throughout 3 successive tick infestations. Gene expression at tick bite-site biopsies was assessed by microarray analysis over 3 time points for each phenotype group. Quantitative reverse transcriptase-PCR expression analysis evaluated 11 candidate genes in tick bite-site biopsies, and 6 in blood leukocytes. Regression curve estimates calculated from the expression values generated by qRT-PCR in tick bite-sites identified correlations between several candidate genes. Increased expression of IGHG1, IL6, IL1α, and IL1RN in bovine tick bite-site biopsies suggests that Th2 differentiation may be important for the local bovine response to A. americanum ticks. Strong correlations in expression for IL1α and IL1β, for IL1α and IL1RN, and for IL1α and TLR4 were found in biopsies from the tick-resistant phenotypes. The up-regulation of IL12 and IL23 in blood leukocytes from Lone Star tick-infested calves of all phenotypes suggests a possible systemic recruitment of memory T cells. This study provides novel insight concerning the bovine immune response to Lone Star ticks and a basis for future investigations to characterize the importance of these factors for tick-resistance in cattle., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S., Methods: We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency., Results: We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635)., Conclusions: These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.

Contamination of cattle peripheral lymph nodes with Salmonella enterica is proposed to occur via a transdermal route of entry. If so, bacteria may be introduced to cattle by biting arthropods. Biting flies, such as horn flies (Haematobia irritans irritans (L.)) (Diptera: Muscidae), are intriguing candidates for transmitting Salmonella to cattle because they provide a route of entry when they breach the skin barrier during blood feeding. Using a green fluorescent protein-expressing strain of Salmonella Montevideo (S. Montevideo-GFP), the current study demonstrated that horn fly grooming subsequent to tactile exposure to the bacteria resulted in acquisition of the bacteria on mouthparts as well as microbial ingestion. Consumption of a bloodmeal containing approximately 10(2), approximately 10(4), or 10(6) S. Montevideo-GFP resulted in horn fly colonization for up to 72 h postingestion (PI). Epifluorescent microscopy indicated that the bacteria were not localized to the crop but were observed within the endoperitrophic space, suggesting that regurgitation is not a primary route of transmission. S. Montevideo-GFP were cultured from excreta of 100% of flies beginning 6-7 h PI of a medium or high dose meal and > 12 h PI in excreta from 60% of flies fed the low-dose meal. Animal hides and manure pats are sources for horn flies to acquire the Salmonella and mechanically transmit them to an animal while feeding. Mean quantities of 5.65-67.5 x 10(2) CFU per fly were cultured from fly excreta passed within 1 d after feeding, suggesting the excreta can provide an additional microbial source on the animal's hide.

Abstract' Background: For >100 years cattle production in the southern United States has been threatened by cattle fever. It is caused by an invasive parasite-vector complex that includes the protozoan hemoparasites Babesia bovis and B. bigemina, which are transmitted among domestic cattle via Rhipicephalus tick vectors of the subgenus Boophilus. In 1906 an eradication effort was started and by 1943 Boophilus ticks had been confined to a narrow tick eradication quarantine area (TEQA) along the Texas-Mexico border. However, a dramatic increase in tick infestations in areas outside the TEQA over the last decade suggests these tick vectors may be poised to re-invade the southern United States. We investigated historical and potential future distributions of climatic habitats of cattle fever ticks to assess the potential for a range expansion., Methods: We built robust spatial predictions of habitat suitability for the vector species Rhipicephalus (Boophilus) microplus and R. (B.) annulatus across the southern United States for three time periods: 1906, present day (2012), and 2050. We used analysis of molecular variance (AMOVA) to identify persistent tick occurrences and analysis of bias in the climate proximate to these occurrences to identify key environmental parameters associated with the ecology of both species. We then used ecological niche modeling algorithms GARP and Maxent to construct models that related known occurrences of ticks in the TEQA during 2001-2011 with geospatial data layers that summarized important climate parameters at all three time periods., Results: We identified persistent tick infestations and specific climate parameters that appear to be drivers of ecological niches of the two tick species. Spatial models projected onto climate data representative of climate in 1906 reproduced historical pre-eradication tick distributions. Present-day predictions, although constrained to areas near the TEQA, extrapolated well onto climate projections for 2050., Conclusions: Our models indicate the potential for range expansion of climate suitable for survival of R. microplus and R. annulatus in the southern United States by mid-century, which increases the risk of reintroduction of these ticks and cattle tick fever into major cattle producing areas.

Background: Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus)., Methods: Using 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections., Results: LPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00-0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas., Conclusions: Two dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established.

The stable fly, Stomoxys calcitrans (L.), is a serious ectoparasite affecting animal production and health of both animals and humans. Stable fly control relies largely on chemical insecticides; however, the development of insecticide resistance as well as environmental considerations requires continued discovery research to develop novel control technologies. MicroRNAs (miRNAs) are a class of short noncoding RNAs that have been shown to be important regulators of gene expression across a wide variety of organisms, and may provide an innovative approach with regard to development of safer more targeted control technologies. The current study reports discovery ad initial comparative analysis of 88 presumptive miRNA sequences from the stable fly, obtained using high-throughput sequencing of small RNAs. The majority of stable fly miRNAs were 22-23 nt in length. Many miRNAs were arthropod specific, and several mature miRNA sequences showed greater sequence identity to miRNAs from other blood-feeding dipterans such as mosquitoes rather than to Drosophilids. This initial step in characterizing the stable fly microRNAome provides a basis for further analyses of life stage-specific and tissue-specific expression to elucidate their functional roles in stable fly biology.

Biting flies are economically important blood-feeding pests of medical and veterinary significance. Chemosensory-based biting fly behaviours, such as host/nutrient source localization and ovipositional site selection, are intriguing targets for the development of supplemental control strategies. In an effort to expand our understanding of biting fly chemosensory pathways, transcripts encoding the highly conserved insect odorant co-receptor (Orco) were isolated from two representative biting fly species, the stable fly (Scal\Orco) and the horn fly (Hirr\Orco). Orco forms a complex with an odour-specific odorant receptor to form an odour-gated ion channel. The biting fly transcripts were predicted to encode proteins with 87-94% amino acid similarity to published insect Orco sequences and were detected in various immature stages as well as in adult structures associated with olfaction, i.e. the antennae and maxillary palps, and gustation, i.e. the proboscis. Further, the relevant proteins were immunolocalized to specific antennal sensilla using anti-serum raised against a peptide sequence conserved between the two fly species. Results from the present study provide a basis for functional evaluation of repellent/attractant effects on as yet uncharacterized stable fly and horn fly conventional odorant receptors., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.

ABSTRACT Acetylcholinesterase cDNAs, BmAChE1, BmAChE2, and BmAChE3 of Rhipicephalus (Boophilus) microplus (Canestrini) were sequenced and found to exhibit significant polymorphism. A portion of the predicted amino acid substitutions in BmAChE1, BmAChE2, and BmAChE3 were found predominantly in organophosphate-resistant strains, but most did not correlate with resistant status. Multiple transcripts were observed from individual ticks, suggesting possible gene duplication or alternative splicing to produce more than two transcripts per individual. BmAChE1 transcript polymorphisms associating with organophosphate-resistant status in laboratory strains were surveyed in laboratory and Mexican strains of R. microplus by sequencing BmAChE1 genomic DNA. Quantitative real-time polymerase chain reaction was used to determine copy numbers of BmAChE1 (eight copies/haploid genome), BmAChE2 (16 copies/haploid genome), and BmAChE3 (four copies/haploid genome). Presence of at least three highly polymorphic amplified genes expressing AChE in tick synganglion suggested that ticks maintain a large and diverse assortment of AChE alleles available for rapid recombination and selection, which potentially reduces fitness costs associated with individual mutations. Elevated copy numbers for each of the BmAChEs may also explain previous failures to identify mutations resulting in insensitivity to organophosphates. It is clear that development of phenotypic resistance to organophosphates is highly complex and may be multigenic in character.

Analysis of doramectin concentration in blood serum of pastured cattle injected repeatedly (12 treatments) at two different dosage rates and 28-day intervals throughout the year was used to predict the probability that cattle fever ticks could successfully feed to repletion during the interval between any two consecutive treatments. Treatment at ~270 μg/kg indicated that serum doramectin concentration dropped below the baseline concentration estimated for tick survival (8 ppb) in 7 of the 12 treatments. However, the longest period between any two treatments during which the doramectin concentration remained below the 8 ppb baseline level for successful tick feeding was 15 days, making it virtually impossible for any ticks to reach ovipositional status prior to a subsequent treatment. At a dosage rate of ~540 μg/kg, the concentration dropped below the baseline tick survival level (8 ppb) only once, following the initial treatment, and the duration during which the concentration remained below the baseline level prior to the subsequent treatment was only 6 days. Thus, at the high dosage rate results indicated, with absolute certainty, that no ticks could successfully feed to repletion between any two consecutive treatments. Based on the data obtained in the study it was concluded that analysis of doramectin concentration in serum of treated animals would be a reliable predictor for assessing the probability that ticks could successfully develop to repletion. More importantly, results demonstrated that the trial policy, instituted by the Cattle Fever Tick Eradication Program, of repeatedly treating cattle with doramectin injections at 25-28 day intervals for eliminating cattle fever ticks would produce little or no risk of any viable ticks developing to repletion and re-infesting the field between treatment applications.

Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The genomic DNA that is spliced to produce G6PDH-A and -B is 8,600-9,000 bases in length and comprises 12 exons. Comparison of the R. microplus G6PDH gene structure with those available from insects and mammals revealed that the tick gene is most like that of humans. Detection of the four transcripts was evaluated by quantitative RT-PCR using template from larvae, unfed adult females and males, salivary gland tissues from 2- to 3-day-fed adult females and males, and salivary gland tissue of 4- to 5-day-fed adult females. The G6PDH-A and -C transcripts were present in all templates, and both displayed induced expression in salivary gland tissue of fed, adult females but not matched males. The G6PDH-D transcript was detected only in unfed adults and in larvae, a stage in which it was most abundant relative to the other three transcripts. The G6PDH-B transcript, while detectable in all templates, was of low copy number suggesting it is a rare transcript. Induced expression of G6PDH-A and G6PDH-C in fed females may play a role in the tolerance of oxidative stress that is induced upon feeding, and the transcript abundance in fed females may be a function of bloodmeal volume and the time adult females spend on the host relative to adult males.

The insect sodium channel is of particular interest for evaluating resistance to pyrethroids because it is the target molecule for this major class of neurotoxic insecticides. The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), sodium channel coding sequence representing domains IS6 through IVS6 was isolated, and the sequence encoding domain II was compared among individuals of a laboratory strain selected for resistance to permethrin and the unselected, parental generation. A point mutation resulting in a leucine-to-histidine amino acid change was identified (Leul014His), and its location corresponded with that observed for knockdown resistance (kdr) mutations in other insects. As a result, the allele was designated kdr-his. A molecular assay was developed to assess the frequency of this mutation in genomic DNA of individual stable flies from the laboratory selections, which provided further evidence that the kdr-his allele accounts for the observed level ofpermethrin resistance in the selected strain. The assay was then used to evaluate the frequency of the mutation from five field-collected populations originating from three horse farms near Ocala, FL; one horse farm near Gainesville, FL; and one dairy farm near Hague, FL. Frequency of the kdr-his allele ranged from 0.46 to 0.78, supporting further investigation of allele prevalence throughout the stable fly season and in response to field insecticide application.

Background: Cattle fever ticks, Rhipicephalus (Boophilus) microplus and R. (B.) annulatus, vector bovine and equine babesiosis, and have significantly expanded beyond the permanent quarantine zone established in South Texas. Currently, there are no vaccines approved for use within the United States for controlling these vectors. Vaccines developed in Australia and Cuba based on the midgut antigen Bm86 have variable efficacy against cattle fever ticks. A possible explanation for this variation in vaccine efficacy is amino acid sequence divergence between the recombinant Bm86 vaccine component and native Bm86 expressed in ticks from different geographical regions of the world., Results: There was 91.8% amino acid sequence identity in Bm86 among R. microplus and R. annulatus sequenced from South Texas infestations. When South Texas isolates were compared to the Australian Yeerongpilly and Cuban Camcord vaccine strains, there was 89.8% and 90.0% identity, respectively. Most of the sequence divergence was focused in one region of the protein, amino acids 206-298. Hydrophilicity profiles revealed that two short regions of Bm86 (amino acids 206-210 and 560-570) appear to be more hydrophilic in South Texas isolates compared to vaccine strains. Only one amino acid difference was found between South Texas and vaccine strains within two previously described B-cell epitopes. A total of 4 amino acid differences were observed within three peptides previously shown to induce protective immune responses in cattle., Conclusions: Sequence differences between South Texas isolates and Yeerongpilly and Camcord strains are spread throughout the entire Bm86 sequence, suggesting that geographic variation does exist. Differences within previously described B-cell epitopes between South Texas isolates and vaccine strains are minimal; however, short regions of hydrophilic amino acids found unique to South Texas isolates suggest that additional unique surface exposed peptides could be targeted.

Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previously identified as presumptively encoding acetylcholinesterases (AChEs), but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization of the recombinant proteins supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates (OPs) malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl-beta-methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent at 10-100 microM choline in agreement with properties reported for the catalytic domain of Anopheles gambiae acetylcholinesterase-1. Substrate affinity and V(max) values were highest for rBmAChE1 proteins, and one rBmAChE1 enzyme (Tx11, derived from the OP-resistant strain Tuxpan), was insensitive to paraoxon and exhibited a greatly reduced V(max) near that of rBmAChE2. To date, recombinant BmAChE1 and BmAChE3 enzymes with reduced sensitivity to OP-inhibition have been cloned and expressed from OP-resistant strains. The presence of at least three genes expressing AChEs in R. (B.) microplus, at least two of which contain mutations expressed as OP-insensitive enzymes, strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character., ((c) 2010 Elsevier B.V. All rights reserved.)

The stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), is one of the most significant pests of livestock in the United States. The identification of targets for the development of novel control for this pest species, focusing on those molecules that play a role in successful feeding and reproduction, is critical to mitigating its impact on confined and rangeland livestock. A database was developed representing genes expressed at the immature and adult life stages of the stable fly, comprising data obtained from pyrosequencing both immature and adult stages and from small-scale sequencing of an antennal/maxillary palp-expressed sequence tag library. The full-length sequence and expression of 21 transcripts that may have a role in chemosensation is presented, including 13 odorant-binding proteins, 6 chemosensory proteins, and 2 odorant receptors. Transcripts with potential roles in sex determination and reproductive behaviors are identified, including evidence for the sex-specific expression of stable fly doublesex- and transformer-like transcripts. The current database will be a valuable tool for target identification and for comparative studies with other Diptera.

Mutations I48L, I54V, R86Q, V137I, I492M, and T548A were identified previously in BmAChE3, a gene encoding acetylcholinesterase, from the organophosphate (OP) acaricide-resistant San Rommn strain of Rhipicephalus (Boophilus) microplus. Recombinant BmAChE3 acetylcholinesterase containing the R86Q mutation was shown to exhibit nearly 20-fold reduction in the rate of phosphorylation by paraoxon relative to the wild-type sequence. In addition, the R86Q mutation was present in resistant laboratory strains at elevated frequency compared with OP-susceptible strains but was insufficient to alone generate the OP-resistant phenotype (J. Med. Entomol. 44: 1013-1018). Here, we developed assays to genotype the remaining five mutations and evaluated frequency of all six BmAChE3 mutations in individual R. microplus ticks from laboratory and Mexican field-collected strains. We found a substantial number of individuals in known OP-susceptible strains that seemed to be homozygous for each of the mutations surveyed, the exception being I48L, which was infrequent in all strains, leading us to conclude that none of the mutations alone were responsible for generation of phenotypic resistance to OP acaricide.

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.

Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Román strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.