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BACKGROUND: A GPCR (G protein‐coupled receptor) target‐based approach was applied to identify antagonists of the arthropod‐specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty‐three structural analogs of previously identified tick kinin antagonists were screened in a 'dual‐addition' calcium fluorescence assay using a CHO‐K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non‐cytotoxic antagonists, four of which were partial (SACC‐0428764, SACC‐0428780, SACC‐0428800, and SACC‐0428803), and three were full antagonists (SACC‐0428799, SACC‐0428801, and SACC‐0428815). Four of these antagonists (SACC‐0428764, SACC‐0428780, SACC‐0428799, and SACC‐0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC‐0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 μm). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: The cattle fever tick, Rhipicephalus (Boophilus) microplus, is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP2b/PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus, Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two. Methods: Expression of the periviscerokinin receptor (Rhimi-CAP2bR) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP2bR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. Results: Rhimi-CAP2bR transcript expression was detected throughout all developmental stages. Rhimi-CAP2bR silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching (P < 0.05). Conclusions: CAP2b/PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP2bR loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP2b/PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control. [ABSTRACT FROM AUTHOR]

Aedes aegypti (L.) is an important mosquito vector of emerging arboviruses such as Zika, dengue, yellow fever, and chikungunya. To quell potential disease outbreaks, its populations are controlled by applying pyrethroid insecticides, which selection pressure may lead to the development of insecticide resistance. Target site insensitivity to pyrethroids caused by non-synonymous knockdown resistance (kdr) mutations in the voltage-gated sodium (NaV) channel is a predominant mechanism of resistance in mosquitoes. To evaluate the potential impact of pyrethroid resistance on vector control, Ae. aegypti eggs were collected from eight mosquito control operational areas in Harris County, Texas, and emerged females were treated in field tests at four different distances from the pyrethroid Permanone 31–66 source. The females were genotyped by melting curve analyses to detect two kdr mutations (V1016I and F1534C) in the NaV channel. Harris County females had higher survivorship rates at each distance than the pyrethroid-susceptible Orlando strain females. Survivorship increased with distance from the pyrethroid source, with 39% of field-collected mosquitoes surviving at 7.62 m and 82.3% at 22.86 m from the treatment source. Both the V1016I and F1534C pyrethroid resistant genotypes were widely distributed and at high frequency, with 77% of the females being double homozygous resistant (II/CC), this being the first report of kdr mutations in Ae. aegypti in Harris County. Analysis of the probability of survival for each mutation site independently indicated that the CC genotype had similar probability of survival as the FC heterozygous, while the II genotype had higher survival than both the VI and VV, that did not differ. The double homozygous resistant genotype (II/CC) had the highest probability of survival. A linear model estimated probability of survival for areas and genotypes. The high frequency and widespread distribution of double-homozygote pyrethroid-resistant Ae. aegypti may jeopardize disease vector control efforts in Harris County. Author summary: Females of the mosquito Aedes aegypti transmit arboviruses of human diseases including Zika, dengue, chikungunya, and yellow fever, and Culex quinquefasciatus transmits West Nile virus (WNV). Upon WNV detection in Harris County, TX, Harris County Public Health Mosquito and Vector Control Division applies pyrethroid or organophosphate insecticides to control Cx. quinquefasciatus mosquitoes. Pyrethroids target the sodium channel and knockdown resistance (kdr) mutations in this channel confer resistance to pyrethroids and increase mosquito survival. While Ae. aegypti is not a current target for public mosquito control in Harris County, we investigated frequency and spatial distribution of two kdr mutations in Ae. aegypti females, and their impact on pyrethroid survival after field tests were conducted with Permanone 31–66. The survival probability of mosquitoes varied depending on distance from application, and on their genotype and area of origin. Survivorship was lowest at 7.62 m, but most females survived at and beyond 15.24 m. Most (98.3%) females genotyped carried at least one kdr allele, and 77% were double homozygous resistant (II/CC). This is the first report of these resistant genotypes in Harris County. The resistant genotype II/CC had the highest survival probability. These results should be useful when modeling the risk of mosquito-borne diseases. [ABSTRACT FROM AUTHOR]

BACKGROUND: The southern cattle tick, Rhipicephalus microplus, is a primary vector of the deadly bovine disease babesiosis. Worldwide populations of ticks have developed resistance to acaricides, underscoring the need for novel target discovery for tick control. The arthropod‐specific R. microplus kinin receptor is such a target, previously validated by silencing, which resulted in female reproductive fitness costs, including a reduced percentage of eggs hatching. RESULTS: In order to identify potent small molecules that bind and activate or inhibit the kinin receptor, a high‐throughput screening (HTS) assay was developed using a CHO‐K1 cell line expressing the recombinant tick kinin receptor (BMLK3). A total of ~20 000 molecules from a random in‐house small molecule library were screened in a 'dual‐addition' calcium fluorescence assay. This was followed by dose–response validation of the hit molecules identified both from HTS and an in silico screen of ~390 000 molecules. We validated 29 antagonists, 11 of them were full antagonists with IC50 values between 0.67 and 8 μmol L–1. To explore the structure–activity relationships (SAR) of the small molecules, we tested the activities of seven analogs of the most potent identified antagonist, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 μmol L–1) were validated in vitro using the recombinant mosquito kinin receptor and showed similar antagonistic activities. In vivo, these three compounds also inhibited the mosquito hindgut contraction rate induced by a myotropic kinin agonist analog 1728. CONCLUSION: Antagonists identified in this study could become pesticide leads and are reagents for probing the kinin signaling system. © 2021 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract Background Insect neuropeptides are involved in diverse physiological functions and can be released as neurotransmitters or neuromodulators acting within the central nervous system, and as circulating neurohormones in insect hemolymph. The insect short neuropeptide F (sNPF) peptides, related to the vertebrate neuropeptide Y (NPY) peptides, have been implicated in the regulation of food intake and body size, and play a gonadotropic role in the ovaries of some insect species. Recently the sNPF peptides were localized in the brain of larval and adult Drosophila. However, the location of the sNPF receptor, a G protein-coupled receptor (GPCR), has not yet been investigated in brains of any adult insect. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in queens of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera), an invasive social insect. Results In the queen brains and subesophageal ganglion about 164 cells distributed in distinctive cell clusters (C1-C9 and C12) or as individual cells (C10, C11) were immuno-positive for the sNPF receptor. Most of these neurons are located in or near important sensory neuropils including the mushroom bodies, the antennal lobes, the central complex, and in different parts of the protocerebrum, as well as in the subesophageal ganglion. The localization of the sNPF receptor broadly links the receptor signaling pathway with circuits regulating learning and feeding behaviors. In ovaries from mated queens, the detection of sNPF receptor signal at the posterior end of oocytes in mid-oogenesis stage suggests that the sNPF signaling pathway may regulate processes at the oocyte pole. Conclusions The analysis of sNPF receptor immunolocalization shows that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). To our knowledge, this is the first report of the cellular localization of a sNPF receptor on the brain and ovaries of adult insects.

Culex quinquefasciatus is one of the most important mosquito vectors of arboviruses. Currently, the fastest approach to control disease transmission is the application of synthetic adulticide insecticides. However, in highly populated urban centers the development of insecticide resistance in mosquito populations could impair insecticide efficacy and therefore, disease control. To assess the effect of resistance on vector control, females of Cx. quinquefasciatus collected from six mosquito control operational areas in Harris County, Texas, were treated in field cage tests at three different distances with the pyrethroid Permanone® 31–66 applied at the operational rate. Females were analyzed by sequencing and/or diagnostic PCR using de novo designed primers for detecting the kdr-like mutation in the voltage-gated sodium channel (L982F; TTA to TTT) (house fly kdr canonical mutation L1014F). Females from the Cx. quinquefasciatus susceptible Sebring strain and those from the six operational areas placed at 30.4 m from the treatment source were killed in the tests, while 14% of field-collected mosquitoes survived at 60.8 m, and 35% at 91.2 m from the source. The diagnostic PCR had a with 97.5% accuracy to detect the kdr-like mutation. Pyrethroid resistant mosquitoes carrying the L982F mutation were broadly distributed in Harris County at high frequency. Among mosquitoes analyzed (n = 1,028), the kdr-kdr genotype was prevalent (81.2%), the kdr-s genotype was 18%, and s-s mosquitoes were less than 1% (n = 8). A logistic regression model estimated an equal probability of survival for the genotypes kdr-kdr and kdr-s in all areas analyzed. Altogether, our results point to a high-risk situation for the pyrethroid-based arboviral disease control in Harris County. Author summary: Female Culex quinquefasciatus mosquitoes blood-feed preferentially on birds but also on mammals including humans, to which they may transmit pathogenic arboviruses. To prevent transmission of West Nile and/or other viruses in Harris County, TX, the vector control agency applies mosquito adulticides when arboviruses are detected. Insecticide resistance can hinder mosquito control, increasing the risk of disease transmission. Pyrethroid resistant mosquitoes carry kdr-like mutation(s) in the sodium channel, the pyrethroid target site in the nervous system, that increases survival probability. We investigated frequency and spatial distribution of the kdr-like mutation in Harris County mosquitoes, and how they affect pyrethroid survivorship. We conducted field cage tests with a commercial pyrethroid formulation, and developed molecular tests to detect the kdr-like mutation in females from 6 operational areas. In field tests, almost no mosquito survived at 30.4 m, but 14% and 35% of mosquitoes survived at 60.8 m and 91.2 m. We found 99% of mosquitoes carried at least one kdr-like allele. At the high rate of pyrethroid, the survival probability of mosquitoes carrying one or two kdr-like alleles was not statistically different in the six areas, but survival increased significantly with distance. These results should be considered when analyzing risk of disease transmission. [ABSTRACT FROM AUTHOR]