Relationship between pharmacokinetics of fluazuron and its efficacy for controlling Rhipicephalus microplus: A comprehensive evaluation of tick drug uptake.

Relationship between fluazuron (FZN) concentrations in cattle plasma and ticks and its therapeutic efficacy percentage (EP) against Rhipicephalus microplus was analyzed. The extent of FZN uptake by ticks after its topical administration was also evaluated. Heifers, naturally infested with R. microplus, were divided in treated and no treated groups. Blood and tick samples were taken to measure FZN concentrations and female ticks were counted to evaluate the EP of the treatment from day 0 to 45 post-treatment (PT). Ticks exposed (TE) and not exposed (TNE) to topical contact with the drug were collected at 12 and 24 h PT. Artificial feeding (AF) of ticks was also performed with blood from a treated heifer. The EP on days 21, 28, 35, 42 and 45 PT was 99.1, 92.3, 81.2, 74.7 and 76.5 %, respectively. Fluazuron disposition within ticks during the first 2 days post-treatment did not mirror the corresponding FZN levels in the bovine plasma, but a statistically significant positive correlation was evident from day 3. Mean FZN concentrations in TE at 12 h and 24 h PT were significantly higher than in TNE. The FZN concentrations in AF ticks were significantly lower than those in TE but comparable to the levels in TNE. The efficacy and pharmacokinetics analysis suggest that maintaining a threshold FZN plasma concentration above 20-25 ng/mL is crucial to prevent the development of larvae into engorged females. The results also indicate that direct absorption of FZN through the tick's integument occurs in the initial days after treatment.

Effects of the novel acaricide acynonapyr on the calcium-activated potassium channel.

Resistance to insecticides and acaricides is a major impediment to effectively controlling insect pests worldwide. These pests include the two-spotted spider mite Tetranychus urticae (T. urticae), which exists globally. This polyphagous herbivore causes major agricultural problems and can develop resistance to the agents above. Therefore, the continuous development of acaricides with new modes of action is important to circumvent the resistance of insects to pesticides. Acynonapyr is a novel class of acaricides containing an azabicyclo ring. In this study, we determined the activity of acynonapyr and its analogs on calcium-activated potassium (KCa2) channels in two-spotted spider mites using electrophysiological techniques (patch-clamp). We also examined their acaricidal efficacy against mites in the laboratory. The acynonapyr and analogs blocked T. urticae KCa2 (TurKCa2) channels in a concentration-dependent manner. A comparison of acaricidal activity against T. urticae with inhibitory activity against TurKCa2 revealed that TurKCa2 channels are the primary toxicological targets. Finally, we examined the effect of acynonapyr on Homo sapiens KCa2 (HsaKCa2.2) channels and demonstrated that the compound at 10 µM had a limited effect on the activity of this channel.

Identification and CRISPR-Cas9 validation of a novel ß-adrenergic-like octopamine receptor mutation associated with amitraz resistance in Varroa destructor.

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the ß-adrenergic-like octopamine receptor (Octß2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octß2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octß2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.

The cytochrome P450 subfamilies CYP392A and CYP392D are key players in acaricide metabolism in Tetranychusurticae.

The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations.

A rapid tick exposure test for monitoring acaricide resistance in Rhipicephalus sanguineus sensu lato ticks on dogs.

BACKGROUND: Brown dog ticks (Rhipicephalus sanguineus sensu lato) are vectors of pathogens adversely affecting the health of dogs in many regions of the world. The three-host life cycle of R. sanguineus s.l., with all stages feeding on dogs, can lead to an uncontrolled build-up of large tick populations if not controlled by acaricides. However, frequent tick control on dogs using acaricides has led to the emergence of resistance to permethrin and fipronil. Currently, the larval packet test (LPT) is the standard tick resistance test, which is laborious, requires laboratory facilities, and takes at least 6 weeks before larvae derived from engorged female ticks can be tested. Our novel approach is to expose semi-engorged adult ticks to acaricides immediately after removing them from dogs, obtaining results within 24 h. METHODS: Adult ticks from three laboratory colonies of R. sanguineus s.l. were tested in RaTexT®, a rapid tick exposure test in which ticks were confined to small compartments and exposed to an acaricide-impregnated, specially designed matrix. Resistance was confirmed by testing larvae derived from the same laboratory colonies using the LPT. RaTexT® was also used to determine the susceptibility of R. sanguineus acaricides in dog shelters. RESULTS: RaTexT® detected resistance to permethrin in adult R. sanguineus s.l. ticks from two Brazilian laboratory colonies compared to a susceptible laboratory strain originating in Greece. Resistance was confirmed by LPT testing of larvae from the same colonies with resistance factors between 2.2 and 3.1. All laboratory strains were susceptible to fipronil. A suspected case of fipronil resistance at a dog shelter in Caxias do Sul, Brazil, was resolved within 24 h by testing adult ticks in RaTexT® and could be attributed to improper treatment. CONCLUSIONS: RaTexT® is a valuable tool for monitoring the development of resistance to synthetic pyrethroids or phenylpyrazoles in tick-infested dogs.

Acaricide effect of plants from the Brazilian savanna on a population of Rhipicephalus microplus with phenotypic resistance to cypermethrin and trichlorfon.

Rhipicephalus microplus is among the most important ectoparasites for livestock. The use of synthetic acaricides has raised some concerns due to the selection of tick populations that are resistant to acaricides and environmental contamination. Therefore, plant extracts have been used as alternatives for the treatment of animals infested with ticks. In this study, R. microplus populations from seven different dairy farms were collected and assessed for their resistance to the acaricides cypermethrin or trichlorfon. Larvae of the most resistant population were used in assays to evaluate the acaricide effect of leaf extracts from plants of the Brazilian savanna. The most active extracts were also tested against fully engorged females. Among seven tick populations, five and three showed resistance level ≥ III for cypermethrin or trichlorfon, respectively. The most resistant tick population was evaluated in mortality assays with the plants Piptadenia viridiflora, Annona crassiflora, Caryocar brasiliense, Ximenia americana, and Schinopsis brasilienses. The ethanolic extracts of C. brasiliense, X. americana and S. brasilienses showed higher larvicidal effects in comparison to the other extracts and cypermethrin. The ethanolic extract of X. americana showed 60.79 % efficacy against fully engorged females of the acaricide resistant tick strain. The ethanolic extracts of C. brasiliense, X. americana, and S. brasilienses showed peaks in HPLC-DAD, indicating the presence of tannins and flavonoids. Three of the plants showed promising results and should be explored in further studies to develop novel tools to control R. microplus in cattle.

Histological changes of oocytes of the cattle tick Rhipicephalus microplus (Canestrini, 1888) treated with copper solutions.

Infections caused by the ectoparasite Rhipicephalus microplus can cause major health problems in cattle, including death. Tick control is regularly made using a range of acaricide products. As a consequence, tick populations have been heavily selected for drug resistance. The objective of this work was to determine the in vitro efficacy of copper chloride and sulfate (CuCl2 and CuSO4) solutions against R. microplus. The adult immersion test (AIT), which measures the egg-laying and egg-hatch effects, was used for the Cu-II solutions at 30, 60, 120, 240, 480, and 1000 mM, in triplicates. Distilled water and the combination of cypermethrin 20% and chlorpyrifos 50% were used as controls. Histological sections were performed from the ovaries of adult engorged female ticks treated with 240, 480, and 1000 mM of CuCl2 and CuSO4. We have established a histological index of the damage caused by the solutions to the tick oocytes. The overall efficacy (egg laying & egg hatch) for CuCl2 and CuSO4 was 81.3, 82.5, 89.8, 84.5, 100.0, and 100%, and 61.7, 43.4, 62.5, 93.1, 100.0, and 98.5% respectively. Smaller oocytes were found in the Cu-II groups compared to the negative control. The histological data showed a concentration-dependent degenerative lesion of oocytes, described as cytoplasmic vacuolation and nuclear disorganization. The combination of cypermethrin and chlorpyriphos showed 100% efficacy. Cu-II solutions showed in vitro efficacy against adult engorged ticks being particularly harmful to oocytes. Thus, bioactive metals could be a complementary biofriendly treatment to control R. microplus and these injuries could be responsible for preventing egg hatch, and reducing pasture contamination. Safety studies are underway demonstrating the Cu-II potential in naturally infected cattle and their persistence in the environment.

Acaricide Flupentiofenox Inhibits the Mitochondrial ß-Oxidation Pathway of Fatty Acids.

A newly developed pesticide, flupentiofenox, has a unique trifluoroethyl phenylsulfoxide structure, and it powerfully affects spider mites, including those with resistance to multiple commercial acaricides. To clarify the mode of action of flupentiofenox, we investigated its effect on mitochondrial energy generation. We observed that flupentiofenox decreased adenosine triphosphate (ATP) levels in two-spotted spider mites (Tetranychus urticae) at a practical dose. Flupentiofenox potently inhibited mitochondrial oxygen consumption under conditions of palmitoyl-carnitine or octanoic acid supply, but not under conditions of pyruvate supply. These results show that flupentiofenox inhibits the mitochondrial fatty acid metabolic pathway between the uptake of long-chain acylcarnitine or medium-chain fatty acid and the synthesis of acetyl-CoA by ß-oxidation, resulting in suppressed mitochondrial energy generation. Our investigations have led us to conclude that flupentiofenox is a pesticide with a novel mode of action.

RaTexT®: a novel rapid tick exposure test for detecting acaricide resistance in Rhipicephalus microplus ticks in Brazil.

BACKGROUND: Acaricide resistance in cattle ticks is a significant concern in (sub)tropical regions, particularly Brazil. The Larval Packet Test (LPT) is the standard laboratory bioassay for resistance diagnosis, which requires triplicates of seven acaricidal dilutions plus controls to cover larval mortalities ranging between 0 and 100%. The value of the LPT lies in providing resistance ratios based on the ratio between the LC50 calculated with potentially resistant and susceptible ticks. However, LC50 ratios are difficult to translate into practical advice for farmers. Moreover, LPT requires laboratory facilities to maintain susceptible tick colonies, and it takes 6 weeks to obtain the larvae to be tested by LPT derived from engorged female ticks collected from cattle in the field. Our novel approach was twofold: first, we upgraded the LPT to the Resistance Intensity Test (RIT) by adopting the latest WHO guidelines for resistance detection in mosquitoes, which combines a 1 × recommended dose with 5 × and 10 × concentrated doses to reveal low, moderate and high resistance intensity, respectively. This reduced the number of test papers and tick larvae and, more importantly, provided relevant information on the resistance level. Our second innovative step was to abolish testing larvae entirely and expose partly engorged adult ticks to the same acaricidal doses immediately after removing them from cattle in the field. This resulted in the Rapid Tick exposure Test (RaTexT®), wherein partly engorged adult ticks were exposed to an acaricide-impregnated, specially designed matrix providing test results within 24 h. This approach directly compared resistance detection in tick larvae in the RIT with resistance in adult ticks in RaTexT®. METHODS: Laboratory validation was conducted in Brazil with resistant and susceptible colonies of Rhipicephalus microplus ticks. For field validation, adult R. microplus ticks collected from different cattle farms in Brazil were evaluated for resistance to RaTexT®, and the results regarding their larval progenies were compared with those for the RIT. Partly engorged adult ticks derived from cattle infested with laboratory and field strains of R. microplus were exposed to deltamethrin in RaTexT® containers, which contained six rows of four interconnected compartments, accommodating five to eight semi-engorged female ticks with a preferred size ranging between 5 and 8 mm. The corresponding larvae of each strain were exposed in the RIT to the same deltamethrin concentrations in filter papers. RESULTS: In RaTexT®, mortality in adult ticks from a resistant strain of R. microplus from Seropédica in Brazil was 38.4%, 54.2% and 75.0% at the 1 ×, 5 × and 10 × doses of deltamethrin, respectively. In RIT, mortality of larvae from the same resistant strain was 2.0%, 4.9% and 19.5% at 1 ×, 5 × and 10 × doses, respectively. The results of RaTexT® and RIT agreed since both tests identified a high level of resistance based on a cut-off of 90% mortality. In RaTexT®, mortality of adult ticks from a susceptible strain originating from Porto Alegre was 73.8%, 92.9% and 97.6% at the 1 ×, 5 × and 10 × doses, respectively. In RIT, mortality of larvae from the susceptible strain was 95.2%, 95.2% and 96.8% at the 1 ×, 5 × and 10 × doses, respectively. Interestingly, both tests identified a low number of unexpected resistant individuals in the susceptible strain since the mortality of neither larvae nor adults reached 100%. This effect remained unnoticed in the LPT, wherein a resistance ratio of 159.5 was found based on the LC50 of the resistant strain divided by the LC50 of the susceptible strain. Next, RaTexT® was compared with RIT using adult and larval ticks derived from three field strains of R. microplus in Brazil. RaTexT® detected high levels of resistance to deltamethrin in adult ticks in all strains, which was confirmed in larvae tested by the RIT. Both tests agreed on the same resistance level with significantly lower mortality rates in larvae than in adult ticks. CONCLUSIONS: RaTexT® is a novel rapid pen-site test for detecting acaricide resistance in adult livestock ticks. It potentially replaces laborious tests using larval ticks and provides results within 24 h relevant to acaricide resistance management of livestock ticks.

Sublethal Effects Induced by a Cyflumetofen Formulation on Honeybee Apis mellifera L. Workers: Assessment of Midgut, Hypopharyngeal Glands, and Fat Body Integrity.

Worldwide, both cultivated and wild plants are pollinated by the honey bee, Apis mellifera. Bee numbers are declining as a result of a variety of factors, including increased pesticide use. Cyflumetofen controls pest mites in some plantations pollinated by bees, which may be contaminated with residual sublethal concentrations of this pesticide, in nectar and pollen. We evaluated the effects of a sublethal concentration of a cyflumetofen formulation on the midgut, hypopharyngeal gland, and fat body of A. mellifera workers orally exposed for 72 h or 10 days. The midgut epithelium of treated bees presented digestive cells with cytoplasm vacuoles and some cell fragmentation, indicating autophagy and cell death. After being exposed to the cyflumetofen formulation for 72 h, the midgut showed a higher injury rate than the control bees, but after 10 days, the organs had recovered. In the hypopharyngeal gland of treated bees, the end apparatus was filled with secretion, suggesting that the acaricide interferes with the secretory regulation of this gland. Histochemical tests revealed differences in the treated bees in both exposure periods in the midgut and hypopharyngeal glands. The acaricide caused cytotoxic effects on the midgut digestive cells, with apical protrusions, plasma membrane rupture, and several vacuoles in the cytoplasm, features of cell degeneration. In the hypopharyngeal glands of the treated bees, the secretory cells presented small electron-dense and large electron-lucent secretory granules. The fat body cells had no changes in comparison with the control bees. In conclusion, the cyflumetofen formulation at sublethal concentrations causes damage to the midgut and the hypopharyngeal glands of honey bee, which may compromise the functions of these organs and colony fitness. Environ Toxicol Chem 2024;00:1-11. © 2024 SETAC.

Molecular assessment of voltage-gated sodium channel (VGSC) gene mutations in Rhipicephalus microplus from Guangxi, China.

BACKGROUND: Pyrethroid chemicals are one of the main acaricides used against ticks. Resistance to these chemicals has been reported to be associated with mutations in the voltage-gated sodium channel (VGSC) gene of the Rhipicephalus microplus. This study investigates R. microplus resistance to pyrethroids in Guangxi region of China, marking one of the first research efforts in this area. The findings are intended to provide vital baseline for the effective implementation of localized tick control strategies. METHODS: From March to July 2021, 447 R. microplus tick samples were collected from five prefecture-level cities in Guangxi. Allele-specific polymerase chain reaction (AS-PCR) was used to amplify segments C190A and G215T of the domain II S4-5 linker and T2134A of domain III S6 in the VGSC, to detect nucleotide mutations associated with resistance to pyrethroid acaricides. Subsequent analyses were conducted to ascertain the prevalence, types of mutations, and genotypic distributions within the sampled populations. RESULTS: Mutations within VGSC gene were identified across all five studied populations of R. microplus, although the mutation rates remained generally low. Specifically, the most prevalent mutation was C190A, observed in 4.9% of the samples (22/447), followed by G215T at 4.0% (18/447), and T2134A at 1.3% (6/447). The distribution of mutations across three critical sites of the VGSC gene revealed four distinct mutation types: C190A, G215T, C190A + G215T, and T2134A. Notably, the single mutation C190A had the highest mutation frequency, accounting for 4.3%, and the C190A + G215T combination had the lowest, at only 0.7%. The analysis further identified seven genotypic combinations, with the wild-type combination C/C + G/G + T/T predominating at a frequency of 90.4%. Subsequently, the C/A + G/G + T/T combination was observed at a frequency of 4.3%, whereas the C/C + T/T + T/T combination exhibited the lowest frequency (0.2%). Additionally, no instances of simultaneous mutations at all three sites were detected. Geographical differences in mutation types were apparent. Both samples from Hechi to Chongzuo cities exhibited the same three mutation types; however, C190A was the most prevalent in Hechi, while G215T dominated in Chongzuo. In contrast, samples from Beihai to Guilin each exhibited only one mutation type: G215T occurred in 12.5% (4/32) of Beihai samples, and C190A in 7.5% (4/53) of Guilin samples. CONCLUSIONS: These findings underscore the relatively low frequency of VGSC gene mutations in R. microplus associated with pyrethroid resistance in the Guangxi, China. Moreover, the variation in mutation types and genotypic distributions across different locales highlights the need for regionalized strategies in monitoring and managing pyrethroid resistance in tick populations. This molecular surveillance is crucial for informing targeted control measures and mitigating the risk of widespread resistance emergence.

Cladistics of Rhipicephalus microplus and laboratory assessment of acaricidal efficacy of different Cassia fistula extracts against R. microplus larvae.

The present study aimed to analyze the cladistics and population structure analysis of Rhipicephalus microplus ticks infesting buffaloes in Haryana, India, as well as the assessment of the anti-tick efficacy of the ethanolic extracts of Cassia fistula (bark, pod pulp, and flowers) against R. microplus larvae. The molecular characterization and population structure analysis were performed by targeting the amplification of the partial mitochondrial cytochrome C oxidase subunit 1 (cox1) gene, whereas anti-tick efficacy was evaluated using a larval packet test. The sequences generated herein revealed a homology of 98.26-100% to the GenBank-archived R. microplus sequences. In population structure analysis, high haplotype (0.500 ± 0.265) and low nucleotide (0.002 ± 0.001) diversities were recorded for the sequences generated in this study. Significantly negative neutrality indices were recorded for the overall dataset. The extracts were found to significantly affect mortality rates in a dose-dependent manner, and the ethanolic extracts of the bark, pod pulp, and flowers of C. fistula exhibited median lethal concentration (LC50) values of 27.989, 40.457, and 49.43 mg/mL, respectively. The LC50 value recorded for the combination of the ethanolic extracts of the bark, flower, and pod pulp of C. fistula was 19.724 mg/mL, whereas the synthetic acaricide ivermectin had an LC50 value of 351.56 mg/mL. In conclusion, R. microplus populations infesting cattle and buffalo hosts in India exhibited negligible genetic differentiation and high gene flow between them. Additionally, the combination of all C. fistula extracts could serve as a potential substitute for the synthetic acaricide.

Varroa destructor mite population dynamics in africanized honeybee (Apis mellifera) colonies in a semi-arid region.

Most published data on mite infestation rates in semi-arid regions have been collected over only 3 or 4 months during a specific period of the year. Therefore, the aim of this study was to observe parasite-host dynamics of hygienic and non-hygienic Africanized bee colonies considering environmental factors that may influence Varroa destructor mite infestation rates in a semi-arid region. To this end, the brood puncture method was applied to 37 colonies, forming two groups, namely G1, encompassing 16 hygienic colonies, and G2, comprising 21 non-hygienic colonies. After forming the groups, 300 worker bees from each colony were examined monthly for mite infestations and the data were correlated with climatological records. The monthly infestation average was considered low, below 10%, except in November, when it reached 12.19% ± 6.45. No statistically significant difference was observed for inter-group infestation rates (P > 0.05). When mite infestation rates were associated with climatic variables, they were linked to colony losses (32%) due to swarming. No significant correlations between hygienic behaviour and parasite infestation rates were noted. Nonetheless, these results support the idea that there is no need to apply acaricides for V. destructor control in Brazil.

Peer review of the pesticide risk assessment of the active substance paraffin oil (CAS 8042-47-5, chain lengths C17-C31).

The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Greece, and co-rapporteur Member State, France, for the pesticide active substance paraffin oil are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of paraffin oil as an acaricide and insecticide on potatoes, ornamentals (flower bulbs) and orchards (pear/apple), on pome fruit and stone fruit, on field and permanent protected fruiting vegetables and on field and permanent protected roses and on citrus. The reliable end points appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

Acaricidal activity of Erigeron acer L. root against Haemaphysalis longicornis and phytochemical profiling by liquid chromatography-tandem mass spectrometry.

The present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.

Sublethal effects of a spiromesifen and abamectin combination on Tetranychus urticae (Acari: Tetranychidae) and its predators Phytoseiulus persimilis and Amblyseius swirskii (Acari: Phytoseiiidae).

The two-spotted spider mite, Tetranychus urticae Koch (TSSM), is an important cosmopolitan pest of agricultural crops that is often managed in greenhouses by augmentation of predatory mites in combination with acaricides. Here we examined the transgenerational effects of low lethal concentrations of a widely-used acaricide, Oberon Speed® (a combination of spiromesifen and abamectin), on the life history traits and population growth of T. urticae and two of its predators, Phytoseiulus persimilis Athias-Henriot and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae). The concentrations employed corresponded to the LC10, LC20 and LC30 values estimated for TSSM protonymphs 48 h post-exposure in a topical bioassay, which yielded an LC50 value of 207.2 ppm. Parental exposure of TSSM to all three low concentrations increased the total developmental time of progeny; both the LC20 and LC30 treatments reduced adult longevity and number of oviposition days, but only the LC30 treatment increased the preoviposition period. Similarly, both the LC20 and LC30 treatments significantly reduced life table parameters (r, R0, λ, and GRR), and increased generation time (T) and population doubling time (DT). Although maternal exposure to the acaricide had various impacts on progeny life history, A. swirskii was less affected than P. persimilis, suggesting the former species would be more compatible for integration with Oberon Speed® for control of T. urticae in greenhouse vegetable production.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Lime for Lyme: Treatment of Leaf Litter with Dolomitic Lime Powder Impairs Activity of Immature Ixodes scapularis Ticks.

Background: Tick-borne diseases are an emerging threat to public health throughout the temperate world, leading to a growing field of research aimed at developing and testing intervention strategies for reducing human-tick encounters or prevalence of infection in ticks. Various wide-spectrum chemical acaricides have proven effective for controlling tick populations, but many of these have potential deleterious side-effects on health and the environment. In addition to chemical acaricides, certain compounds such as diatomaceous earth have been shown to have physical acaricidal properties. We hypothesized that dolomitic lime (CaMg(CO3)2, a corrosive, desiccant mineral that is already used extensively in agricultural and forestry contexts to balance the pH of soils, may affect ticks' locomotory activity, habitat position, or survival and that this should manifest as a reduction in the number of questing ticks collected by dragging. Objective: This study aimed to formally assess this hypothesis in a controlled laboratory setting. Methods: We carried out a microcosm experiment, with one control and three treated microcosm trays, each replicating the natural substrate characterizing I. scapularis habitat in northeastern North America. Each tray was infested with 200 living larvae and 50 nymphs, and then treated with 0 (control), 50, 100, or 500 g/m2 of lime powder. Ticks were collected by microdragging 24 and 72 h postliming. Results: Efficacy of liming at reducing the number of collected questing ticks ranged from 87% to 100% for larvae and 0% to 69% for nymphs 24 h postliming and from 91% to 93% for larvae and -47% to 65% for nymphs 72 postliming. Conclusion: This study provides the first experimental evidence of the potential efficacy of liming for impairing activity of questing immature ticks. Given that lime is a low-cost material, that methods for widespread application in deciduous woodlands already exist, and that it has been documented as having a limited negative impact on the environment, further assessment of lime application as a public health risk reduction intervention for tick-borne diseases is warranted.

Acaricide resistance status of livestock ticks from East and West Africa and in vivo efficacy of acaricides to control them.

Through a collaborative effort across six Sub-Saharan African countries, using recognized international assessment techniques, 23 stocks of three tick species (Rhipicephalus microplus, Rhipicephalus appendiculatus and Amblyomma variegatum) of economic importance for rural small holder farming communities from East and West Africa were collected from cattle, and evaluated in in vitro larval packet tests (LPT). The results demonstrated medium to high resistance to chlorfenvinphos and amitraz across species. Rhipicephalus microplus demonstrated high level alpha-cypermethrin and cypermethrin resistance. Stocks of A. variegatum (West Africa) and R. appendiculatus (Uganda) demonstrated medium level ivermectin resistance. The four least susceptible stocks (East and West African R. microplus, A. variegatum and R. appendiculatus) were taken into in vivo controlled cattle studies where fipronil was found effective against West and East African R. microplus isolates although persistent efficacy failed to reach 90%. Cymiazole and cypermethrin, and ivermectin based acaricides were partially effective against R. microplus without persistent efficacy. Flumethrin spray-on killed A. variegatum within 72 h for up to 10 days posttreatment, however product application was directly to tick attachment sites, which may be impractical under field conditions. A flumethrin pour-on formulation on goats provided persistent efficacy against A. variegatum for up to one-month. Therapeutic control was achieved against R. appendiculatus through weekly spraying cattle with flumethrin, amitraz or combined cymiazole and cypermethrin. A fipronil pour-on product offered four-week residual control against R. appendiculatus (with slow onset of action). Few studies have assessed and directly compared acaricidal activity in vitro and in vivo. There was some discordance between efficacy indicated by LPT and in vivo results. This observation calls for more research into accurate and affordable assessment methods for acaricide resistance. No single active or product was effective against all three tick species, emphasising the need for the development of alternative integrated tick management solutions.

Detrimental effects of amitraz exposure in honey bees (Apis mellifera) infected with Nosema ceranae.

In recent years, there has been growing concern on the potential weakening of honey bees and their increased susceptibility to pathogens due to chronic exposure to xenobiotics. The present work aimed to study the effects on bees undergoing an infection by Nosema ceranae and being exposed to a frequently used in-hive acaricide, amitraz. To achieve this, newly emerged bees were individually infected with N. ceranae spores and/or received a sublethal concentration of amitraz in their diets under laboratory conditions. Mortality, food intake, total volume excrement, body appearance, and parasite development were registered. Bees exposed to both stressors jointly had higher mortality rates compared to bees exposed separately, with no difference in the parasite development. An increase in sugar syrup consumption was observed for all treated bees while infected bees fed with amitraz also showed a diminishment in pollen intake. These results coupled with an increase in the total number of excretion events, alterations in behavior and body surface on individuals that received amitraz could evidence the detrimental action of this molecule. To corroborate these findings under semi-field conditions, worker bees were artificially infected, marked, and released into colonies. Then, they were exposed to a commercial amitraz-based product by contact. The recovered bees showed no differences in the parasite development due to amitraz exposure. This study provides evidence to which extent a honey bee infected with N. ceranae could potentially be weakened by chronic exposure to amitraz treatment.