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.

Comparative effect of ivermectin and amitraz on cellular architecture of ovaries, synganglion and Gené's organ of Rhipicephalus microplus (Acari: Ixodidae).

The present study investigated the effect of ivermectin and amitraz on the cellular architecture of vital organs of Rhipicephalus microplus. Adult female ticks were treated with lethal concentrations (LC95) of ivermectin and amitraz, and the ovaries, synganglion, and Gené's organ were processed 48 h post treatment. In both the treatment groups, the ultra-thin sections of ovary exhibited deformed oocytes, irregular plasmic membrane and chorion layer, extensive vacuolation in the cytoplasm mainly at periphery of the cell and oocyte-pedicel junction. Marked vacuolations in the cortex and neuropile region with significant structural disorganization of the neural fibers were common alterations observed in the synganglion of ticks exposed to ivermectin and amitraz. The tissue sections of Gené's organ revealed deformed tubular glands with severe loss of cellular limit of secretory epithelium and cytoplasmic vacuolations in the ivermectin treated ticks whereas, the alterations were comparatively less severe in amitraz exposed ticks. The cellular deformities in these vital organs probably impaired reproductive function, nerve signal transmission and metabolic activities and thus affected fecundity and survivability of the treated ticks. The findings suggested that the action of ivermectin and amitraz are not restricted to the nervous system of ticks, but also on other vital organs, ovary and Gené's organ affecting the oviposition. The study provided insights into the development of targeted interventions for tick control strategies.

Acaricidal potential of essential oils on Rhipicephalus linnaei: Alternatives and prospects.

The acaricidal potential of various essential oils (EOs) has been evaluated based on their benefits in tick control. This study aimed to investigate the tick-killing activity of Pogostemon cablin "patchouli," Cymbopogon martinii "palmarosa," and Cymbopogon flexuosus "lemongrass" EOs on Rhipicephalus linnaei. Engorged females were collected from domiciled and non-domiciled dogs from Jataí city, Goiás state, to obtain larvae and nymphs in a controlled environment. Two commercial EOs brands were used in this study in different EOs concentrations (2.5, 5, 10, and 20 mg/mL), and was tested by immersion of larvae and nymphs. In the in vitro evaluation of EOs toxicity against R. linnaei larvae, 100 % mortality was achieved with 10 mg/mL of P. cablin oil, whereas mortality rates greater than 98 % were observed with 20 mg/mL of C. martinii and C. flexuosus. In nymphs, high sensitivity was observed, with 100 % mortality achieved using 5 mg/mL of P. cablin and 20 mg/mL of C. martinii and C. flexuosus. The EO of P. cablin "patchouli" demonstrated in vitro toxicity at a lower concentration than the other oils in the two development stages of R. linnaei, and was considered the most efficient and with verified acaricidal activity. Oils of C. martinii "palmarosa" and C. flexuosus "lemongrass" achieved mortality greater than 95 % in larvae and nymphs only at the highest concentration. Therefore, the acaricidal effects of the tested EOs are promising, especially of patchouli oil, which promoted high mortality at a low concentration (LC90 of 2.21 mg/mL).

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.

In vitro evaluation of acaricidal activity of eugenol and benzaldehyde against Rhipicephalus annulatus.

Economic importance of ticks for both humans and animals is significant primarily because of their involvement in disease transmission. The continuous use of synthetic chemical acaricides on animals and/or in the environment has resulted in the emergence of resistant tick populations. Consequently, there is a compelling need to explore newer natural alternatives that can address their adverse effects effectively. Since, there are no earlier reported studies on the acaricidal activity of eugenol and benzaldehyde against Rhipicephalus annulatus, the present study compared the in vitro adulticidal and larvicidal activities of these phytochemicals against R. annulatus. Concentration-dependent mortality and inhibition of fecundity were observed in the adult female ticks treated with both eugenol and benzaldehyde. The LC50 and LC90 values of eugenol against R. annulatus were 162.33 and 467.74 mg/mL for adult female, while it was 1.26 and 1.73 mg/mL for larvae respectively. The LC50 and LC90 values of benzaldehyde against R. annulatus were 178 and 395 mg/mL for adult female and 0.69 and 4.32 mg/mL for larvae respectively.

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.

Current profile of phenotypic pyrethroid resistance in Rhipicephalus microplus (Acari: Ixodidae) populations sampled from Marathwada region of Maharashtra state, India.

This study examined the pattern of resistance to widely applied synthetic pyrethroids, i.e., cypermethrin and deltamethrin, against larvae of Rhipicephalus microplus ticks sampled from Marathwada region in Maharashtra, India. The study also examined the role of α- and ß-esterases and glutathione-S-transferase (GST) in resistance development. All eight R. microplus isolates tested were resistant to deltamethrin (RL IV), having RR50 values from 6.88 to 131.26. LPT analysis exhibited the resistance level II deltamethrin resistance in Beed and Hingoli, III in Dharashiv, and IV in Sambhajinagar, Parbhani, Latur, Jalna, and Nanded isolates. The LIT analysis showed that Dharashiv field isolates had the lowest LC50 value of 229.09 ppm against cypermethrin, while Sambhajinagar field isolates had the highest at 489.78 ppm. The RR50 ranged from 1145.45 to 2448.9. Seven isolates were level I resistant to cypermethrin while the Jalna isolate was level II resistant. In larvae treated with deltamethrin and cypermethrin, the activity of α- and ß-esterase enzymes increased significantly compared to control groups. The enzyme ratios in treated larvae ranged from 0.7533 to 1.7023 for α-esterase and 0.7434 to 3.2054 for ß-esterase. The Hingoli isolate treated with cypermethrin exhibited the highest α-esterase activity (903.261), whereas Sambhajinagar isolate had the highest GST enzyme ratio (2.8224) after deltamethrin exposure. When exposed to cypermethrin, the Hingoli isolate showed the highest GST enzyme ratio, 2.0832. The present study provides the current resistance status in tick populations from Marathwada region indicating deltamethrin and cypermethrin to be ineffective for tick control. The results also suggest that SP compounds should be regulated in this region and alternative control strategies should be introduced.

Drivers and evolution of acaricide resistance and multi-resistance in two Ecuador's subtropical livestock farming areas.

The management of cattle ticks, particularly Rhipicephalus microplus, poses a global challenge in subtropical regions like Ecuador due to its impact on meat and milk productivity, leading to economic losses. Misuse of acaricides has resulted in resistance and multi-resistance, diminishing their effectiveness. This study evaluated resistance to amitraz, alpha-cypermethrin, and ivermectin using the Larval Packet test, laboratory-reared tick larvae collected from cattle were tested. Data on farm management and tick control practices were gathered via a questionnaire in Northwest Pichincha and Quijos River Valley over two years. Resistance rates in the first year (2020-2021) were 67.21% for amitraz, 57.38% for ivermectin, and 67.21% for alpha-cypermethrin. One year later (2021-2022), resistance levels were 59.57% for amitraz, 57.45% for ivermectin, and 68.09% for alpha-cypermethrin, with multi-resistance rates at 67.21% and 65.96% respectively. No significant differences were found between years or locations. Analysis of larval survival data determined lethal doses for tested acaricides. The study emphasizes the association between the lack of acaricide rotation, the incorrect dosage, and the absence of non-chemical measures in tick management could be associated with the development of resistances in ticks. Likewise, this study promotes the need for collaborative efforts to improve control practices and maintain acaricide efficacy.

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.

Target-based discovery of antagonists of the tick (Rhipicephalus microplus) kinin receptor identifies small molecules that inhibit midgut contractions.

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.

Comparative analysis of Rhipicephalus microplus (Canestrini, 1888) infestation in pure breed (Hereford) and cross breed (Braford) cattle herds subjected to the same chemical treatments.

The aim of this work was to analyze the R. microplus (Canestrini, 1888) infestation in two bovine herds with different degrees of natural resistance (i.e., Hereford and Braford) to ticks subjected to an identical chemical treatment scheme to ticks at the same farm, to demonstrate the impact on tick control of the incorporation of a more resistant bovine breed. Two groups of ten Hereford and Braford cows each were subjected to eleven chemical treatments between August 2022 and October 2023 (four fluazuron, two fipronil 1%, one ivermectin 3.15% and four immersion in a dipping vat with a combination of cypermethrin 10% and ethion 40%). Tick population was shown to be susceptible to ivermectin, fluazuron and the mix cypermethrin 10%-ethion 40% and resistant to fipronil according to in vitro tests. Tick infestation was significantly greater in the Hereford cows than in the Braford cows. Tick infestation in both Hereford and Braford breeds was similar when treatment with functional drugs was applied, but when a block of the treatments was done with drugs with decreased functionality due to resistance (i.e. fipronil), treatment failure was manifested more strongly in the most susceptible breed. The incorporation of cattle breeds with moderate or high resistance to R. microplus is instrumental to optimize the efficacy and sustainability of chemical control of ticks in a scenario where resistance to one or more chemical groups is almost ubiquitous, because it favors the biological control of this parasite.

Attractant and repellent properties of Senna didymobotrya plant extracts to Amblyomma variegatum and Rhipicephalus appendiculatus.

The growing challenge of acaricide resistance and geographical range expansion of invasive tick species demands other interventions, like plant-based alternatives, for sustainable tick control. Leaves, flowers, seedpods, and twig branch extracts of Senna didymobotrya were analyzed using coupled gas chromatography mass spectrometry (GC-MS). Response of adult Amblyomma variegatum and Rhipicephalus appendiculatus to extracts was evaluated. The most attractive plant extract was fractionated and ticks' responses to its fractions assessed. Potential tick attractants in the attractive plant part extract and its fractions were identified by GC-MS analysis. Non- significant qualitative and quantitative differences were observed in the plant parts' extract composition (R = 0.6178). Flower extracts attracted both species, with a 0.1-fold higher attraction in A. variegatum compared to the standard attraction aggregation attachment pheromone (AAAP). Leaf and seedpod extracts repelled ticks at various concentrations. Bioassays after fractionating flower extracts identified hexane and ethyl acetate fractions as most attractive to A. variegatum (P < 0.001) and R. appendiculatus (P < 0.001), respectively. Chemical analysis of the most attractive extracts and fractions identified compounds, including documented acarine attractants, squalene and linoleic acid. A squalene and linoleic acid blend (1:1) at 1 mg/mL significantly attracted adult A. variegatum (P < 0.01) and R. appendiculatus (P < 0.001). The results of this study broaden comprehension of how ticks respond to plants in nature, and showcase the promising potential for integrating these insights into effective tick management programs.

Assessment of cattle tick infestation: Molecular insights into Rhipicephalus annulatus and the efficacy of garlic oil and nanoemulsion as acaricidal agents.

Ticks, particularly Rhipicephalus annulatus, pose significant threats to livestock, causing economic losses and transmitting various infectious diseases. This study aimed to evaluate the potential acaricidal properties of garlic oil and its nanoemulsion against ticks infesting cattle, Rhipicephalus annulatus through the evaluation of mortality rate and morphological changes of the treated ticks. The study also included prevalence, risk factors, and molecular confirmation of tick species. Genetic characterization confirmed the identity of R. annulatus. Our results revealed a high prevalence of R. annulatus (46.9%) with a higher risk in male cattle (50%) than females (44.9%) and a nonsignificant high infection (49.1%) in animals ≤ 1 year old. The acaricidal efficiency of garlic oil and its nanoemulsion was concentration and time-dependent. The high concentration of garlic oil (20 mg/L) induced complete mortality within 48 hours. The nanoemulsion formulation enhanced efficacy, particularly at 5 mg/L, which exhibited rapid and substantial acaricidal activity. Scanning electron microscopy revealed morphological alterations induced by garlic oil and its nanoemulsion, including changes to the anterior capitulum, dorsal, and ventral cuticles. The study contributes to the exploration of effective, safe, and eco-friendly alternatives for tick control. Further research is warranted to validate their efficacy under diverse conditions and assess practical strategies.

Cananga odorata L., Illicium verum, and Foeniculum vulgare essential oils impair the reproduction of the cattle tick Rhipicephalus microplus (Acari: Ixodidae).

This study aimed to evaluate the acaricidal potential of the essential oils of Cananga odorata L. (ylang-ylang), Illicium verum (star anise), and Foeniculum vulgare (sweet fennel) in Rhipicephalus microplus engorged females. The essential oils were extracted by hydrodistillation, and the females were immersed in each essential oil at concentrations ranging from 10 µL/mL to 50 µL/mL, using 10% dimethyl sulfoxide as a solvent. Tick mortality was evaluated after seven and 14 days, and the following parameters were determined: female weight before oviposition, preoviposition period, egg mass weight, egg production index, incubation period, hatching rate, fecundity, percentage of reduction in oviposition, percentage of reduction in hatching and product efficacy. Lethal concentration (LC50) values were 34.42 µL/mL (28.34-40.86) for C. odorata and 40.60 µL/mL (32.58-58.21) for F. vulgare. This parameter was not calculated for I. verum because there was insufficient mortality at the concentrations evaluated. The greatest efficacy was observed in the group treated with 40 µL/mL F. vulgare essential oil (97.17%), followed by I. verum (91.35%) and C. odorata (86.19%) at the same concentration. Efficacy results were associated with a greater reduction in egg mass weight and lower hatching rates. Although the essential oils caused a slight acaricidal impact, they showed significant effects on the reproductive performance of R. microplus engorged females. Thus, the results of this study indicate that these essential oils can be used in the development of new acaricidal formulations.

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.

In vitro and in silico studies of the acaricidal and anticholinesterase activities of Randia aculeata seeds against the southern cattle tick Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is a leading cause of significant economic losses in the livestock industry, and tick populations have developed multiple forms of resistance to acaricides; therefore, the potential of novel natural bioactive compounds that are effective for targeting ticks must be addressed. The aim of this study was to evaluate the acaricidal and anticholinesterase activities of R. aculeata seeds and to identify naturally occurring compounds that potentially inhibit anticholinesterase through in silico docking. The acaricidal activity of the extract of R. aculeata seeds against larval and adult R. microplus ticks was assessed through immersion tests. Inhibition of anticholinesterase activity was measured spectrophotometrically. Extracts of R. aculeata seeds showed activity against larvae and engorged females of R. microplus, and a reduction in the reproductive index were also observed. Rutin, chlorogenic acid, quercetin, and epicatechin exhibited noteworthy interactions with the active site residues of RmAChE. These findings could significantly contribute to the exploration of novel natural products that can potentially inhibit RmAChE and could be used in the development of new acaricides for tick control.

Chemical Composition and Acaricidal Activity of Essential Oil of Lavandula dentata L. on Engorged Females of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

PURPOSE: Rhipicephalus (Boophilus) microplus is one the most significant ectoparasite in cattle farming in tropical and subtropical regions, causing problems to livestock health worldwide. The control of this ectoparasite primarily relies on the use of synthetic acaricides. However, the emergence of acaricide resistance has stimulated the search for new control alternatives, including phytocompounds with acaricidal and insecticidal potential. The aim of this study was to evaluate the acaricidal potential of Lavandula dentata essential oil against the engorged females of R. (B.) microplus. METHODS: Engorged females were obtained from infested bovines in dairy farms in Pernambuco, Brazil. L. dentata essential oil was extracted, and adult immersion test assays were performed using the following oil concentrations: 0.2%, 0.4%, 0.6%, 0.8%, and 1%. RESULTS: L. dentata essential oil at a concentration of 1% was lethal to all engorged females, and concentrations of 0.6% and 0.8% caused mortality of 98.6% and 99.1%, respectively. These concentrations disrupted the reproductive capacity of engorged females, reducing oviposition by more than 90% and preventing egg hatching by over 87%. CONCLUSION: The data revealed that L. dentata essential oil possesses effective pharmacological properties against R. (B.) microplus and could be used for tick control following in vivo evaluation, thus contributing to mitigating the negative impacts of synthetic acaricide use.

Effect of ivermectin, amitraz and fipronil on midgut epithelium and digestive enzyme profile in Rhipicephalus microplus ticks (Acari: Ixodidae).

Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 µg/mL and 92.67 ± 3.72 µg/mL, respectively, as compared to the control group (150.0 ± 3.80 µg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 µmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 µmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.

Self-emulsifying drug delivery systems (SEDDS) containing Cymbopogon citratus essential oil: Enhancing the stability and acaricide efficacy against Rhipicephalus (Boophilus) microplus.

The objectives of this study were to develop a self-emulsifying drug delivery system (SEDDS) to enhance the stability and efficacy of Cymbopogon citratus essential oil or lemongrass oil (LEO) against cattle tick larvae and engorged females. The system with the highest oil loading in SEDDS was composed of LEO (23.33%w/w), Tween 80: SGKH 4000 in a 2:1 ratio as surfactant (66.67%w/w), and propylene glycol as co-surfactant (10%w/w). The selected SEDDS-LEO has a particle size of 18.78 nm with a narrow size distribution (polydispersity index of 0.27). Notably, the stability of SEDDS was superior to that of the original oil, both during long-term storage and under accelerated conditions. SEDDS-LEO at oil concentrations ranging from 1.458% to 5.833% w/v showed a significantly higher percentage of egg-laying reduction against adult ticks compared with the original oil at the same concentrations (p < 0.05). Furthermore, SEDDS-LEO demonstrated greater larvicidal efficacy than the original oil, with lower LC50 and LC90 values of 0.91 mg/mL and 1.20 mg/mL, respectively, whereas the original oil's LC50 and LC90 values were 1.17 mg/mL and 1.74 mg/mL, respectively. Our findings indicate that SEDDS-LEO is a promising candidate for use as an acaricide in the control of tick populations in dairy cattle.