Disruption of bacterial interactions and community assembly in Babesia-infected Haemaphysalis longicornis following antibiotic treatment.

BACKGROUND: A previous study highlighted the role of antibiotic-induced dysbiosis in the tick microbiota, facilitating the transstadial transmission of Babesia microti from nymph to adult in Haemaphysalis longicornis. This study builds on previous findings by analyzing sequence data from an earlier study to investigate bacterial interactions that could be linked to enhanced transstadial transmission of Babesia in ticks. The study employed antibiotic-treated (AT) and control-treated (CT) Haemaphysalis longicornis ticks to investigate shifts in microbial community assembly. Network analysis techniques were utilized to assess bacterial interactions, comparing network centrality measures between AT and CT groups, alongside studying network robustness and connectivity loss. Additionally, functional profiling was conducted to evaluate metabolic diversity in response to antibiotic treatment. RESULTS: The analysis revealed notable changes in microbial community assembly in response to antibiotic treatment. Antibiotic-treated (AT) ticks displayed a greater number of connected nodes but fewer correlations compared to control-treated (CT) ticks, indicating a less interactive yet more connected microbial community. Network centrality measures such as degree, betweenness, closeness, and eigenvector centrality, differed significantly between AT and CT groups, suggesting alterations in local network dynamics due to antibiotic intervention. Coxiella and Acinetobacter exhibited disrupted connectivity and roles, with the former showing reduced interactions in AT group and the latter displaying a loss of connected nodes, emphasizing their crucial roles in microbial network stability. Robustness tests against node removal showed decreased stability in AT networks, particularly under directed attacks, confirming a susceptibility of the microbial community to disturbances. Functional profile analysis further indicated a higher diversity and richness in metabolic capabilities in the AT group, reflecting potential shifts in microbial metabolism as a consequence of antimicrobial treatment. CONCLUSIONS: Our findings support that bacterial interaction traits boosting the transstadial transmission of Babesia could be associated with reduced colonization resistance. The disrupted microbial interactions and decreased network robustness in AT ticks suggest critical vulnerabilities that could be targeted for managing tick-borne diseases.

Acaricidal activity of Egyptian crude plant extracts against Haemaphysalis longicornis ticks.

Haemaphysalis longicornis is a common Ixodida tick species found in temperate areas of Asian countries. An anti-tick assay was conducted on adult female H. longicornis ticks. Plant extract solutions were prepared at concentrations of 50, 25, and 10 mg/mL. Tick survival and mortality were assessed by counting the number of dead and live ticks at 24 h, 48 h, 72 h, and 96 h posttreatment. Out of 11 plant extracts screened, Artemisia judaica extract exhibited the highest potency with 100% mortality (5/5) at 48 h when applied at high and moderate concentrations (50 and 25 mg/mL). Similar results were observed at 96 h for the 10 mg/mL group compared to the untreated ticks. Cleome droserifolia extract demonstrated partial activity with 60% (3/5) and 20% (1/5) mortality at 96 h posttreatment at concentrations of 50 and 25 mg/mL, respectively. Forsskaolea tenacissima extract showed a weak effect with 100% tick mortality (5/5) only at the highest treatment concentration after 96 h. To confirm the activity of A. judaica, trial 2 was conducted. A. judaica demonstrated potency within 48 h in high dose and 72 h in moderate dose, with 100% mortality (15/15) at 96 h posttreatment compared to untreated ticks. The median lethal time 50 (LT50) values were 30.37 h for the high and 55.08 h for the moderate doses. Liquid chromatography‒mass spectrometry was performed on the most potent candidate (A. judaica) to identify its phytochemical components. The results revealed the presence of 9 compounds identified through manual annotation and 74 compounds from the Global Natural Products Social library. These compounds included terpenoids, steroids, phenylpropanoids, flavonoid glycosides, flavonoids, and benzenoids. Camphor was identified as the major component via both approaches. These findings suggest the potential use of A. judaica extract in the future development of acaricidal therapeutics.

The contact toxicity and toxic mechanism of essential oils from Pimenta racemosa and Eugenia caryophyllata against Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis, which is widely distributed in China, can transmit various tick-borne diseases such as severe fever with thrombocytopenia syndrome, babesiosis, rickettsia disease and so on, and do great harm to human health and the development of animal husbandry. Chemical acaricides are the most traditional tick control method, but because of its many shortcomings, there is an urgent need to find a substitute with high efficiency, environmental protection and low toxicity. It has been found that some plant essential oils (EOs) have good insecticidal activity and environmental safety. In this study, the components of EOs from Pimenta racemosa and Eugenia caryophyllata were analyzed by gas chromatography-mass spectrometry (GC-MS), and their potential for application in the control of Haemaphysalis longicornis were studied. Gas chromatography-mass spectrometry analysis showed that the main components of P. racemosa EO were eugenol (64.07%), those of E. caryophyllata EO were Hexadecanoic acid, 2-methylpropyl ester (51.84%) and eugenol (39.76%). Larval packet test showed that the EOs of P. racemosa and E. caryophyllata had significant acaricidal activity against unfed larvae of H. longicornis, with LC50 values of 1.20 mg/mL and 0.47 mg/mL and LC90 values of 8.76 mg/mL and 2.91 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed nymph H. longicornis, with LC50 values of 1.65 mg/mL, 2.29 mg/mL and 0.93 mg/mL and LC90 values of 5.03 mg/mL, 11.01 mg/mL and 4.77 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed adults H. longicornis, with LC50 values of 0.51 mg/mL, 2.57 mg/mL and 1.83 mg/mL and LC90 values of 2.44 mg/mL, 11.44 mg/mL and 2.54 mg/mL, respectively. Enzyme assays revealed that the E. caryophyllata EO and eugenol significantly inhibited the activity of carboxylesterase (CarE), eugenol significantly inhibited the activity of catalase (CAT), and two EOs and eugenol had no significant effect on acetylcholinesterase (AchE) (p < 0.05). The above results suggest that the essential oils from P. racemosa and E. caryophyllata have great potential for use as alternatives to synthetic acaricides for tick control.

Comparative analysis of essential oil efficacy against the Asian longhorned tick Haemaphysalis longicornis (Acari: Ixodidae).

This study evaluated the potential repellent and acaricidal effects of 4 essential oils (clove, eucalyptus, lavender, and mint) against the Asian longhorned tick Haemaphysalis longicornis, a vector of various tick-borne diseases in medical and veterinary contexts. Selected for their potential repellent and acaricidal properties, the 4 essential oils were tested on adult and nymph H. longicornis ticks at different concentrations. The experiment assessed mortality rates and repellency, particularly during tick attachment to host skin. There was a significant increase (p<0.05) in tick mortality and repellency scores across all groups. At a 1% concentration, adult tick mortality ranged from 36% to 86%, while nymph mortality ranged from 6% to 97%. Clove oil exhibited notable efficacy, demonstrating high mortality rates of nymphs and adults. Clove oil also displayed strong repellency properties, with a repellency index of 0.05, surpassing those of mint, eucalyptus, and lavender oils. Clove oil showed the highest effectiveness in deterring nonattached adult ticks (90%) and nymphs (95%) when applied to skin. Clove oil was the most effective against adult and nymph ticks, achieving mortality rates of 86% and 97%, respectively, and led to the highest nonattachment rates when applied to skin. In conclusion, essential oils such as clove, eucalyptus, lavender, and mint oils present promising results for tick population control.

Acaricidal Efficacy of Thirty-Five Egyptian Plants Against the Camel Tick, Hyalomma Dromedarii.

PURPOSE: Alternative and affordable tick control strategies are crucial to control and prevent tick bites and tick-borne diseases. METHODS: In this study, we evaluated the acaricidal efficacy of 35 aqueous plant extracts (17%) against the camel tick, Hyalomma dromedarii. RESULTS: The phytochemical profile indicated the presence of various secondary substances. Plants were classified into three groups according to their mortality percentage 15 days post-treatment with 17%. This highly effective group (91%-95%) comprised Ocimum basilicum, Mespilus germanica, and Viola alpine followed by Carum carvi, Cucurbita pepo (peel), and Peganum harmala. A moderately effective group (80%-90%) included Acacia nilotica, Apium graveolens, Capsicum annuum, Ceratonia siliqua, Cucurbita pepo (seeds), Equisetum arvense, Eruca sativa, Ginkgo biloba, Plantago psyllium, Phyllanthus emblica, Punica granatum, and Ziziphus spinachristi. The 20 remaining plants were assigned to the less effective group (< 80%). Viscum album (58.3%), which was the least effective reference plant. The high potency of six plant extracts as acaricides may be attributed to the high content of active principles, e.g., phenols, flavonoids, and tannins. CONCLUSION: All of these highly effective plants are recommended for use as an acaricide, in case of facing acaricidal resistance or limited options for tick control.

Acaricide resistance status of deltamethrin and coumaphos in Hyalomma anatolicum ticks collected from different districts of Haryana.

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, ß-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Laboratory assessment of the acaricidal, repellent and anti-cholinesterase effects of Melaleuca alternifolia and Chamaemelum nobile essential oils against Hyalomma scupense ticks.

In cattle, Hyalomma scupense serves as an important vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. Resistance to chemical acaricides has become widespread affirming the need for new drugs to tick control. The goal of this study was to investigate the acaricidal, repellent activities as well as the putative mode of action of two essential oils (EOs) from Melaleuca alternifolia (Tea tree) and Chamaemelum nobile (Roman chamomile) on Hyalomma scupense. The chemical composition of EOs was also evaluated. Different concentrations of EOs were tested in vitro for their acaricidal property on adults and larvae of H. scupense using adult immersion test (AIT) and larval packet test (LPT). Additionally, using Ellman's spectrophotometric method, the anticholinesterase (AChE) inhibition activity of M. alternifolia and C. nobile EOs was assessed in order to understand their putative mode of action. The main compounds of C. nobile were α-Bisabolene (22.20%) and (E)-ß-Famesene (20.41%). The major components in the analyzed M. alternifolia were Terpinen-4-ol (36.32%) and γ-Terpinene (13.69%). Adulticidal and larvicidal assays demonstrated a promising efficacy of the essential oils against tick H. scupense. The lethal concentration (LC50) values obtained for M. alternifolia and C. nobile oils were 0.84 and 0.96 mg/mL in the AIT and 0.37 and 0.48 mg/mL in the LPT, respectively. Regarding repellent activity, M. alternifolia achieved 100% repellency at the concentration of 1 mg/mL while C. nobile showed 95.98% repellency activity at concentration of 4 mg/mL. Also, M. alternifolia and C. nobile EOs displayed potent AChE inhibition with IC50 value of 91.27 and 100.12 µg/mL, respectively. In the present study, M. alternifolia and, to a lesser degree, C. nobile EOs were found to be effective in vitro acaricides, repellents and acetylcholinesterase inhibitor against H. scupense ticks. These plants may represent an economical and sustainable alternative to toxic synthetic acaricides in the management of ectoparasites of veterinary importance.

Repellent effects of Chinese cinnamon oil on nymphal ticks of Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and Hyalomma asiaticum.

The repellent activity of Chinese cinnamon oil (Cinnamomum cassia) on nymphal ticks (Haemaphysalis longicornis Neumann, Rhipicephalus haemaphysaloides Supino, and Hyalomma asiaticum Schulze and Schlottke) was evaluated in a sample Y-tube bioassay. The results were based on the vertical migration of ticks during the host-seek phase and showed a dose-dependent repellent effect of Chinese cinnamon oil on the tested nymphs after 6 h. For H. longicornis, R. haemaphysaloides, and H. asiaticum at the concentrations (vol/vol) of 3, 3, and 1.5%, the repellent percentages over time were 68-97, 69-94, and 69-93%, respectively, which indicated strong repellent activities against ticks, similar to the positive control DEET (N,N-diethyl-3-methylbenzamide). Chinese cinnamon oil exerted the strongest effect on H. asiaticum nymphs. To our knowledge, this is the first study to investigate the repellent effects of Chinese cinnamon oil on ticks. Chinese cinnamon oil has considerable potential and should be developed as a practical tick repellent.

Persistence Enhancement of a Promising Tick Repellent, Benzyl Isothiocyanate, by Yeast Microcarriers.

Phenethyl isothiocyanate isolated from Armoracia rusticana root oil and its derivatives were tested at different doses in a bioassay designed to evaluate repellency against individual Haemaphysalis longicornis nymphs. Among the tested compounds, benzyl isothiocyanate exhibited repellency against H. longicornis nymphs at the lowest dose of 0.00625 mg/cm2, followed by phenethyl isothiocyanate (0.0125 mg/cm2) and phenyl isothiocyanate (0.025 mg/cm2). The behavioral responses of H. longicornis nymphs exposed to benzyl isothiocyanate and phenethyl isothiocyanate indicated that the mode of action of these compounds can be mainly attributed to the vapor phase. Encapsulated benzyl isothiocyanate showed repellency up to 120 min post-application at 0.1 mg/cm2, whereas pure benzyl isothiocyanate showed repellency up to 60 min post-application at 0.1 mg/cm2. The present study suggests that benzyl isothiocyanate is a potential repellent for protection against H. longicornis nymphs, and encapsulation in yeast cells may enhance the repellency effect.

Ivermectin resistance in the multi-host tick Hyalomma anatolicum (Acari: Ixodidae) in India.

The multi-host tick, Hyalomma anatolicum is a widely distributed vector of many pathogens of veterinary and public health importance. Ivermectin (IVM), as an alternative to control pyrethroid-resistant ticks, has been used extensively for the past 4-6 years in tropical and sub-tropical countries including India resulting in declining tick control efficacy. The present study used adult immersion test (AIT) to examine the resistance status of H. anatolicum collected from three districts in the Indian state of Gujarat against ivermectin. Probit analysis was used for calculation of concentration-mortality regressions; concentrations required for 50% mortality (LC50) and 95% mortality (LC95), along with confidence intervals; slope of mortality; % inhibition of oviposition; and discriminating concentration (DC). The calculated LC50 and LC95 estimates were utilized to determine resistance ratios (RR50, RR95) and the resistance levels (RL) of the field ticks compared to the susceptible population. The DC (2 x LC95) for IVM was calculated as 84.48 ppm, using susceptible H. anatolicum ticks (KHD). Lower estimates of the coefficient of non-determination (1-R2) for AIT ranged from 0.06 to 0.27, and the range of RR50 and RR95 values against IVM was estimated to be from 1.43 to 52.06 and 1.14 to 71.99, respectively, which indicated a varying degree of resistance among the field tick populations. Based on RR50 values, tick populations from Danta and Palanpur showed resistance level IV and II, respectively. Another four populations (Vadgam, Kankrej, Saraswati and Sidhpur) were classified as having level I resistance status against IVM. To our knowledge, this is the first report of ivermectin resistance in H. anatolicum from Gujarat, India.

Acaricidal activity of Cinnamomum cassia (Chinese cinnamon) against the tick Haemaphysalis longicornis is linked to its content of (E)-cinnamaldehyde.

BACKGROUND: The tick Haemaphysalis longicornis (Neumann) is a well-known vector of numerous pathogens of veterinary and medical importance. Various control strategies, including the use of synthetic pesticides, have been developed to control this tick species. However, demand for effective and safe alternative pesticides is increasing due to the adverse effects associated with the intensive and injudicious use of synthetic pesticides, which include undesirable effects on non-target species and environmental pollution. Hence, the acaricidal activity of the extract and the essential oil of Cinnamomum cassia (Chinese cinnamon) and their major components, and the underlying mechanisms of this activity, were evaluated against unfed larvae and nymphs of H. longicornis. METHODS: The components of the extract and essential oil of C. cassia were determined by gas chromatography-mass spectrometry, and their larvicidal and nymphicidal activity were evaluated using the larval and nymphal packet test. The underlying detoxification mechanism was elucidated by targeting in vivo esterase and monooxygenase activity, and the toxicological effect was assessed on non-target Tenebrio molitor and Harmonia axyridis by topical application in open Petri dishes. RESULTS: (E)-cinnamaldehyde was the predominant component of the extract (50.79%) and essential oil (89.95%). The 50% lethal concentration (LC50) for larvae and nymphs treated with the extract was 11.56 and 49.18 mg/mL, respectively. The essential oil, (E)-cinnamaldehyde and fenvalerate exhibited acaricidal activity, with LC50 values of 3.81, 3.15, and 0.14 mg/mL, respectively, against the larvae, and 21.31, 16.93, and 1.89 mg/mL, respectively, against the nymphs. (E)-cinnamaldehyde significantly increased esterase and monooxygenase activity in both larvae and nymphs. Unlike fenvalerate, C. cassia essential oil and (E)-cinnamaldehyde did not cause mortality of T. molitor or H. axyridis adults. CONCLUSIONS: This study demonstrates that C. cassia essential oil and (E)-cinnamaldehyde have the potential to be developed into botanical-based larvicidal and nymphicidal agents for tick control.

Repellent activity of acetylcarvacrol and its effects on salivary gland morphology in unfed Rhipicephalus sanguineus sensu lato ticks (Acari: Ixodidae).

Rhipicephalus sanguineus sensu lato (s.l.), commonly known as brown dog tick, is a widely distributed tick species that is substantially important for human and veterinary medicine. Therefore, it is the target of different control methods. Carvacrol and its semisynthetic derivative, acetylcarvacrol, are promising chemical compounds for alternative tick control. Thus, this study aimed to compare the repellent activities of carvacrol and acetylcarvacrol at different concentrations and drying times. Additionally, morphological alterations found in salivary glands were evaluated through histological techniques after exposure to acetylcarvacrol. The impact of the morphological changes on the development and survival of acini/cells in salivary glands was measured by a semiquantitative analysis. The repellent action of both compounds did not differ when evaluated at different concentrations, although acetylcarvacrol increased its effects as the concentration raised. Regarding the different drying times, acetylcarvacrol maintained its effects after 3 hours of exposure, while the efficacy of carvacrol decreased during this time period. Salivary glands of unfed R. sanguineus s.l. females showed dose-dependent alterations in the size and shape of acini as well as cytoplasmic vacuolization. Loss of the acinar cell limit, rupture of secretory granules and nuclear changes in the cells were also observed in the treated groups. Thus, our results demonstrated the potential of acetylcarvacrol to act as repellent against R. sanguineus s.l. Additionally, the morphological alterations found in salivary glands may interfere with the feeding process of ticks, which contributes to mitigate infestation by this species.

Tick treatment practices in the field: Access to, knowledge about, and on-farm use of acaricides in Laikipia, Kenya.

The prevention of tick-borne diseases is a major challenge for livestock production globally. Tick control strategies include the use of acaricides, but the prescribed strategies do not achieve the desired results in several countries, including Kenya. To better understand how tick treatment practices, contribute to reported tick treatment failures, we assessed livestock owners' acaricide procurement, level of knowledge about acaricides and tick resistance, and how they apply acaricides. We also assessed the quality of the commonly available acaricides. We focused on three livestock systems in Laikipia County, Kenya: two private ranches; one community ranch whose members communally graze their cattle and acquire and apply acaricides; and individual livestock owners in two pastoral communities who individually graze their cattle and acquire and apply acaricides. Through interviews and focus group discussions we assessed; access to acaricides, livestock owners' knowledge, and acaricide use practices; interview data were triangulated with participant observations (n = 107). We analysed nine commonly used acaricides to determine the active ingredient concentration and we determined the concentration of active ingredients in acaricide dilutions collected on farms. All livestock owners had access to and used chemical acaricides for tick control, predominantly amitraz-based. Private ranchers bought one amitraz-based acaricide in bulk directly from the manufacturer, while all other livestock owners bought from agrovet shops. The livestock owners acquired knowledge about acaricides from their own experiences and through experience-based recommendations from peers, but not from the technical information provided by the manufacturers and agrovet shops. All pastoral livestock frequently changed acaricide brand and active ingredient class. A large majority of pastoralists (86%) mixed acaricide brands within and across active ingredient classes; a smaller majority (56%) mixed acaricides with crop pesticides and insecticides. Our lab tests confirmed the content description on the labels bought from agrovet shops. However, on-farm acaricide dilutions from all three livestock systems deviated from the level recommended for effective treatment. If too diluted, the acaricide does not kill ticks, promoting resistance development. If too concentrated, this increases environmental contamination and raises public health concerns. Livestock owners lack a technical understanding of the functioning of acaricides, compromising their use and effectiveness. The widely adopted mixing of acaricides with insecticides and pesticides raises serious health concerns.

Do-It-Yourself Tick Control: Granular Gamma-Cyhalothrin Reduces Ixodes scapularis (Acari: Ixodidae) Nymphs in Residential Backyards.

Lyme disease is the most common vector-borne disease in the United States with hotspots in the Northeast and Midwest. Integrated vector control for mosquito-borne disease prevention is often organized at the community level, but tick control is primarily coordinated at the household and individual level. Management of the blacklegged tick, Ixodes scapularis (Say), the vector of the causative agent of Lyme disease in the Midwest and eastern United States in peridomestic environments may be critical as many tick encounters are reported to occur in the yard. Therefore, we assessed the effectiveness of a widely available and low-cost pesticide that targets common lawn pests and is labeled for use against ticks. In June 2019, we evaluated a granular form of gamma-cyhalothrin in a placebo-controlled residential backyard study (n = 90) in two communities in Wisconsin. The product applied by the research team reduced nymphal blacklegged ticks in plots established in the lawn part of the ecotone by 97% one week after application at both communities and by 89-97% three to four weeks postapplication. The proportion of homes with at least one nymphal tick postapplication was significantly lower at acaricide-treated homes and ranged from 4.2 to 29.2% compared with placebo homes where at least one nymphal tick was found at 50-81.5% of homes. These results support the efficacy of a low-cost do-it-yourself strategy for homeowners seeking to reduce blacklegged ticks in the yard.

Hyalomma anatolicum resistance against ivermectin and fipronil is associated with indiscriminate use of acaricides in southwestern Balochistan, Pakistan.

Ivermectin and fipronil have been used regularly to control the hard tick Hyalomma anatolicum (Acari: Ixodidae) in domestic ruminants for more than a half-decade in Balochistan, Pakistan. Inappropriate and indiscriminate use of these acaricides has resulted in the development of resistances in tick species. In this work, acaricides (ivermectin and fipronil) resistance was evaluated in H. anatolicum through in vitro and in vivo bioassays in a horse farm of Quetta, Balochistan province, Pakistan. A participatory epidemiological survey was conducted to assess potential risk factors associated with the development of acaricide resistance in H. anatolicum. The results of the epidemiological survey revealed that the horse keepers did not follow the manufacturer's instructions for the use of acaricides and applied indiscriminate doses of acaricides. The results of in vitro bioassays (adult immersion test and larval immersion test) showed that fipronil and ivermectin have protective efficacy against H. anatolicum. The results of in vivo bioassay (adult-tick mortality assay) revealed that fipronil had a higher efficacy (78.16%) than ivermectin (49.94%). More than 80% of tick mortality was not achieved in any bioassays, even for the highest acaricide concentration (100 ppm), which suggests the development of acaricide resistance against fipronil and ivermectin. This study highlights the urgency to implement a country-wide awareness about resistance monitoring and effective tick control. Graphical abstract.

Structure-Activity Relationship (SAR) and in vitro Predictions of Mutagenic and Carcinogenic Activities of Ixodicidal Ethyl-Carbamates.

Ethyl-4-bromophenyl-carbamate (LQM 919) and Ethyl-4-chlorophenyl-carbamate (LQM 996) are compounds that inhibit egg-laying and hatching of tick larvae that are resistant to conventional ixodicides. The structure-activity relationship (SAR) to get the endpoint predictions of mutagenicity and carcinogenicity of the LQM 919 and LQM 996 was performed and the absence of mutagenicity was confirmed by Ames test. SAR analysis show no structural alerts indicating the ability of ethyl-carbamates to bind biomolecules or estrogen receptors. Endpoint of mutagenicity with and without metabolic activation showed that the ethyl-carbamates were negative (p <0.05) for mutagenicity induction in strains TA97, TA98, TA102, TA1535, TA1537 and TA1538 of Salmonella typhimurium. Pre-incubation with different ethyl-carbamate concentrations did not increase the number of spontaneously reverting colonies; moreover, the compounds did not induce a concentration-dependent increase in the number of reverting colonies in any of the strains used. This confirmed the absence of mutagenic activity in this test system. Exogenous metabolic activation did not modify these observations; suggesting that no metabolites with mutagenic activity were present. The endpoint of carcinogenicity in rats were negative for LQM 919 (p <0.05,) and LQM 996 (p <0.001). The results of the present study strongly suggest that ethyl-carbamates do not represent a risk for cancer in mammals.

Molecular screening of cattle ticks, tick-borne pathogens and amitraz resistance in ticks of Santo Domingo de los Tsáchilas province in Ecuador.

The province of Santo Domingo de los Tsáchilas in Ecuador is a strategic place for cattle production and trade. The lack of knowledge about tick species, tick control and tick-borne diseases in Ecuador prompted this study with the goal of identifying the cattle-tick species and tick-borne agents present in the area and molecularly determining the potential acaricide resistance to amitraz of the major cattle tick species. Eighty-four cattle farms were visited and in 88 % of them, cattle were infested with ticks. Additionally, 24 historical samples from other surrounding Ecuadorian provinces, were screened as well. Besides morphological keys, PCR-RFLP MspI was used to confirm the presence of the Rhipicephalus ticks. The tick samples were also screened for tick-borne agents using PCR-RFLP BseDI and Hhal tests to identify circulating Babesia sp. and Anaplasma spp. Furthermore, the PCR-RFLP EciI technique was used to identify the amitraz resistance gene in populations of Rhipicephalus microplus in the province. Pooled testing was used to determine prevalence at individual-tick level. The presence of R. microplus and Amblyomma cajennense sensu lato (s.l.) ticks was found in 83 % and 21 % of the cattle farms respectively, showing R. microplus is widespread in the province of Santo Domingo de los Tsáchilas. Regarding tick-borne agents, only Anaplasma marginale was observed in 50 % of the visited farms of the province, while about 27 % of the ticks tested positive according to estimations from the data of the tick pools. The presence of Babesia bigemina was only confirmed in samples collected outside the province. The amitraz resistance allele in R. microplus was found in 62 % of the farms, but the percentage of farms with cattle ticks completely resistant to this acaricide was low (2%). The findings of this study should prompt cattle producers and animal health authorities to monitor control strategies, which address the management of resistant tick populations and the epidemiologically-unstable areas of tick-borne diseases.

Expression and function assessment of two serpin-type serine protease inhibitors from Haemaphysalis doenitzi.

Serine protease inhibitors (serpins) in ticks are implicated in the modulation of the vertebrate host response to the tick bite. Experimentally, it has been demonstrated that serpins interfere with tick-borne pathogen transmission. However, knowledge on serpins in the tick Haemaphysalis doenitzi is lacking. In this study, the expression of two serpin genes, named HDS1 and HDS2, were assessed in H. doenitzi, and their roles in immune regulation were further investigated. The expression of HDS1 and HDS2 showed no tissue specificity, with maximum expression levels detected in the hemolymph and salivary gland, respectively. Among the developmental stages, the highest expression of HDS1 and HDS2 were detected in larvae and adults, respectively. The recombinant protein rHDS1 displayed obvious inhibitory effects on trypsin and thrombin, whereas rHDS2 clearly inhibited thrombin only. In addition, rHDS1 and rHDS2 showed certain inhibitory activities against bacteria and fungi. The female engorgement body weight, female engorgement rate, and egg hatchability were significantly decreased after injection of double-stranded RNA (dsRNA) of HDS1 gene, whereas no significant effects were observed concerning the feeding period or attachment rate at 24 h after introduction via rabbit ears. When injected with dsRNA of HDS2 gene, no significant effect was observed on the attachment rate at 24 h after introduction into the rabbit ears, but the engorgement body weight and engorgement rate of female ticks were significantly decreased, and no egg hatchment occurred. The above results contribute to better understanding the function of serpins in the development and innate immunity of H. doenitzi.

Metarhizium anisopliae sensu lato (s.l.) oil-in-water emulsions drastically reduced Rhipicephalus microplus larvae outbreak population on artificially infested grass.

Rhipicephalus microplus Canestrini, 1887 (Arachnida: Ixodidae) is a mandatory bloodsucking ectoparasite, and it is considered one of the main sanitary problems in livestock. In the development of new technologies for controlling ticks, Metarhizium anisopliae complex (M. anisopliae s. l.) Sorokin, 1883 (Ascomycota: Clavicipitaceae) have been shown to be an effective and safe alternative. However, when applied in the field, abiotic factors may directly influence their microbial activity, and different oily adjuvants enhance fungal efficacy, stability and viability under environmental conditions. Here, the efficacy of two Metarhizium anisopliae s.l. isolates (CG 148 and CG 347) were compared in mineral oil (MO) or vegetable oil (VO) emulsions to control cattle ticks under semi-natural conditions, as well as their persistence in the soil over time. Engorged female ticks were placed on the soil of grass pots previously treated or not with conidial oil-in-water emulsions. Both M. anisopliae s.l. oil-in-water emulsions tested were able to control R. microplus, achieving up to 100% daily efficacy, and remained viable in the soil for up to 60 days after treatment. These results show the prospective applicability of Metarhizium spp. to control the cattle tick and a real possibility for its control in the environment.

Liquid water intake of the lone star tick, Amblyomma americanum: Implications for tick survival and management.

Ixodid ticks are ectoparasites that feed exclusively on blood as their source of nutrients. Although ticks spend most of their life off the host, until now it has been assumed that the blood and the water vapor are the only sources of water to maintain water balance and prevent desiccation. Here we report for the first time that adult lone star ticks, Amblyomma americanum, also actively drink nutrient-free water, which greatly increases their survival. The volume of ingested water is greater in females than males (0.55 ± 0.06 vs 0.44 ± 0.07 µl) and most likely due to differences in tick size. Water uptake occurs through mouthparts and it can be later observed in the salivary glands and the midgut. We also exploited this behavior by adding a variety of inorganic compounds and microorganisms to water. Addition of inorganic salts to drinking water such as KH2PO4 + NaCl+KNO3 resulted in 100% tick mortality within 3 days. As a proof of concept for using the water drinking as a delivery route of toxic reagents for ticks, we also show that adding Pseudomonas aeruginosa to drinking water quickly leads to tick death. This tick behavior can be exploited to target important physiological systems, which would make ticks vulnerable to dehydration and microbial dysbiosis.