Description of Rhipicephalus hibericus sp. nov. (Ixodoidea: Ixodidae), a species of the Rhipicephalus sanguineus group in southwestern Europe.

We describe all the life stages of Rhipicephalus hibericus n. sp., provide the types, and present molecular support for a new species of the Rhipicephalus sanguineus sensu lato group, present in southwestern Europe, that has been historically confused with Rhipicephalus turanicus Pomerantzev, 1940. A new name is proposed for this taxon because it was impossible to ascribe to types of already described species in the group, deposited for more than 100 years in natural history institutions. The males have a dorsum showing deep and coarse punctations (absent in Rhipicephalus sanguineus sensu stricto) and adanal plates with large punctations (absent in R. sanguineus s.s.); the tail of the spiracular plate is as wide as the closest festoon (half the width in R. sanguineus s.s.). Females have large punctations in dorsal fields, a wide spiracular plate, and a "V" shaped genital opening; such a combination of characters cannot be found in other species of the group. Immatures are described from specimens collected on hosts (Rodentia and Eulipotyphla). Both larvae and nymphs are markedly smaller than R. sanguineus s.s. Nymphs display long, backward-projected auriculae; larvae are almost half the size of R. sanguineus s.s. The new species can hybridize with R. sanguineus s.s. in laboratory colonies producing an unfertile F2, laying brown and dry eggs that did not hatch. Phylogenetic analysis of partial coxI gene sequences placed R. hibericus in a well-supported clade with other sequences of R. sanguineus s.l. from Portugal, as a sister clade of R. sanguineus s.s. The new species does not belong to the R. turanicus group of species. Both 12S and 16S partial gene sequences were not as precise in the correct phylogenetic placement of R. hibericus, in part probably due to the existence of erroneously identified sequences in GenBank©. This description, together with the previous reinstatement of Rhipicephalus secundus and Rhipicephalus rutilus, and the description of the neotypes of R. sanguineus s.s. should help researchers to adequately identify their collections. Our findings demonstrate that R. turanicus is absent in southwestern Europe. Old collections should be re-examined to provide the actual range of the new species.

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.

Characterization of deltamethrin, cypermethrin, coumaphos and ivermectin resistance in populations of Rhipicephalus microplus in India and efficacy of an antitick natural formulation prepared from Ageratum conyzoides.

Rhipicephalus microplus is posing a serious threat to productive animal husbandry. Excessive use of synthetic chemicals in tick management has led to the development of resistant tick populations. Characterization of resistance to deltamethrin, cypermethrin, coumaphos and ivermectin in ticks is necessary to develop a suitable and sustainable control strategy. Based on adult immersion test and larval packet test, the resistance ratios (RR50) for adults and larvae of R. microplus populations from two Indian states ranged from 3.8 to 19.4 and 1.35-25.0 against deltamethrin, 0.061-26.3 and 0.22-19.2 against cypermethrin, and 0.2-9.5 and 0.01-3.1 against coumaphos, respectively, were recorded. Moreover, the RR50 for adults ranged from 0.212 to 3.87 against ivermectin. The RR50 for different acaricides was significantly (p<0.01) correlated with esterases, Glutathione S-transferase and monooxygenase activity. A point mutation at the 190th position of the domain II S4-5 linker region of the sodium channel gene in synthetic pyrethroids (SP) resistant populations was also detected. An antitick natural formulation prepared from the plant Azeratum conyzoides and containing two major compounds, Precocene-I (7­methoxy-2, 2-dimethyl 2H-chromene) and Precocene II (6, 7-dimethoxy-2, 2-dimethyl- 3-chromene), was developed and tested against the resistant ticks. The LC50 values of the natural formulation against the resistant populations were in the range of 4.31-5.33% irrespective of their RR50 values. Multi-acaricide resistant populations of R. microplus are established in India and the A. conyzoides based natural formulation can be used for its management.

Interfering with cholesterol metabolism impairs tick embryo development and turns eggs susceptible to bacterial colonization.

Cholesterol is a known precursor of arthropod molecules such as the hormone 20-hydroxyecdysone and the antimicrobial boophiline, a component of tick egg wax coat. Because the cholesterol biosynthetic pathway is absent in ticks, it is necessarily obtained from the blood meal, in a still poorly understood process. In contrast, dietary cholesterol absorption is better studied in insects, and many proteins are involved in its metabolism, including Niemann-Pick C (NPC) transporter and acyl-CoA:cholesterol acyltransferase (ACAT), as well as enzymes to convert between free cholesterol and esterified cholesterol. The present work addresses the hypothesis that tick viability can be impaired by interfering with cholesterol metabolism, proposing this route as a target for novel tick control methods. Two drugs, ezetimibe (NPC inhibitor) and avasimibe (ACAT inhibitor) were added to calf blood and used to artificially feed Rhipicephalus microplus females. Results show that, after ingesting avasimibe, tick reproductive ability and egg development are impaired. Also, eggs laid by females fed with avasimibe did not hatch and were susceptible to Pseudomonas aeruginosa adhesion and biofilm formation in their surfaces. The immunoprotective potential of ACAT against ticks was also accessed using two selected ACAT peptides. Antibodies against these peptides were used to artificially feed female ticks, but no deleterious effects were observed. Taken together, data presented here support the hypothesis that enzymes and other proteins involved in cholesterol metabolism are suitable as targets for tick control methods.

Five ixodid tick species including two morphotypes of Rhipicephalus turanicus on nestlings of Eurasian eagle owl (Bubo bubo) from south-eastern Bulgaria.

BACKGROUND: Birds are major hosts for many tick species (Acari: Ixodidae, Argasidae), and their role is especially important in transporting ticks over large distances along their seasonal migratory routes. Accordingly, most studies across Europe focus on the importance of avian hosts in tick dispersal, and less emphasis is laid on resident birds and their role in supporting tick life cycles. Eurasian eagle owls (Bubo bubo) exemplify the latter, but all the few studies on their tick infestation were carried out in Western Europe and even those did not involve a large sample size and did not assess infestation prevalence in natural habitats. METHODS: In this study, 320 ixodid ticks were collected from nestlings of Eurasian eagle owls during the period 2018-2020 in Bulgaria in south-eastern Europe. These ticks were analysed morphologically, and selected specimens molecularly based on cytochrome c oxidase subunit I (cox1) gene. The effects of environmental and habitat-related conditions and of the species of prey eaten by eagle owls on tick infestation were also evaluated. RESULTS: The majority of ticks were identified as adults of Rhipicephalus turanicus (n = 296). In addition, 15 Hyalomma marginatum (three males, 11 nymphs and a larva), one female of Haemaphysalis erinacei and of Ha. punctata, and a nymph of Ixodes ricinus were found. Among R. turanicus, two distinct morphotypes were observed, but they do not form a monophyletic clade in the phylogenetic tree based on the mitochondrial gene cox1. We found a positive correlation between the total number of ticks on nestlings from a particular nest and the number of medium-sized to large prey mammals brought to the nestling owls. Also, the most important predictor for tick abundance was the effect of the extent of arable land (negative), while forests and grasslands contributed less, with no effect observed in case of urbanized areas and watercourses. CONCLUSIONS: The intensity of tick infestation can be high on nestling Eurasian eagle owls (mean intensity 16.59 ticks/nestling). In this study, five different tick species were recorded, among which R. turanicus dominated. Two male morphotypes of this tick species were found, but their morphological differences were not reflected by genetic diversity or phylogenetic clustering. The most important factor determining tick abundance was the land-use structure.

Efficacy of a water-based botanical acaricide formulation applied in portable spray box against the southern cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), infesting cattle.

Ectoparasitism result in annual losses to livestock producers estimated to reach billions of dollars in tropical and subtropical parts of the world where the southern cattle tick, Rhipicephalus (Boophilus) microplus, is established because this invasive pest is also a vector of pathogens causing bovine babesiosis and anaplasmosis. Reintroduction of R. microplus could be economically devastating for the cattle industry in the United States. Novel technologies are necessary to manage populations of R. microplus that are resistant to multiple classes of synthetic chemicals widely used as acaricides to control ticks infesting livestock, and to mitigate the environmental impact of these synthetic acaricides. Bioassays with a water-based formulation of a commercially available botanical acaricide (Essentria® IC3) at 6.25 % against a laboratory strain of R. microplus was 100 % lethal against unfed larvae, and 94 % mortality was recorded against engorged female ticks. These results prompted documentation of efficacy on infested cattle using an acaricide delivery system mimicking field conditions, which is required to consider the use of a product by integrated tick management programs. Twelve tick naïve cattle were artificially infested with unfed larvae on days -19, -12 and -5. On day -2, ticks from the left-side of the body of each animal were manually counted. Depending on pre-treatment tick counts the animals were assigned into three separate treatment groups: i) water (untreated control); ii) coumaphos 0.3 % in water (positive control); and iii) botanical acaricide 6.25 % in water. Five days after the last infestation, cattle were doused in a spray box and moved to individual stalls to collect tick data for calculation of treatment efficacy. The percentage control, based on the reproduction of surviving engorged female ticks after treatment with the 6.25 % Essentria in water-based spray was 70 % against R. microplus. Coumaphos treatment had a mean percent control of 100 %. Whereas this application of Essentria® IC3 cannot be recommended as a stand-alone method to eradicate R. microplus, the 6.25 % botanical acaricide water-based spray could be part of integrated tick management to control populations of R. microplus resistant to organophosphates like coumaphos and other classes of commercially available synthetic acaricides.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Innovative granular formulation of Metarhizium robertsii microsclerotia and blastospores for cattle tick control.

The tick Rhipicephalus microplus poses a serious threat to the cattle industry, resulting in economic losses aggravated by tick resistance to chemical acaricides. Strains of Metarhizium spp., a well-known group of entomopathogenic fungi, can contribute to managing this ectoparasite. We explored two novel granular, microsclerotia- or blastospores-based formulations of Metarhizium robertsii for R. microplus control under semi-field conditions. Fungal persistence in soil was also observed for 336 days. The experiment used pots of Urochloa decumbens cv. Basilisk grass, treated with 0.25 or 0.5 mg of granular formulation/cm2 (25 or 50 kg/ha) applied to the soil surface prior to transferring engorged tick females onto the treated soil. The fungal granules yielded more conidia with subsequent sporulation under controlled indoor conditions than in the outdoor environment, where the levels of fungus rapidly declined over time. Metarhizium-root colonization ranged from 25 to 66.7% depending on the propagule and rate. Fungal formulations significantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efficacy. Microsclerotia or blastospores-granular formulations of M. robertsii can reduce the impact of R. microplus, and thus prove to be a promising tool in the control of ticks.

In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus.

Rhipicephalus microplus is responsible for high economic losses in livestock and its control has become difficult due to the establishment of tick populations resistant to commercial acaricides. This study aimed to evaluate the in vitro larvicidal effect of the alkaloids berberine and piperine, and also to investigate their inhibitory mechanisms against the acetylcholinesterase enzyme. The effects of the alkaloids on larvae were observed through the immersion test at the following concentrations: 1.5; 3; 6; 12; 16 and 24 mM. Berberine and piperine presented larvicidal activity greater than 95 %, not differing from 100 % for the positive fipronil control (p > 0.05). Of the two alkaloids, piperine had a lower effective concentration (EC), with an EC50 of 6.04 mM. The acetylcholinesterase enzyme used in the study was obtained from R. microplus larvae (RmAChE) and the anticholinesterase activity was determined spectrophotometrically. The highest anticholinesterase activity, measured as inhibition concentration (IC), was observed for berberine (IC50 = 88.13 µM), while piperine showed lower activity (IC50 > 200 µM). Docking studies in RmAChE, followed by 10 ns molecular dynamics simulation, suggest that berberine stabilizes the RmAChE at lower Root-Mean-Square Deviation (RMSD) than Apo protein. Few hydrogen-bond interactions between berberine and RmAChE residues were balanced by hydrophobic and π-type interactions. Berberine fills preferentially the peripheral anionic site (PAS), which correlates with its non-competitive mechanism. These results suggest that berberine and piperine alkaloids have an in vitro acaricidal action on R. microplus larvae, and the likely mechanism of action of berberine is related to RmAChE inhibition when accessing the PAS residues. These findings could help the study of new natural products that could inhibit RmAChE and aid in the development of new acaricides.

An update on amitraz efficacy against Rhipicephalus microplus after 15 years of disuse.

Amitraz is an acaricide that is widely used in veterinary medicine to control the cattle tick Rhipicephalus microplus. However, controversy exists in the literature regarding the resistance of R. microplus to this product. The present work provides an update on the acaricidal efficacy of amitraz (Triatox®, 12.5 % amitraz) after 15 years without its use on a property. Two in vivo (bovines treated with amitraz and submitted to tick counts, n = 20 animals) and one in vitro (adult immersion test, n = 40 ticks) assays were performed to determine product efficacy. The efficacy of the commercial formulation tested in the first in vivo trial ranged from 14.1 to 47.0%, and in the second from 3.6 to 35.1%, for the 28 days of the experiments. Efficacy for the in vitro trial was 47.38%. The dose recommended by the manufacturer of the product did not cause mortality to most of the ticks of this strain, and efficacy/resistance was not reverted or modified after 15 years (estimated 60 tick generations).

Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock.

Animal production has a key role in global economic development and food security. Ticks, specifically Rhipicephalus microplus cause substantial economic and health impacts on more than eighty percent of the world cattle population. Though synthetic acaricides play a major role in tick management, their injudicious usage has caused environmental pollution and also promote the establishment of multi-acaricide resistant tick populations which is a matter of great concern. To provide an effective tool for controlling these resistant ticks, the present work was aimed to develop safe and inexpensive antitick natural formulations. Our bioprospection studies of Ageratum conyzoides plant established it as a species potentially having strong acaricidal activity due to the presence of potent acaricidal phyto-chemicals. To develop a suitable antitick natural formulation, 41 samples/fractions/formulations were prepared from the dry powder of the whole aerial part of the A. conyzoides plant using different techniques and delivery matrices. The strongest antitick effect was recorded for formulation ACF6, which demonstrated 87 ± 6% mean mortality with 57 % inhibition of oviposition in treated female ticks. Ticks treated with the ACF6 formulation showed a significant (p < 0.001) reduction in cuticular protein (1.238 ± 0.01 mg/mL) as compared to control ticks (2.928 ± 0.01 mg/mL) but no significant difference in chitin content of treated ticks and control ticks was observed. The formulation was found safe in a rat model as no significant differences in biochemical and haematological parameters among treated and control rats were noted. Histopathological studies indicated no sign of hepatocellular necrosis and no significant changes in the weights of liver and spleen was recorded. The overall in vivo efficacy of the formulation was 85 % for experimentally infested cattle with direct mortality of more than 80 % within 96 h post-application. The lethal effect of the formulation was in the form of drying and dead ticks 1-2 d after application. The developed formulation has the potential to be adopted as an alternative tick control measure in an ecofriendly manner.

The molecular prevalence, distribution and risk factors associated with Babesia bigemina infection in Peninsular Malaysia.

Babesia bigemina is a tick-borne protozoan that affects cattle in almost all regions of the world. Despite its importance, there is no report of its prevalence in cattle using molecular detection methods in Peninsular Malaysia. This study describes the prevalence, distribution, and risk factors associated with B. bigemina infection using molecular diagnostic methods. Also, the species of ticks infesting cattle and the attitude of cattle farmers towards tick control in Peninsular Malaysia were studied. Blood samples were collected from 1045 cattle from 43 herds throughout the country, and were subjected to molecular studies to detect B. bigemina. Tick samples for entomological studies were also collected and identified. Epidemiological information of each cattle and farm were obtained using a well-structured questionnaire containing open-ended and closed-ended questions. Data were statistically analyzed using Univariate and Multivariate models. The 211-base pair of AMA-1 gene of B. bigemina was amplified and confirmed in 30.5 % (319/1045; 95 % CI = 27.8-33.4) of the sampled population, with the haemoprotozoan detected in all the sampled herds. Breed, age, physiological status, management type, rate of de-ticking, and closeness to human settlement were the risk factors significantly (p < 0.05) associated with the prevalence of B. bigemina in cattle. Rhipicephalus (Boophilus) microplus and Haemaphysalis bispinosa were the species of ticks collected from cattle, with the former been more prevalent. A large number of cattle farmers (12/43; 28 %) do not control ticks in their herds. The findings of this study will create baseline data on the epidemiology of the haemoprotozoan and control patterns of its tick vectors that will guide the government in enacting policies that will improve food security and the economy of the nation.

Genotyping amitraz resistance profiles in Rhipicephalus microplus Canestrini (Acari: Ixodidae) ticks from Punjab, India.

Acaricide resistance is one of the greatest threats to sustainable and effective control of vector ticks worldwide. The amitraz resistance status in cattle tick, Rhipicephalus microplus populations collected from 18 districts of Punjab in north-western India were characterized using bioassay and molecular assays. The modified larval packet test was used and the resistance factors (RF) against amitraz for the field populations were in the range of 0.36-4.85, indicating level I resistance status in ten populations. Characterization of a partial segment of the octopamine/tyramine (OCT/Tyr) receptor gene of R. microplus field populations from Punjab revealed a total of 18 nucleotide substitutions in the coding region out of which 5 were non-synonymous substitutions. Three of these non-synonymous substitutions (T8P, V15I and A20 T) were earlier reported in American and South African populations of R. microplus. Among the two single nucleotide polymorphisms (A22C-T8P; T65C-L22S) potentially linked to amitraz resistance in American, South African and Zimbabwean resistant populations, only the T8P substitution was recorded from the Barnala population. The PCR-RFLP assay using EciI restriction enzyme was used for genotyping of the larvae as homozygous resistant (RR), homozygous susceptible (SS) and heterozygous (SR). Genotyping of 514 larval DNA samples from 18 field populations revealed 92.8 % larval population as SR and the remaining 7.2 % as RR genotypes. The percentage of resistant alleles in the tick populations was 53.6 (range 50.0-57.2) indicating its moderate distribution in the region. The present study is the pioneer report establishing the hypothesis that amitraz-resistance is recessively inherited and heterozygous individuals show phenotypic susceptibility to the drug in the Indian tick populations.

A transcriptome and proteome of the tick Rhipicephalus microplus shaped by the genetic composition of its hosts and developmental stage.

The cattle tick, Rhipicephalus microplus, is a monoxenous tick that co-evolved with indicine cattle on the Indian subcontinent. It causes massive damage to livestock worldwide. Cattle breeds present heritable, contrasting phenotypes of tick loads, taurine breeds carrying higher loads of the parasite than indicine breeds. Thus, a useful model is available to analyze mechanisms that determine outcomes of parasitism. We sought to gain insights on these mechanisms and used RNA sequencing and Multidimensional Protein Identification Technology (MudPIT) to generate a transcriptome from whole larvae and salivary glands from nymphs, males and females feeding on genetically susceptible and resistant bovine hosts and their corresponding proteomes. 931,698 reads were annotated into 11,676 coding sequences (CDS), which were manually curated into 116 different protein families. Male ticks presented the most diverse armamentarium of mediators of parasitism. In addition, levels of expression of many genes encoding mediators of parasitism were significantly associated with the level and stage of host immunity and/or were temporally restricted to developmental stages of the tick. These insights should assist in developing novel, sustainable technologies for tick control.

Multiple orthonairoviruses including Crimean-Congo hemorrhagic fever virus, Tamdy virus and the novel Meram virus in Anatolia.

We conducted orthonairovirus RNA screening of 7043 tick specimens-representing 16 species-collected from various regions of Anatolia. In 602 pools, Crimean-Congo hemorrhagic fever virus (CCHFV) Europe 1 and 2 lineages were detected in seven pools (1.1 %) comprising Hyalomma marginatum, Hyalomma scupense, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks. In pools of Hyalomma aegyptium, we detected Tamdy virus (TAMV) and an unclassified nairovirus sequence. Next-generation sequencing revealed complete coding regions of three CCHFV Europe 2 (AP92-like) viruses, TAMV and the novel orthonairovirus, tentatively named herein as Meram virus. We further performed in silico functional analysis of all available CCHFV Europe 2, TAMV, Meram and related virus genomes. The CCHFV Europe 2 viruses possessed several conserved motifs, including those with OTU-like cysteine protease activity. Probable recombinations were identified in L genome segments of CCHFV and TAMV. Through phylogeny reconstruction using individual genome segments, Meram virus emerged as a distinct virus among species within the Orthonairovirus genus. It further exhibited conserved motifs associated with RNA binding, encapsidation, signal peptidase cleavage, post-translational modification, RNA-dependent RNA polymerase and OTU-like activities. Bole tick virus 3 was also detected in two pools with CCHFV reactivity. Hereby, we describe a novel tick-associated orthonairovirus, in a CCHFV-endemic region with confirmed TAMV activity. Human and animal health impact of these viruses need to be addressed.

Repellent and acaricidal activity of essential oils and their components against Rhipicephalus ticks in cattle.

Ticks, particularly the Rhipicephalus which are the most prevalent and invasive affect 80 % of the cattle population worldwide. Through transmission of pathogens, tick worry and physical damage to the hides, ticks cause economic loss of billions of dollars each year with 1 billion US dollars loss per annum reported only in Latin-America. These losses can be minimized only by successful management of Rhipicephalus ticks. Various strategies like chemical control, vaccination and biological control are aimed at control of Rhipicephalus ticks. There are some serious limitations associated with them like tick resistance, drug toxicity, antigenic variations etc. In contrast to these issues related with chemical tick control, the botanicals particularly the essential oils obtained from aromatic plants of medicinal importance are eco-friendly and non-toxic to most host. In recent years, essential oils-based control of cattle ticks has gained considerable attraction of scientists all over the world as depicted from this review. A comprehensive effort has been made to critically analyze the role of essential oils in controlling Rhipicephalus ticks with particular emphasis on the mode of action of bioactive compounds both as repellents and acaricides. Furthermore, we have pointed out the most important challenges which need to be addressed for development and commercialization of an essential oil based anti-tick product.

Acaricidal activity of Acmella oleracea (Asteraceae) extract against Rhipicephalus microplus: What is the influence of spilanthol?

The present study was carried out to evaluate and compare the acaricidal activity of different fractions of Acmella oleracea methanolic extract, containing 0.0 % (F1), 24.5 % (F2), 48.0 % (F3) and 100 % (F4) of spilanthol, on unfed larvae and engorged females from the same Rhipicephalus microplus population. To obtain these fractions, the crude extract was subjected to different extraction procedures using increasingly polarized solvents to isolate the spilanthol compound. The Larval Packet Test was used to evaluate acaricidal activity in unfed larvae at concentrations ranging from 0.2 to 25.0 mg/mL, while for engorged females, the Adult Immersion Test was performed at concentrations from 3.1 to 25.0 mg/mL. The F1 fraction showed no activity on unfed larvae, while a control percentage of 44.6 % was observed at a concentration of 25.0 mg/mL for engorged females. For unfed larvae, the F2 fraction resulted in 95.7 % mortality at a concentration of 1.6 mg/mL, with a control percentage of 92.7 % for engorged females at a concentration of 12.5 mg/mL. Fractions F3 and F4 had similar activity against unfed larvae, with mortality >84.0 % from the concentration of 0.8 mg/mL. This similarity between the fractions was also observed for engorged females from a concentration of 12.5 mg/mL, resulting a control percentage >94.0 %. These results demonstrate that the presence of spilanthol is an important factor for the acaricidal activity of A. oleracea extract. Fraction extracts with 24.5, 48 and 100 % of spilanthol have similar acaricidal activity on R. microplus.

A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides.

Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.

1,8-cineole and castor oil in sodium lauryl ether sulphate disrupt reproduction and ovarian tissue of Rhipicephalus (Boophilus) microplus.

Essential and fixed oils have been researched as alternatives to chemical acaricides. The activity of volatile compounds from essential oils (1,8-cineole, citral and eugenol) at 1.0% (w/v) and fixed oil (castor oil) at 0.3% (w/v) dissolved in 2.0% (v/v) dimethyl sulfoxide (DMSO) + 0.2% (w/v) Tween 80® was assessed against Rhipicephalus microplus using immersion tests. 1,8-cineole (29.0%) and castor oil (30.2%) had the highest reproductive inhibition rate. A second experiment was performed to verify the effect of the 1,8-cineole (10.0% w/v) and, or castor oil (0.3% w/v) on tick reproduction using different solubilizing agents. The highest reproductive inhibition was observed for the combination of 1,8-cineole/castor oil (94.1%) and 1,8-cineole in 2.0% (w/v) sodium lauryl ether sulphate (SLES) (92.8%). A third experiment showed morphological changes in R. microplus oocytes at different stages of development, as well as in pedicel cells. The most intense effects were observed when ticks were immersed in the formulation containing 1,8-cineole (10.0% w/v) and castor oil (0.3% w/v) dissolved in 2% (w/v) SLES. These findings highlight the potential of this formulation as an alternative for managing cattle ticks as their cytotoxic effects can reduce R. microplus reproductive success.

Clinical safety of lauric acid for cattle and its in vitro and in vivo efficacy against Rhipicephalus microplus.

The aim of the present study was to test the in vitro acaricidal activity of saturated fatty acids (hexanoic, octanoic, decanoic, lauric, myristic, palmitic, octadecanoic, eicosanoic, docosanoic and tetracosanoic) against Rhipicephalus microplus and select a candidate compound for the subsequent determination of its clinical safety for mice and bovines as well as its in vivo efficacy (ethical clearance number 507/2013). None of the compounds exhibited in vitro larvicidal effectiveness, but acaricidal effectiveness was greater than 95 % in the adult immersion test at 40 mg/ml (hexanoic, octanoic, decanoic, lauric, myristic, palmitic and eicosanoic acids). After a second AIT evaluation of serial concentrations of the fatty acids, lauric and myristic acids were selected for the safety and in vivo efficacy assays. No adverse effect was found in the local lymph node assay in mice treated with lauric or myristic acid. Moreover, no clinical signs of systemic poisoning or dermatological, hematological or biochemical abnormalities were found in cattle after the topical application of 1 % lauric acid. In the dose determination test, the 1% solution of this compound exhibited 86% efficacy in cattle naturally infested by a field population of Rhipicephalus microplus susceptible to all chemical groups, except synthetic pyrethroids. The efficacy of 1 % lauric acid was 53.4 % in the dose confirmation test performed on another herd with a field R. microplus population resistant to all chemical groups of acaricides. In conclusion, fatty acids are potential bioactive compounds for the control of R. microplus. Topically applied lauric acid (C12) exhibits in vivo acaricide activity against adults, nymphs and larvae of R. (B) microplus and is safe for cattle.