Current perspectives and difficulties in the design of acaricides and repellents from plant-derived compounds for tick control.

Ticks and tick-borne diseases have gained increasing attention in recent years due to their impact on public health and significant losses in livestock production. The use of synthetic compounds for tick control is becoming problematic, mainly due to the resistance to commercially available products as well as their toxicity. Therefore, new alternative control methods are required. For this purpose, plant-derived extracts may be considered as effective repellents and/or acaricides. The present literature review focuses on studies evaluating the acaricidal and repellent activity of plant-derived extracts and plant secondary metabolites. We also noted recent advances in protein-ligand-docking simulation to examine the possible toxic effect of natural chemical compounds on ticks. In conclusion, plant-derived repellents/acaricides can be effective against ticks, especially in rural areas and livestock farms.

Natural Products as Mite Control Agents in Animals: A Review.

Mites have been a persistent infectious disease affecting both humans and animals since ancient times. In veterinary clinics, the primary approach for treating and managing mite infestations has long been the use of chemical acaricides. However, the widespread use of these chemicals has resulted in significant problems, including drug resistance, drug residues, and environmental pollution, limiting their effectiveness. To address these challenges, researchers have shifted their focus towards natural products that have shown promise both in the laboratory and real-world settings against mite infestations. Natural products have a wide variety of chemical structures and biological activities, including acaricidal properties. This article offers a comprehensive review of the acaricidal capabilities and mechanisms of action of natural products like plant extracts, natural compounds, algae, and microbial metabolites against common animal mites.

Assessment of weight gain and control of parasitism by Rhipicephalus microplus in dairy cattle in the field using organosynthetic parasiticide, phytotherapics and homeopathy / Avaliação do ganho de peso e controle do parasitismo por Rhipicephalus microplus em bovinos leiteiros a campo usando parasiticida organosintético, fitoterápicos e homeopatia

The goal of this research was to compare the effect of the following products on dairy cattle, parasitized by Rhipicephalus microplus: organosynthetics - Clorfenvinfós and Ivermectina (OG); phytotherapics - Eucalyptus oil (EG) and Neem cake (NG); Homeopathy (HG); The control group (CG) received no treatment. Infestation was by R. microplus (MIC) and weight gain (GP) were evaluated. The study included 60 animals (5 groups) from ­ Dutch / Zebu, aged between 25-44 months and initial weight between 211-477kg. EG and NG showed MIC 84.9% and 14.0% greater than CG, respectively (P <0.05; P = 0). HG and OG had MIC less than CG 24.4% and 16.9%, respectively (P<0.05; P <0.05). EG, NG and OG obtained GP lower than CG in 7.9%, 8.9% and 8.06% (P <0.05) respectively. The HG GP was 4.9% higher (P <0.05) than CG. This was the first research to prove parasitism control by R. microplus in a field test, using homeopathy. The methodology for choosing Homeopathy for the control of enzooties, developed and tested for the first time in this study, proved to be adequate and efficient, opening the possibility for establishing a new methodology for strategic control of parasitism by R. microplus.(AU)

O objetivo desta pesquisa foi comparar o efeito dos seguintes produtos sobre bovinos leiteiros, parasitados por Rhipicephalus microplus: organossintéticos - clorfenvinfós e ivermectina (OG); fitoterápicos - óleo de eucalipto (EG) e torta de Neem (NG) e homeopatia (HG). O grupo controle (CG) não recebeu tratamento. Foram avaliados infestação por R. microplus (MIC) e ganho de peso/grupo (GP) em 60 animais (5 grupos), a partir de ­ holandês/zebu, com idade entre 25 e 44 meses e peso inicial entre 211 e 477kg. EG e NG apresentaram MIC 84,9% e 14,0% maior que CG, respectivamente (P<0,05; P=0). HG e OG apresentaram MIC menor que CG 24,4% e 16,9%, respectivamente (P<0,05; P<0,05). EG, NG e OG obtiveram GP menor que CG em 7,9%, 8,9% e 8,06% (P<0,05), respectivamente. O GP do HG foi 4,9% maior (P<0,05) que o CG. Esta foi a primeira pesquisa a comprovar controle do parasitismo por R. microplus em teste a campo, usando homeopatia. A metodologia para escolha de medicamentos homeopáticos para controle de enzootias, desenvolvida e testada pela primeira vez neste estudo, mostrou-se adequada e eficiente, abrindo a possibilidade para estabelecimento de nova metodologia para controle estratégico do parasitismo por R. microplus.(AU)

Exposure of Rhipicephalus sanguineus sensu lato Latreille, 1806 (Acari: Ixodidae) to hexane extract of Acmella oleracea (Jambu): semi-engorged and engorged ticks.

The present study analyzed the efficacy of hexane extract of Acmella oleracea against Rhipicephalus sanguineus sensu lato. After exposure of R. sanguineus s. l. female ticks to 14 different concentrations of the hexane extract of A. oleracea, the LC50 was established as 18.135 mg/mL (limits: 16.251-22.675). The effects of the extract were more significant in the ovary of the semi-engorged females, that presented damages in most oocytes. Since such changes would not be repaired, the oocytes could not advance to further stages of development (I-V), thereby causing the inhibition of ovary development, interruption of vitellogenesis, oocyte death and, consequently, infertility. The semi-engorged females also presented extensive damages in the midgut cells, which would prevent (totally or partially) these cells from functioning properly (blood intake and release of nutrients), impairing the ectoparasite nutrition and the viability of the individual. Thus, the hexane extract of A. oleracea can be considered as a promising alternative to chemical control of pests of medical and veterinary importance, due to its efficient acaricidal activity and lesser environmental impact, when used against female ticks in the middle feeding stage.

Lithium chloride outperformed oxalic acid sublimation in a preliminary experiment for Varroa mite control in pre-wintering honey bee colonies.

Since lithium salts were demonstrated to be very effective for the potential control of Varroa destructor, a highly detrimental parasite of honey bee (Apis mellifera), no studies have been reported on their comparison with any commonly used varroicides in commercial bee colonies. In this study we compared the effectiveness of lithium chloride to that of oxalic acid, a widely used miticide. The results of the present study confirm that lithium has superior efficacy to oxalic acid sublimation both as a main or a supplementary pre-wintering treatment at moderate infestation levels, restricted to certain pre-wintering conditions. Considering its easy implementation in apicultural practice and its twofold mode of action, trickling would be the preferred way of administration after the use of lithium salts as varroicides is authorised.

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.

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.

Synthetic Pyrethroid, Natural Product, and Entomopathogenic Fungal Acaricide Product Formulations for Sustained Early Season Suppression of Host-Seeking Ixodes scapularis (Acari: Ixodidae) and Amblyomma americanum Nymphs.

We compared the ability of product formulations representing a synthetic pyrethroid acaricide (Talstar P Professional Insecticide), a natural product-based acaricide (Essentria IC3), and an entomopathogenic fungal acaricide (Met52 EC Bioinsecticide) to suppress Ixodes scapularis Say and Amblyomma americanum (L.) nymphs when applied following USEPA approved manufacturers' label recommendations for tick control using hand-pumped knapsack sprayers before the beginning of their seasonal activity period in the spring. We applied Met52 EC Bioinsecticide (11% Metarhizium anisopliae Strain F52) to five 100 m2 plots (10.6 ml AI/plot) in mid-April 2020. Two weeks later at the end of April 2020, we treated an additional five 100 m2 plots each with either Talstar P Professional Insecticide (7.9% bifenthrin @ 2.5 ml AI/plot) or Essentria IC3 (10% rosemary oil, 5% geraniol, and 2% peppermint oil @ 86.6 ml AI/plot). Weekly sampling of all plots through the end of June 2020 showed that both Met52 EC Bioinsecticide and Essentria IC3 failed to maintain a 90% suppression threshold for I. scapularis, compared to control plots, and required two additional applications over the course of the trial. In contrast, Talstar P Professional Insecticide suppressed 100% of I. scapularis nymphs and ≥96 and 100% of A. americanum nymphs and adults, respectively. Such pre-season applications of synthetic pyrethroids significantly reduce the early season acarological risk for exposure to host-seeking ticks as well as the frequency of acaricide applications.

Ultrasound-assisted extraction extracts from Stemona japonica (Blume) Miq. and Cnidium monnieri (L.) Cuss. could be used as potential Rhipicephalus sanguineus control agents.

Nicotiana tabacum, Stemona japonica, and Cnidium monnieri are common plants that are widely used for their anti-parasitic properties. The purpose of this study was to evaluate the acaricidal activity of extracts from these plants against the brown dog tick, Rhipicephalus sanguineus. A composition analysis of crude extracts by GC-MS was conducted to discover compounds with acaricidal effects. The toxicity of extraction against the engorged nymphs of R. sanguineus was evaluated by an immersion test. The results showed that the crude extracts of S. japonica and C. monnieri in varying ratios, concentrations, and from different extraction methods, had a killing effect on R. sanguineus. Lethality reached 76.67% ± 0.04410 when using a 1:1 extract of S. japonica:C. monnieri in 75% ethanol with ultrasonic extraction; the crude extract was determined at a concentration of 0.5 g/mL. GC-MS results showed that osthole and 5-hydroxymethylfurfural (5-HMF) are the main components of the extract. These results suggested that ultrasound-assisted extraction (UAE) extracts contained acaricidal components acting against R. sanguineus, which may result in the development of effective extracts of S. japonica and C. monnieri as a source of low-toxicity, plant-based, natural acaricidal drugs.

Repellency effect of Pilocarpus spicatus A. St.-Hil essential oil and nanoemulsion against Rhipicephalus microplus larvae.

Rhipicephalus microplus, the cattle tick, is a major cause of economic losses in bovine production. Due to the widespread acaricidal resistance to commercially available products, as well as their toxicity and environmental impact, alternative control methods are required. Nanoformulations produced from plant extracts as bioactive substances are very promising as innovative acaricidal agents. Thus, the aim of this study was to evaluate the in vitro repellent activity of Pilocarpus spicatus essential oil and its nanoemulsion against R. microplus, using larval repellent test (RT). The essential oil was extracted by hydrodistillation, using a Clevenger-type apparatus. The nanoemulsion was prepared with 5% essential oil, 5% tween 80, and 90% water, using the phase inversion method (50 mg/mL). Limonene was the major component (46.8%) of the essential oil, as determined by gas chromatography-mass spectrometry (GC/MS) and confirmed by flame ionization detection (GC/FID). According to the RT results, the essential oil had a repellent activity greater than 69%, from concentrations of 3.12 mg/mL (69.81 ± 10%) to 50 mg/mL (98.10 ± 0.6%), whereas the nanoemulsion at 50 mg/mL presented repellent activities of 97.14 ± 1.37% and 97.89 ± 0.52% 6 and 10 h after treatment, respectively. These values regarding to total repellency were very close to those calculated for mortality corrected by Abbott's formula. The phase inversion method preserved the chemical and physical characteristics of the essential oil since both reached an equal repellent effect at the same concentration. Therefore, P. spicatus essential oil and nanoemulsion had excellent repellent activities against R. microplus larvae, demonstrating its potential for future use as an alternative for tick control.

Control of Dermanyssus gallinae (De Geer 1778) and other mites with volatile organic compounds, a review.

Dermanyssus gallinae (De Geer 1778), commonly named the poultry red mite (PRM), is considered to be the most harmful ectoparasite in poultry farms in Europe. This species feeds on the blood of laying hens, but spends most of its time hidden in cracks and crevices around hen nests. To control PRM populations in poultry houses, chemical pesticides are currently used; however, concern is growing regarding the harmful residues found in eggs and hens, along with the increased resistance of mites against several compounds. Alternatives to synthetic compounds are now being explored, including vaccines, biological control, physical control and semiochemical control based on the chemical ecology of PRM. This review focused on the different volatile organic compounds (VOCs) identified from D. gallinae and other mite species that have been discovered to control them. Pheromones (aggregation pheromone, sex pheromone and alarm pheromone) and kairomones promoting attraction behaviour in D. gallinae and other mite species are presented, while VOCs from essential oils and plant extracts with repellent properties are also explored. Finally, devices using VOCs on PRM in the field are described, with devices that have been tested on other Acari species being mentioned as potential directions for the future control of PRM.

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.

Botanical acaricides and repellents in tick control: current status and future directions.

Ticks are obligate blood-sucking ectoparasites and notorious as vectors of a great diversity of, in many instances, zoonotic pathogens which can cause considerable damage to animal and human health. The most commonly used approach for the control of ticks is the application of synthetic acaricides. However, the negative impacts of synthetic acaricides on the treated animals and the environment, in addition to its documented role in the development of resistance has led to the search for safer and more environmentally friendly alternative methods without compromising efficacy. An emerging promising approach for the control of ticks which has attracted much attention in recent years is the use of botanicals. Indeed, botanicals have been widely reported to show diverse effects and great potential as tick repellent and control. Although several excellent reviews have previously focused on this topic, studies on the exploration and application of botanicals to control ticks have expanded rapidly. Herein, we provide an update on the current understanding and status of botanical acaricides and repellents in tick control using recently published articles between 2017 and 2019. We also discuss the challenges and future directions in the application of botanicals in tick control, with a view of providing important clues for designing new integrated tick control methods.

Utilisation of indigenous knowledge to control ticks in goats: a case of KwaZulu-Natal Province, South Africa.

Local indigenous knowledge (IK) informs decision-making about fundamental aspects of life. The inclusion of indigenous knowledge is increasingly becoming a topical subject to enhance livestock veterinary care. The objective of the current study was to explore the extent of utilisation of indigenous knowledge to control ticks in goats. There was an association (P < 0.05) between the use of IK and gender, with males using IK (76.58%) more than females. The association between age distribution and IK use was (P < 0.05); however, farmers above the age of 50 years were using IK more than all group ages. Farmers ranked the purposes of using IK differently (P < 0.05). Ectoparasites were ranked as the most important constraint limiting goat productivity. Ticks were ranked as the most important external parasites. Amblyomma tick species were ranked as the most important amongst the tick species, followed by Rhipicephalus evertsi evertsi ticks. A significant population of farmers (80.7%) are dependent on the use of tick sprays, whereas others used injections (3.3%). Cissus quadrangularis L. (Inhlashwana) was singled out as the most used ethno-veterinary plant to control ticks with a frequency of (64%), followed by Gomphocarpus physocarpus E. Mey (Uphehlacwathi) (55.9%). The probability of keeping goats in wet rangelands (P < 0.05) was 3.04 times more likely to influence the extent of IK use compared to their contemporaries in the dry rangeland. Male farmers keeping goats (P < 0.01) were 2.95 more likely to influence the extent of use of IK than females. The type of rangeland, gender, age, residing on farm and also having the herbalist in the locality were the most common factors that influenced the extent of IK utilisation.

Acaricidal efficacy of Calotropis procera (Asclepiadaceae) and Taraxacum officinale (Asteraceae) against Rhipicephalus microplus from Mardan, Pakistan.

Medicinal plants are used by traditional folk healers, modern physicians, and veterinarians as an alternative to conventional drugs to treat a wide range of disorders including parasitic diseases. Some compounds from these plants have been shown to have acaricidal activity and repel arthropods. The cattle tick Rhipicephalus microplus is one of the most destructive pests to the livestock industry in tropical and subtropical parts of the world. The potential to develop herbal acaricides to control R. microplus infestations is critical in maintaining cattle herd productivity, reducing economic losses, and curtailing the overuse of synthetic chemical acaricides. Calotropis procera, the apple of Sodom, and Taraxacum officinale, the common dandelion, were evaluated for acaricidal activity against R. microplus larvae and adults in vitro. Both plant species tested are common indigenous species of Pakistan where R. microplus infestations are widespread across livestock species including cattle, sheep, and goats. Whole-plant extracts derived from C. procera and T. officinale significantly reduced the index of egg laying (P < 0.01) and increased the percent inhibition of oviposition of adult female ticks at a concentration of 40 mg/mL when assessed by the adult immersion test (AIT). Calotropis procera and T. officinale treatments at the same concentration also resulted in larval mortality of 96.0% ± 0.57 and 96.7% ± 0.88, respectively, as measured using the larval packet test (LPT). An increasing range of extract concentrations was tested to determine the LD50 and LD90 for C. procera, 3.21 and 21.15 mg/mL, respectively, and T. officinale, 4.04 and 18.92 mg/mL, respectively. These results indicate that further studies are warranted to determine the relative contribution of individual phytochemicals from whole-plant extracts on acaricidal activity. This information will guide the design of further acaricidal efficacy tests using livestock infested with R. microplus.

Toxicity and effects of essential oils and their components on Dermanyssus gallinae (Acari: Dermanyssidae).

The acaricidal activity of 30 essential oils against the poultry red mite, Dermanyssus gallinae, female adults and behavioral responses of the mites to these essential oils were investigated. Cinnamon bark oil and clove bud oil showed 100% acaricidal activity after 24 h in the 1.3 µg/m2 treatment. In addition, four components in cinnamon bark oil and three components in clove bud oil were identified using gas chromatography-mass spectrometry. Cinnamon bark oil showed the highest LD50 value among all of the components, and eugenol showed 0.97-fold higher relative toxicity (RT) than the other components of clove bud oil. The fumigant effects of both essential oils and their seven components were observed using a vapor phase toxicity bioassay. All the substances showed repellent activity except for cinnamyl acetate, which did not show any repellent response even in the > 10 µg treatment. In the experiment using the T-tube olfactometer with the 10 µg treatment of each substance, D. gallinae female adults responded to all the substances except cinnamyl acetate. However, eugenol and eugenol acetate showed an attractant effect after 240 and 120 min of treatment, respectively. These results suggest that the two studied essential oils and their components may be used as control agents against D. gallinae.

Evaluation of Tagetes patula (Asteraceae) as an ecological alternative in the search for natural control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle tick Rhipicephalus microplus is a great sanitary problem and causes huge losses to livestock, being a vector of important diseases. The aim of this work was to verify the action of plant-derived material obtained from Tagetes patula on the life cycle of R. microplus, as well as to analyze the action of these extracts on ovary cells of engorged females. In the Adult Immersion Test, the crude ethanolic extract (TpEtOH) showed an efficacy of 99.2% (LC50 = 18.60 mg mL-1). From the repellency test, it was found that both the TpEtOH and essential oil were 100% efficient on the larval stage. In addition, in the Larval Package Test, a mortality of 98.37% was obtained with TpEtOH (LC50 = 3.798 mg mL-1). From the microscopic analysis of ovary cells, morphological changes in the chorion and cytoplasm of oocytes were observed, with vacuolization around the germinal vesicle and disorganization of the pedicular cells, suggesting an interference in the normal embryogenic formation of the larvae. These results demonstrate that T. patula extracts interfere at all stages of development of the ixodid, from eggs to adults forms, and have a pronounced repellent effect. In addition, the results of the cytotoxicity assays performed on keratinocytes, as well as previous information on oral and dermal acute toxicity (LD50 > 4000 mg kg-1), attest that T. patula can be a safe alternative for ectoparasitic control, representing an alternative for development of a formulation used to help control populations of ticks in the field.

Development of essential oil-based phyto-formulations to control the cattle tick Rhipicephalus microplus using a mixture design approach.

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most important ectoparasites for livestock in tropical and subtropical areas around the world. This tick economically impacts cattle production by reducing weight gain and milk production. Moreover, it is a vector of pathogens causing diseases such as babesiosis and anaplasmosis. Conventional tick control relies mainly on the use of chemical acaricides; however, their intensive use has led to the rapid appearance of resistant tick populations. It is therefore necessary to look for alternative tick control products. In that sense, plant extracts might represent a promising source of new acaricides. Previously, we reported a significant acaricide effect of essential oils from selected plant species. In the present study, we used a mixture design approach to develop phyto-formulations by combining individual essential oils. We produced several mixtures at 10% containing different proportions of individual essential oils (ranging from 0 to 1) from cinnamon (Cinnamomum zeylanicum), cumin (Cuminum cyminum) and allspice (Pimenta dioica) and tested their acaricidal activity against R. microplus ticks by means of larval packet test (LPT) and adult immersion test (AIT) assays. The optimal mixture predicted against R. microplus was composed of 66%, 17% and 17% of essential oils from C. zeylanicum, C. cyminum and P. dioica, respectively. We generated an estimated response surface contour plot that estimates 80%-100% acaricidal efficacy. In the optimal mixture 34 compounds were identified, which represent 98.65% of the total composition, with cinnamaldehyde (37.77%), ß-caryophyllene (13.92%), methyl eugenol (12.27%) and cuminaldehyde (8.99%) being the major components. Next, we developed emulsions by combining the optimal mixture with several surfactants and determined particle size, Zeta potential, stability and bioactivity. Emulsions containing 2% and 5% Tween 20 or Tween 80 remain stable after 14 days at 54 °C. Finally, optimized emulsion retained a high acaricidal activity against larval and adult R. microplus ticks. Taken together, our findings showed the usefulness of mixture design method for the development of essential oil mixtures with potent acaricidal activity. These formulations have the potential to successfully control tick infestations.

Sublethal concentrations of acetylcarvacrol strongly impact oocyte development of engorged female cattle ticks Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae).

Rhipicephalus microplus, commonly known as southern cattle tick, causes huge economic losses in the cattle industry. Its infestation affects the production of meat and milk and causes discomfort to hosts. In addition, it is the vector of Babesia spp. and Anaplasma spp. The most frequent method used to control these parasites is through synthetic acaricides. However, their indiscriminate use can be toxic for hosts and environment as well as cause selection of resistant ticks. Plant extracts and essential oils emerge as promising alternatives to manage tick infestation. Carvacrol, an aromatic monoterpene extracted from plants, has recognized antimicrobial, antioxidant, insecticidal, repellent and acaricidal activities. Acetylation of carvacrol is believed to enhance its nematicidal and acaricidal activities and to decrease its toxicity to hosts. Thus, the aim of this study was to evaluate the effect of different concentrations of acetylcarvacrol in the morphology of ovaries of engorged R. microplus ticks. The most remarkable morphological alterations found in the female germ cells were irregular and thicker chorion, decreasing in size and irregular shape of female germ cells (oocytes), cytoplasmatic vacuolization as well as ring-shaped nucleoli. These alterations were analyzed through a semi-quantitative method proposed in this study for ixodid ticks. Treatment group IV, which was exposed to 4.5 µL/mL of carvacrol acetate, showed the most significant alterations, and it was also statistically different when compared to control groups. Therefore, sublethal concentrations of acetylcarvacrol demonstrated to impact the reproductive system of R. microplus by causing several damages in the female germ cells. This would hinder the generation of new individuals, probably contributing for a long-term control of tick infestation.

In vitro bioassays used in evaluating plant extracts for tick repellent and acaricidal properties: A critical review.

Ticks are haematophagous arthropods which rank closely with mosquitoes in their capacity to transmit disease pathogens of importance to animals and humans. Current control of ticks is based on the routine use of synthetic chemicals administered to animals or their environment. However, years of use and overuse of these chemicals have resulted in the development of resistance in these parasites and negative environmental impacts, hence the need for cheaper, safer and more environmentally friendly alternatives with alternate modes of action. There has been a large interest in using plants for these purposes. Peer-reviewed articles on plants evaluated for their tick repellent and/or acaricidal activities against immature and adult stages of ticks were collected from nine scientific databases with the aim of reviewing the bioassays employed. Search words included "acaricidal", "tick repellent", "antitick assays" and "phytomedicine". Many different methods were used to determine repellency and acaricidal activity. These include, among a few others, petri dish, tick climbing, olfactometer, larval packet and immersion bioassays. Tick climbing repellency and adult immersion bioassays were most commonly used. Ethanol was the most widely used solvent and Rhipicephalus (Boophilus) microplus was the most commonly studied tick across all the reviewed papers. It is unclear whether the outcome of these experiments on a one-host tick can be applied to other species of ticks that infest animals and humans. Also, most of the assays on repellency did not discriminate between olfaction and tactile chemoreception-based repellency and though some of the observed methods were similar, results differ significantly. These aspects will need further evaluation. Standardized laboratory methods are required to enable the valid comparisons between results from different laboratories.