In-vitro efficacy of a botanical acaricide and its active ingredients against larvae of susceptible and acaricide-resistant strains of Rhipicephalus (Boophilus) microplus Canestrini (Acari: Ixodidae).

Ticks and tick-borne diseases are a major constraint for a sustainable cattle industry in the tropical and subtropical regions of the world. The development of resistance to most of the commonly used acaricides has led to an assessment of herbal products for acaricidal activity as an eco-friendly tick control alternative. A botanical product, Essentria® IC-3 insect concentrate containing rosemary oil (10%), geraniol (5%) and peppermint oil (2%), acts on target pests by blocking octopamine. Essentria® IC-3 and its active components were evaluated for larvicidal activity against several susceptible and acaricide-resistant strains of the cattle fever tick, Rhipicephalus (Boophilus) microplus Canestrini by Larval Packet test using 14-21 d old unfed larvae. The efficacy was assessed by measuring percent larval mortality and estimating lethal concentrations at 50% (LC50) and 95% (LC95) with 95% confidence limits (CL) using probit analysis. The LC50 and LC95 (95% CL) values for Essentria® IC-3 against the susceptible strain were estimated as 0.647% (0.59-0.69) and 1.033% (0.94-1.19), respectively, whereas, LC50 and LC95 values for other strains were variable, ranging from 0.597-1.674% and 0.927-2.236%, respectively. Among the various active ingredients, the larvicidal property of Essentria® IC-3 seem to be attributable mainly to geraniol and the LC50 and LC95 (95% CL) values for geraniol against the susceptible Deutch strain were estimated as 0.656% (0.61-0.69) and 1.114% (1.03-1.25), respectively. The comparison of LC50 and LC95 values of acaricide-resistant strains showed susceptibility comparable to Deutch against geraniol except for the Las Palmas strain. We report a low level of resistance in some of the acaricide-resistant strains against the herbal acaricide in the cattle tick for the first time, possibly due to cross-resistance to chemical acaricides.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.

The novel isoxazoline ectoparasiticide fluralaner: selective inhibition of arthropod γ-aminobutyric acid- and L-glutamate-gated chloride channels and insecticidal/acaricidal activity.

Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.

Acaricidal properties of Ricinus communis leaf extracts against organophosphate and pyrethroids resistant Rhipicephalus (Boophilus) microplus.

Indian cattle ticks have developed resistance to commonly used acaricides and an attempt has been made to formulate an ecofriendly herbal preparation for the control of acaricide resistant ticks. A 95% ethanolic extract of Ricinus communis was used to test the efficacy against reference acaricide resistant lines by in vitro assay. In in vitro assay, the extract significantly affects the mortality rate of ticks in dose-dependent manner ranging from 35.0 ± 5.0 to 95.0 ± 5.0% with an additional effect on reproductive physiology of ticks by inhibiting 36.4-63.1% of oviposition. The leaf extract was found effective in killing 48.0, 56.7 and 60.0% diazinon, deltamethrin and multi-acaricide resistant ticks, respectively. However, the cidal and oviposition limiting properties of the extract were separated when the extract was fractionated with hexane, chloroform, n-butanol and water. The HPTLC finger printing profile of R. communis leaf extract under λ(max.) - 254 showed presence of quercetin, gallic acid, flavone and kaempferol which seemed to have synergistic acaricidal action. In vivo experiment resulted in 59.9% efficacy on Ist challenge, however, following 2nd challenge the efficacy was reduced to 48.5%. The results indicated that the 95% ethanolic leaf extract of R. communis can be used effectively in integrated format for the control of acaricide resistant ticks.

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse.

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD(65) and GAD(67) genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD(65) and GAD(67) resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.

Serial analysis of gene expression in the southern cattle tick following acaricide treatment of larvae from organophosphate resistant and susceptible strains.

Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos. Serial analysis of gene expression (SAGE) was used to analyse differential gene expression in response to OP treatment and to compare the responses of OP-treated and -untreated resistant and susceptible tick larvae. An R. microplus Gene Index was used as an EST database to identify genes which corresponded to SAGE tags whose abundance changed in response to acaricide exposure. Relative quantitative RT-PCR was used to confirm the differential expression results from the SAGE experiments. Of particular interest is a SAGE tag which corresponds to a cytochrome P450-like EST in the Gene Index which was more abundant in untreated OP resistant larvae compared to untreated OP susceptible larvae. This SAGE tag was also more abundant in OP resistant larvae treated with OP compared to OP susceptible larvae treated with OP.