Expressed sequenced tags and new genes from the cattle tick, Boophilus microplus

We used the expressed sequenced tags (ESTs) approach to study the genome of the cattle tick Boophilus microplus. One hundred and forty-two of our 234 unique ESTs were from genes not previously identified from ticks, mites or any other arachnids. The largest class of identified ESTs (29%) was from genes involved in transcription and translation. Ninety-one ESTs (39% of all ESTs) did not match any sequences in international databases; some of these may be specific to ticks. Thirteen percent of our ESTs were from ribosomal proteins and two ESTs were for genes implicated in resistance to pesticides. (C) 1998 Chapman & Hall Ltd.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Identification and characterisation of a cytochrome P450 gene and processed pseudogene from an arachnid: the cattle tick, Boophilus microplus

We isolated and sequenced the first known cytochrome P450 gene and pseudogene from an arachnid, the cattle tick, Boophilus microplus. Bath the gene and pseudogene belong to the family CYP4, but a new subfamily, CYP4W, had to be created for these genes because they are substantially different to other CYP4 genes. The gene, CPP4W1, has greatest homology with CYP4C1 from a cockroach, Blaberus discoidalis. The predicted molecular weight of the protein encoded by CYP4W1 (63 KDa) is greater than that of the other CYP4 genes. The pseudogene, CYP4W1P, is probably a processed pseudogene derived from the functional gene CYP4W1. This is only the third CYP processed pseudogene to be identified. The pseudogene is 98% identical to the functional gene, CYP4W1, therefore we hypothesise that this pseudogene evolved recently from the functional gene. The CYP4 genes from arthropods have diverged from each other more than those of mammals; consequently the phylogeny of the arthropod genes could not be resolved. (C) 1999 Elsevier Science Ltd. All rights reserved.

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B, microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations

The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. (C) 2009 Elsevier Ltd. All rights reserved.

Larvicidal potential of Sapindus saponaria to control the cattle tick Boophilus microplus

The objective of this study was to evaluate the larvicidal potential of a crude ethanol extract (CEE) of soapberry Sapindus saponaria stem peel on the cattle tick Boophilus microplus. Tick larvae obtained by incubating engorged females, collected from naturally infested cattle, were placed in envelopes of filter paper impregnated with different concentrations of CEE in the test group, and distilled water in the control group. Four repetitions were made with each solution (n>120). Mortality was observed after 48 hours. Lethal concentration values of 1,258 ppm (LC50) and 6,360 ppm (LC99) were obtained.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Morphological, histological, and ultrastructural characterization of degenerating salivary glands in females of the cattle-tick Rhipicephalus (Boophilus) microplus (CANESTRINI, 1887) (Acari : Ixodidae)

The present study describes the morphology and ultrastructure of the salivary glands of semi-engorged females of the southern cattle-tick Rhipicephalus (Boophilus) microplus. The acini that compose these glands, at that specific feeding stage, show cells featuring degenerative process of the salivary glands, such as: vacuolated cytoplasm, condensed chromatin, fragmented nuclei, and presence of apoptotic bodies. In addition, the presence of microorganisms was detected, with morphology typical of protozoa, inside these organs. (c) 2005 Elsevier Ltd. All rights reserved.

Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus microplus (Canestrini, 1887) (Acari : Ixodidae)

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-tick Boophilus microplus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a lumen delimitated by a wall of small epithelial cells with rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of the cellular components would be undergoing a degenerative process and/or reabsorption. (c) 2004 Elsevier B.V. All rights reserved.

Esterase profile in a pyrethroid-resistant Brazilian strain of the cattle tick Boophilus microplus (Acari, Ixodidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of macrocyclic lactone acaricides (MLs). Technical and commercial MLs were used in larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT). Tn LIT and AIT the toxicity of MLs was much higher than for LPT. In the AIT, diluting the injectable formulation of MLs in water was as effective as dilution in ethanol + Triton X-100. LC50, LC99.9 and 95% confidence limits were determined so that a discriminating dose (DD) could be set for larval and adult tests in order to diagnose potential resistance to MLs in field samples of the tick. These DDs are for Australian strains of B. microplus and may not be suitable for other strains until further work is carried out.The value of these diagnostic tests can only be verified if or when resistance to MLs emerges in ticks. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Gene discovery in Boophilus microplus, the cattle tick - the transcriptomes of ovaries, salivary glands, and hemocytes

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.