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Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13 000 probes, having been derived from a previously described R. microplus gene index (BmiGI Version 2; Wang et al., 2007 ). Relative quantitative reverse transcriptase-PCR, real time PCR, and serial analysis of gene expression data was used to verify microarray data. Among the differentially expressed genes with informative annotation were legumain, glutathione S-transferase, and a putative salivary gland-associated protein. [ABSTRACT FROM AUTHOR]

Genome sizes and the organization of repetitive DNA were determined in the hard ticksIxodes scapularisandBoophilus microplususing reassociation kinetics. TheI. scapularisgenome contains∼2.15 pg (2.1 × 103 Mbp) of DNA and consists of no foldback (FB), 27% highly repetitive (HR), 39% moderately repetitive (MR), and 34% unique DNA. TheB. microplusgenome contains 7.5 pg (7.1 × 103 Mbp) DNA, and consists of 0.82% FB, 31% HR, 38% MR, and 30% unique DNA. In both species, repetitive sequences occur in a mixture of long and short period interspersion but most (65–80%) of the DNA follows a pattern of short period interspersion. Genome size and organization in the three tick species so far examined are distinct from other arthropods in having a greater proportion of MR, a lower proportion of unique and HR DNA of very low sequence complexity. [ABSTRACT FROM AUTHOR]

Resistance to synthetic pyrethroids (SP) was first recorded in buffalo flies in Australia in 1980, associated with previous use of DDT and fenvalerate. By the 1990s, resistance was widespread. Resistance to SP in the related horn fly of the Americas is associated with kdr and super-kdr mutations in a gene encoding for a voltage-gated sodium channel. We describe 7-20-fold resistance to SP in buffalo flies from south-east Queensland, present evidence of flies that are heterozygous resistant at the kdr locus and show an increase in the frequency of the resistant allele 1 month after treatment of cattle with SP., (© 2011 The Authors. Australian Veterinary Journal © 2011 Australian Veterinary Association.)

The southern cattle tick, Rhip,cephalus (Boophilus) microplus, the horn fly, Haematobia irritans, and the New World screwworm, Cochl,omyia hominivorax, are economically important parasites of cattle throughout the world. Understanding the biology and genomics of these pests is critical to developing novel control methods. We developed EST sequence databases for these parasites and used bioinformatic analysis to identify conceptual open reading frames and assigned Gene Ontology (GO) terms to those database transcripts that had informative BlasIX hits. Bloinformatic analysis showed that a large fraction of the tick database entries have no clear matches in other sequenced genomes. Several putative tick G protein-coupled receptors (GPCRs) were identified that mightserve as starting points to develop novel control technologies.

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.

Between 1999 and 2002, the effect of mid-season doramectin treatments on the level of resistance in pyrethroid-resistant horn fly populations was examined at three separate Louisiana State University Agricultural Center research stations. The cattle were treated with pyrethroid ear tags in all years at all farms, and each farm received a mid-season doramectin treatment in 1 year. The number of weeks of control at Red River was 11 weeks higher in the year following the mid-season treatment of doramectin. At Macon Ridge, the number of weeks of control was 2 weeks higher in the year following the doramectin treatment. No change was observed at St. Joseph. The LC50s for fly populations tested at Macon Ridge and St. Joseph were found to increase for pyrethroids from the spring populations to the fall populations between 2000 and 2002. The LC50s for fly populations at Red River followed the same trends except in 2000, the year when the doramectin treatment was administered. Flies collected pre and post-treatment each year from St. Joseph and Red River were assayed for two alleles (kdr and skdr) associated with target site resistance to pyrethroids. Flies collected pretreatment at Macon Ridge in 1999 also were assayed for the kdr and skdr, and this population of flies had a frequency of 85.6% R-kdr alleles. At St. Joseph and Red River there was a general decline in the frequency of homozygous susceptible skdr (SS-skdr) and homozygous susceptible kdr (SS-kdr) individuals, as well as a general increase in homozygous resistant skdr (RR-skdr) and homozygous resistant kdr (RR-kdr) individuals, during the 4-year study. At both sites, the frequency of R-kdr alleles increased significantly in flies collected in the fall compared to flies collected in the spring with the exception of Red River in 2000, when dormacetin was applied. The frequency of the R-kdr alleles was significantly higher in flies collected in the fall compared to flies collected in the spring in the following year at both sites in two out of three comparisons. The frequency of R-skdr alleles was significantly lower in fly populations tested in the spring compared to fly populations tested in the fall at both farms in years when doramectin was not applied but there were no differences in the years when doramectin was applied. The frequency of R-skdr alleles was significantly higher in fly populations tested in the fall compared to in the spring the following year during all three comparisons at Red River and in one of three comparisons at St. Joseph.

We used an expressed sequence tag approach to initiate a study of the genome of the southern cattle tick, Boophilus microplus. A normalized cDNA library was synthesized from pooled RNA purified from tick larvae which had been subjected to different treatments, including acaricide exposure, heat shock, cold shock, host odor, and infection with Babesia bovis. For the acaricide exposure experiments, we used several strains of ticks, which varied in their levels of susceptibility to pyrethroid, organophosphate and amitraz. We also included RNA purified from samples of eggs, nymphs and adult ticks and dissected tick organs. Plasmid DNA was prepared from 11,520 cDNA clones and both 5' and 3' sequencing performed on each clone. The sequence data was used to search public protein databases and a B. microplus gene index was constructed, consisting of 8270 unique sequences whose associated putative functional assignments, when available, can be viewed at the TIGR website (http://www.tigr.org/tdb/tgi). A number of novel sequences were identified which possessed significant sequence similarity to genes, which might be involved in resistance to acaricides.

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions.

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na(+) channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-borne diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-borne diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-borne disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-borne diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/chlorfenvinphos.

We utilized RNA Northern blot analysis and ribonuclease protection assays (RPA) to study the mRNA expression level of a putative carboxylesterase-encoding gene from several strains of Boophilus microplus (Canestrini). Both the Northern analysis and RPAs indicated that an esterase transcript was more abundant in the pyrethroid resistant strain, Coatzacoalcos (Cz), compared to a susceptible control strain and a resistant strain whose pyrethroid resistance is mediated through a target site insensitivity mechanism. A PCR-based assay was designed to identify the presence of a previously reported point mutation in this B. microplus esterase gene. The reported G-->A substitution at nucleotide 1120 creates an EcoR I site in the mutant allele which can be detected by EcoR I digestion of the amplification products. The PCR assays showed that the frequency of the mutant allele was highest in the Cz-resistant strain, which has been shown to have an esterase-mediated resistance mechanism. The PCR assay can be performed either on individual tick larvae or hemolymph from adults.

A polymerase chain reaction-based assay was developed to detect the presence of a pyrethroid resistance-associated amino acid substitution in Boophilus microplus (Canestrini). The assay uses a simple method for the extraction of genomic DNA from individual larvae and genotypes individuals for the presence of a Phe-->Ile amino acid substitution in the S6 transmembrane segment of domain III of the para-like sodium channel, clearly distinguishing heterozygotes from homozygotes. High frequencies for this amino acid substitution were found in the Corrales and San Felipe strains, which have target site insensitivity mechanisms for pyrethroid resistance. The Caporal resistant strain contained lower yet substantial numbers of amino acid-substituted alleles. Low amino acid substitution frequencies were found in the susceptible reference Gonzales strain and the Coatzacoalcos strain, which has metabolic esterase-mediated pyrethroid resistance. The amino acid substitution was not found in six other strains that were susceptible to pyrethroids.

In this study we describe a nonradioactive single-fly microassay for permethrin hydrolysis. We used this assay with a microplate assay for general esterase activity to evaluate the permethrin hydrolyzing and general esterase activities of aging pyrethroid-susceptible male and female horn flies, Haematobia irritans (L.). We found substantial gender- and age-related differences regarding general esterase activity, permethrin sensitivity, and permethrin hydrolyzing activity within the colony. Extracts of female flies collected 48 h after receiving their first blood meal yielded significantly greater esterase activity than male extracts. Aging female flies were more tolerant of permethrin than were male flies. In addition, a positive correlation was found to exist between the general esterase activity of aging females and their ability to hydrolyze permethrin.

A polymerase chain reaction (PCR)-based rapid screening procedure was developed to test individual horn flies, Haematobia irritans irritans (L.), for the presence of a specific nucleotide substitution in the sodium channel gene sequence that has been associated with pyrethroid resistance. By a systematic optimization of reaction conditions and judicious choice of PCR primers differing in DNA sequence by a single nucleotide, we identified pyrethroid-susceptible or resistant sodium channel alleles in individual flies. Laboratory and field populations were examined by both the PCR assay and conventional filter paper bioassays with the pyrethroid cyhalothrin to verify that populations containing greater proportions of individuals with the resistant sodium channel allele DNA sequence also had higher bioassay LC50 values. The PCR assay for resistance alleles gave definitive information on the genotype of an individual fly and detected the presence of heterozygous individuals that might serve as reservoirs of resistance genes in field populations.

A study of the molecular genetics of the horn fly, Haematobia irritans irritans (L.), has resulted in the isolation and sequencing of a 2316-bp complementary DNA (cDNA) clone, HFN2b. An open reading frame was found, the deduced 464 amino acid sequence of which encodes a protein with 40% identity to AFG1, a member of a yeast family of ATPases; it contains an ATP- or GTP-binding P-loop motif. Northern and Southern blot analyses do not reveal significant differences in the expression of the gene homologous to HFN2b when comparing laboratory-reared horn flies that are susceptible or resistant to pyrethroid insecticides.

We report the isolation of a turgor-responsive gene of pea, trg-31, whose transcription induced within 30 min after the loss of leaf turgor. Structure of the coding region and 1.4 kb of 5' untranslated region was determined by DNA sequencing. A 3 kb promoter fragment from trg-31 was fused to a beta-glucuronidase (GUS) reporter gene in pBI101, tobacco leaf disks and mature plants analyzed for turgor-responsive induction of GUS mRNA. Significant amino acid sequence homology exists between trg-31 and putative transport proteins of bovine, Phaseolus, soybean and Escherichia coli membranes.

A set of 5' promoter deletions from Zmg13, a genomic clone of a pollen-specific gene of maize, has been transcriptionally fused to a beta-glucuronidase (GUS) reporter gene in the binary vector pBI101. Tobacco leaf disks were transformed and mature plants analyzed for GUS activity directed by the Zmg13 promoter constructs. Transgenic plants containing the 375 bp Zmg13 sequence from -314 to +61 relative to the transcription start site transcribed GUS RNA and expressed active GUS enzyme in mature pollen but not in leaves. Plants transformed with a 35S CaMV promoter-GUS transcriptional fusion expressed GUS RNA in leaves but not in pollen. Neither GUS RNA or active enzyme could be detected in pollen or leaves from plants containing a 124 bp Zmg13-GUS transcriptional fusion missing the putative Zmg13 TATA box. No GUS RNA or enzyme expression was not detected in non-transformed tobacco. RNA and GUS histochemical analysis of the T1 generation confirmed that the temporal expression pattern of Zmg13-GUS transcription in tobacco followed that of the native gene in maize and that the Zmg13 promoter sequences from the maize gene are able correctly to direct genetically stable, tissue-specific gene expression in transgenic tobacco plants.

Reduction of turgor in pea shoots caused the accumulation of several poly(A) RNAs. cDNA clones derived from three different poly(A) RNAs which accumulate in wilted pea shoots were isolated, sequenced and expression of the corresponding genes examined. Clone 7a encoded a 289 amino acid protein. The C-terminal 180 amino acids of this protein were homologous to soybean nodulin-26. RNA hybridizing to cDNA 7a was abundant in roots, and induced in shoots by dehydration, heat shock and to a small extent by ABA. Hydropathic plots indicate that the protein encoded by cDNA 7a contains six potential membrane spanning domains similar to proteins which form ion channels. Clone 15a encoded a 363 amino acid protein with high homology to cysteine proteases. RNA hybridizing to cDNA 15a was more abundant in roots than shoots of control plants. Dehydration of pea shoots induced cDNA 15a mRNA levels whereas heat shock or ABA treatment did not. Clone 26g encoded a 508 amino acid protein with 30% residue identity to several aldehyde dehydrogenases. RNA hybridizing to cDNA 26g was induced by dehydration of shoots but not roots and heat shock and ABA did not modulate RNA levels. Levels of the three poly(A) RNAs increased 4-6-fold by 4 h after wilting and this increase was not altered by pretreatment of shoots with cycloheximide. When wilted shoots were rehydrated, RNA hybridizing to cDNA 26g declined to pre-stress levels within 2 h. Run-on transcription experiments using nuclei from pea shoots showed that transcription of the genes which encode the three poly(A) RNAs was induced within 30 min following reduction of shoot turgor. One of the genes showed a further increase in transcription by 4 h after dehydration whereas transcription of the other 2 genes declined. These results indicate that plant cells respond to changes in cell turgor by rapidly increasing transcription of several genes. Furthermore, the expression of the turgor-responsive genes varies with respect to the time course of induction and reversibility of the wilting-induced changes.

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23 degrees C air for 20 min. Poly(A)(+) RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)(+) RNAs increased in wilted plants. Most of the poly(A)(+) RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A lambdagt10 cDNA library was constructed using poly(A)(+) RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)(+) RNAs which are induced in wilted shoots.

We report here the cloning and sequence analysis of cDNAs for a pair of closely related proteins from soybean (Glycine max [L.] Merr. cv. Williams 82) stems. Both proteins are abundant in soluble extracts of seedling stems but not of roots. One of these proteins (M r=28 kDa) is also foundd in the cell wall fraction of stems and actumulates there when seedlings are exposed to mild water deficit for 48 h. The mRNA for these proteins is most abundant in the stem region which contains dividing cells, less abundant in elongating and mature stem cells, and rare in roots. Using antiserum against the 28 kDa protein, we isolated cDNA clones encoding it and an antigenically related 31 kDa protein. The two cDNAs are 80% homologous in nucleotide and amino acid coding sequence. The predicted proteins have similar hydropathy profiles, and contain putative NH2-terminal signal sequences and a single putative N-linked glycosylation site. The two proteins differ significantly in calculated pI (28 kDa=8.6; 31 kDa=5.8), and the charge difference is demonstrated on two-dimensional gels. The proteins described here may function as somatic storage proteins during early seedling development, and are closely related to glycoproteins which accumulate in vacuoles of paraveinal mesophyll cells of fully expanded soybean leaves when plants are depodded.