Kevin B. Temeyer

Identification of a Third Boophilus microplus (Acari: Ixodidae) cDNA Presumptively Encoding an Acetylcholinesterase

Abstract Oligodeoxynucleotide primers, based on amino acid sequences conserved in known acetylcholinesterases (AChEs), were used in reverse-transcription polymerase chain reaction (RT-PCR) with mRNA from Boophilus microplus (Canestrini) as the template. Primer walking and rapid amplification of cDNA ends (RACE) techniques were used to complete the cDNA sequence identified by RT-PCR. The complete B. microplus cDNA sequence contained an open reading frame encoding a 620 amino acid protein with a 20 amino acid signal peptide at the N-terminus targeting the protein for the secretion pathway. BLAST searches of GenBank using the presumptively encoded protein revealed highest sequence similarity to AChEs. The presumptively encoded protein was of similar size and structural properties to other identified AChEs, including the presence of the catalytic triad (Ser, Glu, His) and appropriate placement of internal cysteines to yield three internal disulfide bonds corresponding to those of known AChEs. Putative conserved domains identified the sequence as a member of the carboxylesterase family, pfam00135.8, of which AChE is a member. This cDNA therefore presumptively encodes a third transcribed AChE (AChE3) cDNA of B. microplus. Comparison of the three AChE cDNA sequences expressed in B. microplus demonstrated no discernible nucleotide sequence homology and relatively low amino acid sequence homology, strongly suggesting that they are not alleles of one another. The potential presence of multiple expressed AChEs in B. microplus suggests alternative mechanisms for development of resistance to pesticides that target AChE. The homology-based identification of a third expressed AChE in B. microplus is a surprising result and strongly implies the need for confirmation of gene identity for presumptive AChEs.

Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus

Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previously identified as presumptively encoding acetylcholinesterases (AChEs), but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization of the recombinant proteins supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates (OPs) malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl-beta-methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent at 10-100 microM choline in agreement with properties reported for the catalytic domain of Anopheles gambiae acetylcholinesterase-1. Substrate affinity and V(max) values were highest for rBmAChE1 proteins, and one rBmAChE1 enzyme (Tx11, derived from the OP-resistant strain Tuxpan), was insensitive to paraoxon and exhibited a greatly reduced V(max) near that of rBmAChE2. To date, recombinant BmAChE1 and BmAChE3 enzymes with reduced sensitivity to OP-inhibition have been cloned and expressed from OP-resistant strains. The presence of at least three genes expressing AChEs in R. (B.) microplus, at least two of which contain mutations expressed as OP-insensitive enzymes, strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character.

R86Q, a Mutation in BmAChE3 Yielding a Rhipicephalus microplus Organophosphate-Insensitive Acetylcholinesterase

Abstract Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Román strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.

Antigenicity and immunogenicity of Hypoderma lineatum soluble proteins in the bovine host

Protein species found in soluble crude extracts of Hypoderma lineatum (common cattle grub) 1st-instar larvae (HL1) were separated by non-denaturing and denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed for antigenicity by Western blotting using serum from H. lineatum-infested and vaccinated cattle. All HL1 proteins resolved by non-denaturing PAGE were found to be antigenic in the infested bovine host. Treatment of the proteins with sodium dodecyl sulfate and 2-mercaptoethanol destroyed the ability of hypodermin B and the Peak 2 proteins from DEAE-ion exchange HPLC to be bound by antibody. The principal proteins, hypodermin A and hypodermin C (collagenase), appear to be the most immunogenic of the larval proteins. Although having similar amino acid composition, hypodermin A did not appear to share an antigenic epitope with the most prevalent protein, hypodermin C. These results may allow for the selection of proteins to be used in vaccine trials and studies of protective immunological mechanisms associated with acquired resistance to H. lineatum infestation in the bovine host.

A Survey of Rhipicephalus microplus Populations for Mutations Associated With Pyrethroid Resistance

ABSTRACT Mutations associated with pyrethroid resistance were found in Mexican strains of Rhipicephalus microplus (Canestrini). A mutation in the sodium channel gene was reported in strains highly resistant to permethrin and another mutation in an esterase gene in a strain that shows moderate resistance to the same pesticide. Methods based on the melting temperature difference of amplified allele-specific DNA fragments were developed that can detect these mutations rapidly in individual larvae. When these methods were applied to ticks from various strains of R. microplus from Australia, neither of these mutations could be demonstrated. Different resistance mechanisms have apparently developed independently between Australian and Mexican strains of R. microplus.

Cloning and sequence analysis of a cDNA encoding Pso o II, a mite group II allergen of the sheep scab mite (Acari: Psoroptidae).

Abstract Psoroptes ovis (Hering), the sheep scab mite, is responsible for psoroptic scabies of cattle and sheep. Reverse translation of 30 N-terminal amino acids of the major P. ovis allergen, previously chosen as a candidate immunogen and identified as a 16 kDa protein yielded a degenerate sequence used to design oligodeoxynucleotide polymerase chain reaction (PCR) primers. Use of the PCR primers with a P. ovis cDNA library succeeded in amplification of a 90 bp cDNA gene fragment that was cloned, sequenced, and used to select unique sequencing/PCR primers. Primer walking generated overlapping subclones which yielded the 588 nucleotide consensus sequence of the cDNA encoding the 143 amino acid P. ovis allergen precursor. Nucleotide and translated sequences of the cDNA were compared with sequences in GenBank and found to be homologous to mite group II allergens Lep d II (formerly Lep d I) of Lepidoglyphus destructor Schrank, Der f II of Dermatophagoides farinae Hughes, Der p II of Dermatophagoides pteronyssinus (Trouessart), Tyr p II of Tyrophagus putrescentiae (Schrank), Eur m II of Euroglyphus maynei (Cooreman) and Gly d II of Glycophagus domesticus (De Geer). The mature P. ovis allergen is composed of 126 amino acids with a calculated molecular mass of 13,468 Da, three disulfide bonds, and pI of 6.06 with one potential o-glycosylation site at Thr116. We designate the P. ovis 16 kDa protein as Pso o II in conformity with nomenclature for mite group II allergens. RESUMEN Psoroptes ovis (Hering), el ácaro de la sarna de los ovinos, es responsable de la sarna psoróptica del ganado y las ovejas. Traducción reversa de 30 amino ácidos de la región N-terminal del alérgeno mayor de P. ovis, fue previamente escogido como un candidato a immunógeno e identificado como una proteína de 16 kDa, de la cual se diseñaron oligonucleótidos iniciadores degenerados para PCR. El uso de los iniciadores con una geneteca de cDNA de P. ovis, fueron exitosos para amplificar un fragmentode cDNA de 90 pares de bases, los cuales fueron clonados, secuenciados y usados para seleccionar iniciadores específicos para PCR. Iniciadores detraslape de secuencias generaron subclonas, las cuales resultaron en una secuencia consenso de cDNA de 588 nucleótidos, que codifica el alergeno precursor de 143 amino ácidos de P. ovis. Las secuencias traducidas de nucleótidos de cDNA fueron comparadas con secuencias en el GenBank y se encontraron homólogas al grupo II de alergenos de ácaros Lep d II (antes Lep d I) de Lepidoglyphus destructor (Schrank), Der f II de Dermatophagoides farinae (Hughes), Der p II de Dermatophagoides pteronyssinus (Trouessart), Tyr p II de Tyrophagus putrescentiae (Schrank), Eur m II de Euroglyphus maynei (Cooreman) y Gly d II de Glycophagus domesticus (De Geer). El alergeno maduro de P. ovis esta compuesto de 126 amino ácidos con una masamolecular calculada de 13,468 Da, three puentes disulfuro y un pI de 6.06, con un sitio potencial de o-glicosilación en Thr116. Nosotros designamos la proteínade 16 kDa de P. ovis como Pso o II para concordar con la nomenclatura para el grupo II de alergenos de ácaros.

Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae)

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.

Baculovirus Expression of BmAChE3, a cDNA Encoding an Acetylcholinesterase of Boophilus microplus (Acari: Ixodidae)

Abstract The complete cDNA sequence encoding a Boophilus microplus (Canestrini) (Acari: Ixodidae) acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable with that of adult, organophosphate-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE.

Acetylcholinesterases of blood-feeding flies and ticks

Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimers disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to elucidate structure-activity relationships for AChE that are important for advances in targeted pest control, as well as potential applications for medicine and biosecurity.

Genotyping Mutations in BmAChE3: A Survey of Organophosphate-Resistant and -Susceptible Strains of Rhipicephalus (Boophilus) microplus

ABSTRACT Mutations I48L, I54V, R86Q, V137I, I492M, and T548A were identified previously in BmAChE3, a gene encoding acetylcholinesterase, from the organophosphate (OP) acaricide-resistant San Román strain of Rhipicephalus (Boophilus) microplus. Recombinant BmAChE3 acetylcholinesterase containing the R86Q mutation was shown to exhibit nearly 20-fold reduction in the rate of phosphorylation by paraoxon relative to the wild-type sequence. In addition, the R86Q mutation was present in resistant laboratory strains at elevated frequency compared with OP-susceptible strains but was insufficient to alone generate the OP-resistant phenotype (J. Med. Entomol. 44:1013–1018). Here, we developed assays to genotype the remaining five mutations and evaluated frequency of all six BmAChE3 mutations in individual R. microplus ticks from laboratory and Mexican field-collected strains. We found a substantial number of individuals in known OP-susceptible strains that seemed to be homozygous for each of the mutations surveyed, the exception being I48L, which was infrequent in all strains, leading us to conclude that none of the mutations alone were responsible for generation of phenotypic resistance to OP acaricide.