Andrew C. Chen

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.

Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus

We have isolated and sequenced a novel P450 gene (CYP319A1) from the cattle tick, Boophilus microplus. The CYP319A1 cDNA encodes a protein of 531 amino acids with an estimated molecular weight of 60.9k. It contains all highly conserved motifs characteristic of P450 enzymes. Comparison of deduced amino acid sequence with other CYP members shows that the CYP319A1 is more closely related to CYP4 family, but its overall identity to the CYP4 family is less than 40%. Therefore, it was assigned to a new P450 family by the P450 nomenclature committee. A pseudogene which shares high homology with the CYP319A1 was identified. Analysis of genomic sequence of the pseudogene indicated that the pseudogene contains two additional DNA inserts in the coding region, which disrupt the open reading frame. RT-PCR analysis showed that CYP319A1 is expressed in both susceptible and acaricide-resistant ticks.

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.

The partial purification of a Trichogramma pretiosum pupation factor from hemolymph of Manduca sexta

Abstract Factors responsible for pupation of Trichogramma pretiosum Riley reared in vitro were partially purified. The active material was extracted with 76% ethanol from Manduca sexta (L.) hemolymph. The water soluble active fraction was chromatographed first on a Sephadex G-10® column and then on a C 18 cartridge (reversed phase). After removing more than 99% of the original protein, 20% of the original pupation activity remained; this fraction, however, also contained about 20% of the original hemolymph carbohydrate. The pupation factor was further separated by DEAE-HPLC into two active carbohydrate containing fractions. HPLC of the active fraction from the C 18 cartridge on an amino column produced some separation of the original carbohydrate, but no pupation activity was found in single fractions after this separation. However, when certain fractions were re-combined, weak pupation activity was obtained. The results indicate that when T. pretiosum were fed nutritionally rich artificial diets, at least two and possibly several polar, low molecular weight chemicals in M. sexta hemolymph were also needed and were responsible for growth and development of the parasitoids to the pupal stage. This is the first report describing the partial purification of insect growth factors affecting the growth and development of parasitoids.

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.

Demonstration of a Human Atrial Natriuretic Peptide-Like Material in the Stable Fly, Stomoxys Calcitrans

Atrial natriuretic peptide (ANP) is a peptide first reported in humans (hANP) and later in other mammals (Flynn, et al., 1987). More recently, similar peptides have been isolated from lower vertebrates (Miyata, et al., 1988; Sakata, et al., 1988). Although the precise mechanism of action of this peptide is not well understood, the gross effects when administered in these animals are vasodilation and increases in the rates of excretion of water and electrolytes, particularly sodium. The mammalian ANP’s are extremely well conserved 28 amino acid peptides with only one amino acid substitution at the 12th position. Due to my interest in stable fly diuresis, I decided to examine the possible existence of similar peptides in this insect. In this communication I shall report evidence for the presence of hANP-immunoreactivity in the stable fly.