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Published on: April 20, 2011
Identification of a third Boophilus microplus (Acari: Ixodidae) cDNA presumptively encoding an acetylcholinesterase
Kevin B Temeyer1, Ronald B Davey, Andrew C Chen
1Knipling-Bushland United States Livestock Insects Research Laboratory, United States Department of Agriculture-Agricultural Research Service, Kerrville, TX 78028-9184, USA.
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 eDNA 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.
Insights
Researchers identified a third acetylcholinesterase (AChE3) gene in the Boophilus microplus tick using molecular techniques. This discovery suggests multiple AChE forms may contribute to pesticide resistance in ticks.
Area of Science:
- Molecular Biology
- Entomology
- Biochemistry
Background:
- Acetylcholinesterases (AChEs) are crucial enzymes targeted by pesticides.
- Understanding AChE diversity in pests like Boophilus microplus is vital for effective pest control.
Purpose of the Study:
- To identify and characterize novel acetylcholinesterase (AChE) genes in the Boophilus microplus tick.
- To investigate the potential for multiple AChE forms in B. microplus and their implications for pesticide resistance.
Main Methods:
- Reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers.
- Primer walking and rapid amplification of cDNA ends (RACE) to obtain full-length cDNA.
- BLAST searches and conserved domain analysis for sequence identification and characterization.
Main Results:
- A novel cDNA sequence, presumptively encoding a third AChE (AChE3), was identified in B. microplus.
- The encoded protein shares structural similarities with known AChEs, including the catalytic triad and disulfide bonds.
- Sequence comparisons revealed low homology between the three identified AChE sequences, suggesting they are distinct genes.
Conclusions:
- The identification of AChE3 in B. microplus indicates the presence of multiple expressed AChE forms.
- This diversity may offer alternative mechanisms for pesticide resistance development in ticks.
- Further confirmation of the gene identity for presumptive AChEs is warranted.
