The nicotinic acetylcholine receptor subunits Mdα5 and Mdβ3 on autosome 1 of Musca domestica are not involved in spinosad resistance

Abstract

Spinosad is a relatively new insecticide that exerts its toxic action via nicotinic acetylcholine receptors (nAChRs). Spinosad resistance in house flies appears to be due to an altered target site. To evaluate the molecular basis of spinosad resistance, two nAChR subunit genes, Mdalpha5 and Mdbeta3 were cloned and characterized from an insecticide-susceptible (aabys) and spinosad resistant (rspin) strain of the house fly, Musca domestica. The Mdalpha5 and Mdbeta3 cDNAs encode proteins of 781 and 432 amino acid residues, respectively. Phylogenetic analysis with insect nAChR subunits suggested that Mdalpha5 and Mdbeta3 are most closely related to Dalpha5 and Dbeta3 of Drosophila melanogaster, respectively. Mdbeta3 is intronless, which is unique among all previously described nAChR genes. A-to-I RNA editing was found at 13 sites in Mdalpha5, eleven of which resulted in amino acid substitutions. No evidence for A-to-I RNA editing was found in Mdbeta3. Mdalpha5 expression, quantified by real time PCR, was 340- and 23-fold higher in the head and thorax than in the abdomen. Mdbeta3 expression was more uniform, being only 2.4-fold higher in the head and 1.4-fold lower in the thorax, compared to the abdomen. There was no difference in the expression of Mdalpha5 and Mdbeta3 between the aabys and rspin strains. Although Mdalpha5 and Mdbeta3 both map to the same chromosome as spinosad resistance, there were no unique features of either gene in rspin, relative to the aabys strain. This suggests neither Mdalpha5 nor Mdbeta3 is responsible for spinosad resistance in house flies.

Unraveling the mystery of spinosad resistance in insects

“…We previously identified 12, 13, one and zero A-to-I RNA editing sites in house fly Mda6, Mda5, Mda2, Mdb3, respectively. [38][39][40] The sites of RNA editing for a6 subunit genes are conserved between D. melanogaster, M. domestica and H. virescens. 34,40,41) RNA editing is not randomly distributed across nAChR subunits, nor are the regions of the protein that have resulting amino acid substitutions the same between different subunits.…”

“…For example, A-to-I editing ranged from 3% to 100% at different sites within Mda5. 38) Grauso et al first investigated A-to-I editing in the nAChR family and found seven edited sites in Da6 from D. melanogaster. 41) Hoopengardner et al 45) further identified seven, four and two A-to-I RNA editing sites in Da5, Db1 and Db2, respectively.…”

“…The biological function of these shortened transcripts is not known, but they have been observed in both D. melanogaster 29) and M. domestica. 38,40) The nAChR with the most alternative transcripts in D. melanogaster is Da6 (two alternative exon 3s and three alternative exon 8s), where five different full length transcripts have been identified. 41) It appears use of alternative transcription may be conserved between species, as the same alternative exon use noted for Da6 was found in M. domestica, 40) and analysis of a6 nAChRs in An.…”

“…37) In the Caenorhabditis elegans genome, 42 possible nAChR subunits were predicted from which 27 nAChR subunit transcripts have been determined. 36) The relatively small number of nAChR subunits in insects is compensated for by diversification due to alternative exon use and RNA editing 29,[38][39][40] as described below. Alternative splicing plays an important role in expanding protein diversity and is commonly employed to expand diversity of insect nAChRs.…”

“…However, given that rspin and aabys express many of the same transcripts, suggests that changes in the sequence of Mda5 are not responsible for spinosad resistance. 38) The 2343-bp ORF of Mda5 encodes a 781-amino acid proprotein that possesses a 27-aa signal peptide. 38) Besides the signal peptide, the protein possesses typical characteristics of nAChR a subunits including a long N-terminal extracellular domain having the a subunit signature YXCC motif, the Cys loop domain and four hydrophobic transmembrane domains (TM1-4).…”