Defence mechanisms against insecticides temephos and diflubenzuron in the mosquitoAedes caspius: the P‐glycoprotein efflux pumps

Abstract

P-glycoproteins (P-gps) are efflux transporters found in cells of a broad range of both procaryotic and eukaryotic taxa, whose action is to relieve the cells of multiple, structurally dissimilar, toxic compounds. The possible role of P-gps in defence against the insecticides temephos and diflubenzuron was investigated in the mosquito Aedes caspius (Pallas), also known as Ochlerotatus (Aedes) caspius (Diptera: Culicidae), and the genomic DNA sequences encoding for P-gp transporters were isolated to provide molecular instruments for future research into the expression and characterization of genes codifying for P-gps in this mosquito species. Mosquito larvae were treated with insecticides alone and in conjunction with a sublethal dose of the P-gp inhibitor verapamil. The inhibition of P-gps reduced the LD(50) values of temephos and diflubenzuron by factors of 3.5 and 16.4, respectively, suggesting the potential involvement of P-gps in insecticide defence. Using a polymerase chain reaction (PCR)-based approach, a 481-bp sequence was isolated. The inferred nucleotide sequence shows high homology with the C-terminal sequence of known P-gps. The isolation and characterization of a putative P-gp sequence from Ae. caspius is the first step towards a better molecular understanding of the role played by multidrug transporters in the defence against insecticides in this species. This knowledge may open the way to a novel control strategy based on the inhibition of pest defences. The beneficial consequences of the inhibition of efflux pumps in improving insecticide performance are discussed.

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

“…P-glycoproteins have been associated with resistance to cancer therapy drugs, conferring a multi-drug resistance phenotype when overproduced (Wang et al, 1995). ABC transporters have been shown to participate in insecticide resistance in various species, including mosquitoes (Porretta et al, 2008;Gahan et al, 2010;Bariami et al, 2012). ABC transporter mRNAs were also found overexpressed in Plutella xylostella strains resistant to chlorpyrifos (You et al, 2013).…”

Putative role of an ABC transporter in Fonsecaea pedrosoi multidrug resistance

“…Subsequently, by using a degenerate primer set based on the conserved regions of the Walker A and Walker B motifs of ABC transporters [18], it was possible to detect and to sequence an mRNA partial sequence of an ABC transporter in F. pedrosoi, containing the conserved regions Walker A, Walker B and the ABC signature [19]. The same cDNA sequence was detected both in the laboratory and patient strain of F. pedrosoi used in this study.…”

“…RT-PCR was carried out in a final volume of 50 L containing 3 L of the first-strand cDNA template (see above), 5 L of 10× Taq Platinum buffer, 0.2 mM dNTP, 4 mM MgCl 2 , 1.25 U of Taq Platinum DNA polymerase (Invitrogen) and 50 pmol of either degenerated primers ABC Cas 1 (forward, 5 -GTY GGT TCH TCH GGH TGY GGW AA-3 ) and ABC Cas 2 (reverse, 5 -RTC YAA AGC DGA DGT DGC YTC ATC-3 ) designed for the Walker motifs of ABC-type transporters [18], or 10 pmol of primers Fon-F (forward, 5 -TAA TGC GGG TGT TGC CTC TG-3 ) and Fon-R (reverse, 5 -AGG GGT GGA AAG TGT GAA CT-3 ) designed for the internal transcribed spacer (ITS) regions of rDNA from fungi of the Fonsecaea genus [19]. Amplifications were done on a GeneAmp ® PCR System 9700 (Applied Biosystems, Foster City, CA).…”

“…Degenerate primers [18] designed to the highly conserved Walker A and Walker B motifs of ATPase proteins [27] were used to PCR-amplify two major DNA fragments (ca. 400 bp and ca.…”

“…Agarose gel of reverse transcription polymerase chain reaction (RT-PCR) fragments amplified from total RNA extracts with the degenerate primers ABC Cas 1 and ABC Cas 2[18] (lanes 3 and 5) and Fon-F and Fon-R primers[19] (lanes 4 and 6).Lane 1, DNA molecular weight standard; lane 2, negative control; lanes 3 and 4, laboratory strain; lanes 5 and 6, patient strain; lane 7, positive RT-PCR control (from SuperScript TM III Kit; Invitrogen, Carlsbad, CA).…”