Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

Abstract

P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) geneediting technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, betacypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

“…In contrast, a number of publications have examined target gene mutation mediated resistance. To date, at least ten studies (five from D. melanogaster , three from H. armigera and two from S. exigua ) edited nAChRα6 , RyR , CHS1 , CAD , ABCA2 , TSPAN1 and P‐gp genes . Overall, the CRISPR/Cas9 manipulate techniques are already advanced in the model insect D. melanogaster and agricultural pest H. armigera , and this system provides a useful genome editing tool for other interesting insect species.…”

“…To date, at least ten studies (five from D. melanogaster, three from H. armigera and two from S. exigua) edited nAChR 6, RyR, CHS1, CAD, ABCA2, TSPAN1 and P-gp genes. 24,[35][36][37][38][40][41][42][43][44] Overall, the CRISPR/Cas9 manipulate techniques are already advanced in the model insect D. melanogaster and agricultural pest H. armigera, and this system provides a useful genome editing tool for other interesting insect species. The current study demonstrates the efficient generation of Px 6 subunit modification via the CRISPR/Cas9 system in P. xylostella, but it is likely that the editing system can be further optimized.…”

“…34 To date, by using CRISPR/Cas9 coupled with non-homologous end joining (NHEJ) and/or homology directed repair (HDR) approaches, the following mutation(s) edited in the nAChR 6 subunit (G275E and P146S of D. melanogaster), chitin synthase 1 (CHS1, I1015M/F/L of D. melanogaster), ryanodine receptors (RyR, G4946E/V & M4790I of D. melanogaster and Spodoptera exigua), cytochrome P450 gene (CYP9M10, KO in Culex quinquefasciatus), cadherin (CAD, KO in Helicoverpa armigera), P-glycoprotein (P-gp, KO in S. exigua), ABCA2 (KO & KI in H. armigera), and tetraspanin (TSPAN1, KO & KI in H. armigera) were confirmed in vivo to be involved in insect susceptibility to chemical insecticides or Bacillus thuringiensis Cry toxins. 24,[35][36][37][38][39][40][41][42][43][44] In our previous study, a three amino acid deletion identified in TM4 of P. xylostella 6 (Px 6) was associated with a high level of resistance to spinosad in the SZ-SpinR strain. We then in vitro introduced a three amino acid deletion into an easily expressed prototype human nAChR 7, and found this mutation leads to a complete loss of acetylcholine activation and significantly lower binding affinity of the [3H] -bungarotoxin, compared to the wild-type receptor.…”

“…The CRISPR/Cas9 system has been successfully applied in a number of insect species, and has recently been used to investigate insecticide resistance mechanisms . To date, by using CRISPR/Cas9 coupled with non‐homologous end joining (NHEJ) and/or homology directed repair (HDR) approaches, the following mutation(s) edited in the nAChRα6 subunit (G275E and P146S of D. melanogaster ), chitin synthase 1 ( CHS1 , I1015M/F/L of D. melanogaster ), ryanodine receptors ( RyR , G4946E/V & M4790I of D. melanogaster and Spodoptera exigua ), cytochrome P450 gene ( CYP9M10 , KO in Culex quinquefasciatus ), cadherin ( CAD , KO in Helicoverpa armigera ), P‐glycoprotein ( P‐gp , KO in S. exigua ), ABCA2 (KO & KI in H. armigera ), and tetraspanin ( TSPAN1 , KO & KI in H. armigera ) were confirmed in vivo to be involved in insect susceptibility to chemical insecticides or Bacillus thuringiensis Cry toxins …”

The ABCB Multidrug Resistance Proteins Do Not Contribute to Ivermectin Detoxification in the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

“…In contrast, MDR gene expression and protein activity have been identified as important factors in detoxification of ivermectin and other macrocyclic lactones in a multitude of other species, including dogs [36] and mice [57,58], where MDRs help prevent ivermectin penetration through the blood-brain barrier and into nerve tissue. MDR expression was also linked to ivermectin resistance in the nematodes Caenorhabditis elegans [65] and Haemonchus contortus [66], and in many arthropods, including the tick Rhipicephalus (Boophilus) microplus [30] and the model insect D. melanogaster [29], while in the agricultural pests H. armigera [67] and Spodoptera exigua [68], either gene expression or protein activity has been linked to ivermectin or abamectin resistance. In L. decemlineata, genome analysis has shown there is significant expansion of the ABCC subfamily of ABC proteins [38], making these multidrug resistance-associated proteins (MRPs) also candidates for providing defense against an insecticide such as ivermectin.…”