Striking diflubenzuron resistance in Culex pipiens, the prime vector of West Nile Virus

Abstract

Culex pipiens mosquitoes cause severe nuisance and transmit human diseases including West Nile. Vector control by insecticides is the main tool to prevent these diseases and diflubenzuron is one of the most effective mosquito larvicides used in many places. Here, high levels of resistance were identified in Cx. pipiens from Italy, with a Resistance Ratio of 128 fold. The phenotype was associated with mutations at amino acid I1043 (I1043M and I1043L) of the Chitin synthase gene, which showed significantly higher frequency in bioassay survivors. Both mutations have been introduced in the Drosophila melanogaster chitin synthase gene using the genome editing method CRISPR/Cas9 and validated to confer significant levels of resistance, although at different levels. The I→M mutation results in a Resistance Ratio >2,900 fold and the I→L mutation >20 fold. Two PCR based diagnostics were developed for monitoring of the resistant mutations in field populations. The findings are of major concern for public health given the importance of diflubenzuron in mosquito control in many places, the intensity of the resistance phenotype and the limited availability of alternative larvicides.

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 …”

Fitness costs of key point mutations that underlie acaricide target‐site resistance in the two‐spotted spider miteTetranychus urticae

“…Resistance to mite growth inhibitors and chitin synthase inhibitors has been attributed to the I1017F mutation in chitin synthase (Demaeght et al., 2014; Douris et al., 2016; Grigoraki et al., 2017; Riga et al., 2017; Van Leeuwen et al., 2012). As the I1017F mutation is located in a highly conserved region of the CHS1 gene (Douris et al., 2016; Grigoraki et al., 2017; Suzuki, Shiotsuki, Jouraku, Miura, & Minakuchi, 2017; Yamamoto, Yoneda, Hatano, & Asada, 1995) , this substitution could impair the enzymatic function. In the current study, the impact of I1017F on spider mite fitness was quantified by analyzing single‐generation life‐history traits, fertility life table parameters of individual near‐isogenic lines as well as by an interline competition experiment.…”