Pesticide Biochemistry and Physiology 73 (2002) 157–163 www.academicpress.com
Metabolic detoxication and the kdr mutation in pyrethroid resistant house flies, Musca domestica (L.) Nannan Liu* and Julia W. Pridgeon Department of Entomology and Plant Pathology, 301 Funchess Hall, Auburn University, Auburn, AL 36849-5413, USA Received 5 April 2002; accepted 2 July 2002
Abstract Two house fly strains, ALHF and SeALHF, were collected from Alabama after control failures with pyrethroids. While pyrethroid resistance in ALHF partially conferred by P450 monooxygenase- and hydrolase-mediated metabolism has been reported, no studies have been conducted on resistance of SeALHF. The current study was carried out to investigate mechanisms of pyrethroid resistance in SeALHF and the possible role of target site insensitivity, due to kdr mutation, in pyrethroid resistance of ALHF. Resistance to permethrin in SeALHF was dramatically and partially suppressed by PBO and DEF, respectively, suggesting that P450 monooxygenase-mediated metabolism plays a major role in permethrin resistance in this strain, while hydrolytic metabolism has a minor contribution to resistance. Incomplete suppression of permethrin resistance by PBO and DEF suggests that one or more additional minor mechanisms are involved in overall resistance of SeALHF. Injection did not decrease levels of resistance to permethrin in SeALHF, implying that a decreased rate of cuticular penetration (pen) does not play a role in permethrin resistance in this strain. A 392 bp para-type sodium channel gene fragment, where kdr (L1014F) and superkdr (M918T) mutations reside, was generated by RT-PCR from ALHF and SeALHF. The M918T mutation was not detected in ALHF or SeALHF, suggesting that the super-kdr mutation is not important in permethrin resistance of these two house fly strains even though ALHF possesses a much higher level of resistance than SeALHF. The L1014F mutation was present in ALHF, but not in SeALHF, suggesting that the kdr mutation is an important factor in pyrethroid resistance in ALHF. A leucine to histidine (L1014H) substitution at the position corresponding to kdr mutation was detected in SeALHF. The importance of this mutation in resistance is discussed. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction The house fly, Musca domestica L., is not only a serious pest at livestock and poultry facilities, but also a public health pest that acts as a mechanical vector of human and animal pathogens
*
Corresponding author. Fax: 1-334-844-5005. E-mail address:
[email protected] (N. Liu).
[1,2]. The effectiveness and low mammalian toxicity of pyrethroid insecticides has made these the preferred compounds for house fly control. Intensive use of pyrethroids has resulted in development of resistance in house flies [3,4]. Mechanisms of resistance in house flies have attracted attention, since they elucidate pathways of resistance development and help in designing novel strategies to prevent, or minimize the spread and evolution of resistance. The voltage-gated
0048-3575/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 4 8 - 3 5 7 5 ( 0 2 ) 0 0 1 0 1 - 3
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N. Liu, J.W. Pridgeon / Pesticide Biochemistry and Physiology 73 (2002) 157–163
sodium channel is the primary target of pyrethroid insecticides [5] and its insensitivity has been associated with pyrethroids resistance in knockdown resistant (kdr) house fly strains [6–10]. A mutation of leucine at position 1014 to phenylalanine (L1014F), termed the kdr mutation, in the para-type sodium channel gene is consistently associated with knockdown resistance in house flies [11–13]. An additional mutation of methionine at position 918 to threonine (M918T) occurs in combination with the kdr mutation in super-kdr house fly strains with higher levels of resistance to pyrethroid than kdr house flies. Increased metabolic detoxication mediated by P450 monooxygenases and decreased rate of cuticular penetration (pen) are two other important mechanisms in house fly pyrethroid resistance [9,10,14–18]. Multiple mechanisms can interact to increase the levels of resistance [9,10,14,19,20]. Two house fly strains, ALHF and SeALHF, were collected from Alabama in 1998 after control failures with pyrethroid insecticides. ALHF exhibits high levels of resistance to pyrethroids [14,21]. Incomplete suppression of permethrin resistance by PBO or DEF and no effect of pen on resistance [14,21] reveal that P450 monooxygenases, hydrolases and one or more mechanisms largely unaffected by PBO and DEF are involved in overall resistance of ALHF. A factor(s) on autosome 3 plays a major role in pyrethroid resistance in ALHF; however, this factor(s) is not related to P450-mediated detoxication or decreased rate of cuticular penetration [21]. Because kdr and super-kdr traits are on autosome 3 of house flies [22–24], the possibility of the involvement of target site resistance in ALHF merits investigation. No studies have been conducted on pyrethroid resistance of SeALHF.
2. Materials and methods 2.1. House fly strains ALHF, a pyrethroid resistant strain collected from a poultry farm in Marshall County, Alabama, in 1998 after control failures with the use of pyrethroids [14] and maintained under biannual selection with permethrin; SeALHF, collected from a poultry farm in Lee County, Alabama in 1998 and maintained in the laboratory without insecticide selection; aabys, an insecticide-susceptible strain with recessive morphological markers ali-curve (ac), aristapedia (ar), brown body (bwb),
yellow eyes (ye), and snipped wings (sw) on autosomes 1, 2, 3, 4, and 5, respectively, obtained from Dr. J.G. Scott (Cornell University); and CS, a wild-type insecticide-susceptible strain obtained from Dr. J.G. Scott. Flies were reared as described by Wheelock and Scott [25]. 2.2. Bioassays Topical application was performed by delivering a 0:5 ll of insecticide (in acetone) to 2- to 3day-old female flies [18] using a 25 ll Hamilton gastight syringe (Fisher Scientific). Injection was performed by injecting 0:5 ll of insecticide (in acetone) into the intersegmental membrane between thoracic and abdominal notum of female flies using a 10 ll Hamilton gastight syringe. PBO and/or DEF were applied topically at the maximum sublethal dose ð10 lg=flyÞ to the thoracic notum 1 h before the insecticide treatment as described previously [14,18]. Each bioassay consisted of 20 flies per dose and four or five doses that give >0 and
