Metabolic detoxication and the kdr mutation in pyrethroid resistant house flies, Musca domestica (L.)

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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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 <100% mortality. Control groups received acetone or synergists alone. Treated flies were put in Sweetheart ice cream cups (Sweetheart Cup, Owings Mills, MD) with a piece of dental wick saturated in 15% sugar water. Mortality was assessed after 24 h and immobile flies were scored as dead. All tests were run at 25  2 °C and replicated at least three times on different days. Bioassay data were pooled and probit analysis was conducted. Statistical analysis of LD50 s was based on nonoverlap of 95% confidence intervals. Synergism ratios (SRs) were calculated (the LD50 of insecticide alone divided by the LD50 of synergist + insecticide). 2.3. Cloning and sequencing of the para-type cDNA fragment from house flies To investigate the kdr and super-kdr mutations in pyrethroid resistant house flies, total RNA was extracted from heads and thoraces of 20 house flies of each strain using the acidic guanidine thiocyanate–phenol–chloroform method [26]. mRNA from each sample was isolated with oligotex(dT) suspension as described by the manufacturer (Qiagen). cDNA synthesis was carried out by reverse transcription-mediated polymerase chain reaction (RT-PCR). First strand cDNA was synthesized with Superscript II reverse transcriptase (Gibco-BRL) and an antisense 50 -anchored oligo(dT) primer 50 - TAATACGACTCACTAT AGGGAGATTTTTTTTTTTTTTTT-30 ) [27] us-

2.8 (0.4) 3.9 (0.5) 1.4 (1.2–1.7) 3000 (2600–3700) 260 240 7.9 240 11 24 59 2200

– 520 – 16 – 220 – 14 – –

(0.7) (0.4) (0.5) (0.4) (0.4) (0.5) (0.7) (0.5) b

a

Permethrin + PBO & DEF

Permethrin + DEF

Permethrin + PBO

abbys SeALHF aabys SeALHF aabys SeALHF aabys SeALHF Permethrin alone

LD50 values in nanograms per house fly. Synergist ratio, LD50 of insecticide alone/LD50 of insecticide + synergist. c Resistance ratio, LD50 of the resistance strain/LD50 of the susceptible strain.

5.0 1.8 3.2 2.2 2.6 3.5 4.6 3.7 5.9 (4.9–7.0) 3100 (2100–6500) 0.8 (0.6–0.9) 13 (7.6–1.7) 0.6 (0.4–0.7) 130 (100–160) 0.1 (0.08–0.1) 1.4 (1.3–1.6)

SRb Slope (SE) LD50 a (95% CI) n

Topical application Strains

A decrease in the rate of cuticular penetration (pen) is a pyrethroid resistance mechanism in house flies [9,15–17,22]. To investigate whether pen is involved in permethrin resistance in SeALHF, permethrin (in acetone) was injected directly into the intersegmental membrane between thoracic and abdominal notum of female

Insecticides

3.2. Role of the decreased rate of cuticular penetration (pen) in pyrethroid resistance

Table 1 Toxicity of permethrin with and without PBO and DEF to SeALHF and aabys house fly strains

SeALHF had elevated levels of resistance to permethrin with a resistance ratio of 520 compared with aabys (Table 1). When house flies were pretreated with PBO, an inhibitor of cytochrome P450 monooxygenases, 240- and 7.9-fold increases in toxicity of permethrin to SeALHF and aabys, respectively (Table 1), were observed, resulting in a decrease in permethrin resistance of SeALHF (Table 1). This result suggests that P450 monooxygenase-mediated detoxification contributes to permethrin resistance in SeALHF. DEF, an inhibitor of hydrolases, enhanced toxicity of permethrin to SeALHF and aabys by 24- and 11-fold, respectively, and decreased resistance to 220-fold in SeALHF (Table 1), indicating that hydrolytic metabolism has a minor contribution to resistance. Neither PBO or DEF alone (Table 1), or PBO + DEF (resistance ratio ¼ 14, Table 1) could completely abolish resistance to permethrin in SeALHF. These results suggest that one or more additional minor mechanisms are involved in overall resistance of SeALHF.

RRc

3.1. Role of metabolic detoxication in pyrethroid resistance of SeALHF

300 160 240 240 240 240 300 260

3. Results

n

Injection

LD50 (95% CI)

Slope (SE) RR

ing house fly mRNA as template. The first strand cDNA products were amplified by PCR with two internal primers, sense primer F1 (50 -CATGG CCCACACTGAATTTACTC-30 ) and antisense primer R1 (50 -AGTCGGGGCTGATAAACTAGATG-30 ), based on sequences of the wild-type house fly para-type sodium channel gene (Accession No. X96668) [12]. PCR products were purified from agarose gel using QIAquick Gel Extraction Kit (Qiagen) and cloned into PCR 2.1 Original TA cloning vector (Invitrogen). Sequences of cDNA fragments from TA clones were verified by automated sequencing (Research Instrumentation Facilities, Auburn University).

159

– 2100

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flies. Injection caused a 4-fold increase in toxicities of permethrin to aabys compared with topical application (Table 1), suggesting that the cuticular penetration barrier provides protection from permethrin in this strain. In contrast, injection increased levels of resistance to permethrin by 4-fold in SeALHF (Table 1). This suggests that decreased cuticular penetration does not play a role in permethrin resistance in SeALHF. 3.3. Role of the kdr and/or super-kdr mutations in pyrethroid resistance Target site insensitivity, due to a mutation of leucine at position 1014 to phenylalanine (L1014F), termed the kdr mutation, in the parahomologous sodium channel gene is associated with knockdown resistance of house flies [11–13]. In addition, a mutation of methionine at position 918 to threonine (M918T), termed super-kdr mutation, in combination with the kdr mutation process higher levels of resistance to pyrethroids. To investigate whether these mutations in ALHF and SeALHF strains, a 392 bp para-type sodium channel gene fragment, where kdr and super-kdr mutations reside, was generated by RT-PCR from aabys, CS, ALHF, and SeALHF and cloned into TA cloning vector. Sequences of cDNA fragments from five TA clones of each house sample were analyzed. The L1014F substitution was not present in aabys, CS, and SeALHF strains (Fig. 1). However, a T to A mutation resulting in a leucine 1014 to histidine amino acid change (L1014H), was detected in SeALHF (Fig. 1), and the classic L1014F was present in ALHF. Two silence nucleotide changes, T to C at nt 2914 and G to C at nt 2944, were detected in ALHF. Neither ALHF nor SeALHF carried the super-kdr mutation (M918T).

4. Discussion Our study showed that resistance to permethrin in SeALHF was partially suppressed by PBO, suggesting that P450 monooxygenase-mediated metabolism plays a major role in permethrin resistance. In contrast, although a 2-fold decrease in resistance to permethrin in SeALHF by DEF can be easily attributed to the effect of DEF on hydrolytic metabolism of permethrin in SeALHF and imply that hydrolytic metabolism has a minor contribution to resistance, these data should be interpreted with caution. It has been proposed

that DEF is not a completely specific inhibitor of hydrolases and that it can inhibit microsomal oxidases at a high concentration [17]. Incomplete suppression of permethrin resistance by PBO and DEF suggests that one or more additional mechanisms are involved in overall resistance. Multiple mechanism interactions are common in house fly pyrethroid resistance [9,14,19]. The current study reveals that permethrin resistance in SeALHF is conferred by the interaction of a major mechanism (e.g., cytochrome P450 mediated detoxication) and possibly more than one minor mechanism. Although P450 mediated detoxication is an important mechanism in pyrethroid resistance in ALHF [14,21], it appears that there may be another major mechanism responsible for a high level (1800-fold) of permethrin resistance in ALHF that is largely unaffected by PBO (resistance ratio ¼ 100) and DEF (resistance ratio ¼ 350) [14]. Injection did not decrease levels of permethrin resistance in SeALHF, indicating that a decreased rate of cuticular penetration (pen) does not play an important role in resistance. This is similar to the ALHF strain [21] although in other strains [11,17,19,22] decreased cuticular penetration (pen) does contribute to pyrethroid resistance. Nevertheless, injection caused a 4-fold increase in permethrin resistance of SeALHF, which is similar to that reported in pyrethroid resistant German cockroaches [28]. Differences in the physical structures and chemical components of the cuticle between the susceptible and SeALHF strains may result in faster cuticular penetration of permethrin in SeALHF compared with the susceptible strain [28,29] and cause an increased level of resistance by injection. In addition, the tissue(s) of SeALHF where permethrin has been injected may have higher concentrations of detoxication enzymes resulting in more rapid metabolism of permethrin in SeALHF than in the susceptible strain. Target site insensitivity due to kdr (L1014F) and super-kdr (M918T) mutations has been associated with pyrethroid resistance of house flies [11–13]. The same mutation in the para-type sodium channel gene associated with pyrethroid resistance has also been found in horn flies (Haematobia irritans [30], diamondback moths (Plutella xylostella) [31], aphids (Myzus persicae) [32], mosquitoes (Anopheles gambiae) [33,34], German cockroaches (Blattella germanica) [11,35,36] and Colorado potato beetles (Leptinotarsa decemlineata) [37]. In our study, the kdr mutation (L1014F) was present in ALHF, sug-

N. Liu, J.W. Pridgeon / Pesticide Biochemistry and Physiology 73 (2002) 157–163 Fig. 1. Alignment of the cDNA/deduced amino acid sequences of the 392 bp para-type sodium channel gene fragment where the kdr and super-kdr mutations reside from aabys, CS, ALHF, and SeALHF. Sequence alignment is carried out with wild type house fly para-type sodium channel gene (X96668) and numbering is according to the sequence of Williamson et al. [12]. The nucleotide changes and corresponding amino acid substitutions are in bold and underlined. 161

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gesting that target site insensitivity caused by the kdr mutation is a mechanism of resistance in this strain. The L1014F substitution was not present in SeALHF, however, a T to A substitution resulting in a leucine to histidine (L1014H) amino acid change was present. This study is the first to report a different mutation occurred at the position corresponding to kdr mutation in pyrethroid resistant house flies although the same mutation (L1014H) has been reported in pyrethroid resistant tobacco budworms (Heliothis virescens) [38]. In addition, a substitution of leucine to serine (L1014S) has been detected in pyrethroid resistant mosquitoes (Culex pipiens) [39]. These results suggest that the mutation at leucine residue corresponding to Leu1014 of the house fly sodium channel plays an important role in pyrethroid resistance. The super-kdr mutation (M918T) was not detected in ALHF or SeALHF, indicating that the M918T substitution is not important in pyrethroid resistance of these two house fly strains even though ALHF possesses much higher levels of resistance [21] than SeALHF. The present study has indicated that resistance in SeALHF was conferred by P450 monooxygenase-mediated detoxication (major), hydrolytic metabolism (minor) and one or more additional minor mechanisms. While the L1014F mutation is not an important contributing factor to permethrin resistance in SeALHF, the importance of the L1014H substitution in resistance deserves more detailed investigation. ALHF, on the other hand, carries the kdr mutation. Whether the kdr mutation confers a 400-fold level of permethrin resistance that is largely unaffected by PBO and DEF [21] in ALHF remains further investigation. Acknowledgments This study was supported by funds from the Biogrants program at Auburn University and Hatch Project ALA08-029. References [1] N.R.H. Burgess, Houseflies, bluebottles and related flies, in: N.R.H. Burgess (Ed.), Public Health pests, Chapman & Hall, London, 1990, pp. 55–59. [2] D.S. Kettle, Muscidae (houseflies, stableflies), in: D.S. Kettle (Ed.), Medical and Veterinary Entomology, Croom Helin, London, 1984, pp. 229– 231.

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