Research Article Received: 19 February 2014

Revised: 17 June 2014

Accepted article published: 21 July 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/ps.3861

Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia Kai Dang,a,b Cheryl S Toi,a David G Lilly,a Wenjun Bub and Stephen L Doggetta* Abstract: BACKGROUND: Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown resistance (kdr) mutations, notably V419L and L925I. Information regarding this kdr-type resistance mechanism is unknown in Australia. This study aims to examine the status of kdr mutations in Australian C. lectularius strains. RESULTS: Several modern field-collected strains and museum-preserved reference collections of Australian C. lectularius were examined. Of the field strains (2007–2013), 96% had the known kdr mutations (L925I or both V419L/L925I). The ‘Adelaide’ strain (2013) and samples from the preserved reference collections (1994–2002) revealed no known kdr mutations. A novel mutation I936F was apparent in the insecticide-resistant ‘Adelaide’ strain, one strain from Perth (with L925I) and the majority of the reference collection specimens. The laboratory insecticide-resistant ‘Sydney’ strain showed a mixture of no kdr mutations (20%) and L925I (80%). CONCLUSION: The novel mutation I936F may be a kdr mutation but appeared to contribute less resistance to the pyrethroids than the V419L and L925I mutations. The detection of high frequencies of kdr mutations indicates that kdr-type resistance is widespread across Australia. Hence, there should be a reduced reliance on pyrethroid insecticides and an integrated management approach for the control of C. lectularius infestations. © 2014 Society of Chemical Industry Keywords: Cimex lectularius; knockdown mutations; pyrethroid resistance; bed bug

1

INTRODUCTION

Bed bugs (Cimex spp.) are blood-sucking insects that readily attack humans. While once common and widespread, bed bug infestations became rare in the latter half of the twentieth century, particularly in developed nations.1 Over the last 15 years, infestations of bed bugs worldwide, including Australia, have undergone a dramatic reappearance.2 – 6 The control of bed bugs has relied on indoor residual spraying of insecticides, especially the pyrethroids.4,7,8 However, bed bugs have developed high resistance to this insecticide class, and pyrethroid resistance has globally spread.9 – 16 Various factors have been postulated as the cause of the bed bug global resurgence, but the widespread resistance to the pyrethroids (and other insecticide classes) is postulated as the most likely key trigger.1,6 Pyrethroids, such as permethrin, deltamethrin and lambdacyhalothrin, are a major class of neurotoxic insecticides. These products have been widely used in the control of insect pests because they have low mammalian toxicity, have some degree of residual activity, and are relatively inexpensive. Like DDT, pyrethroids influence the insect nervous system by targeting the voltage-gated sodium channel (VGSC). They stimulate nerve cells to produce repetitive discharges, which leads to paralysis and death, an effect known as knockdown.17 – 20 An important Pest Manag Sci (2014)

resistance mechanism against pyrethroids (and DDT) is known as ‘knockdown resistance’ (kdr), and it has been shown that various mutations in the VGSC gene are responsible for increased resistance in many insect pests.19,21 These mutations act by substituting the amino acid sequence of the VGSC protein that prevents the insecticide from acting on the nervous system. Recently in the United States, two kdr mutations (V419L and L925I) were found to be strongly associated with high pyrethroid resistance in the common bed bug, Cimex lectularius L. (the NY-BB strain), whereas other resistance mechanisms such as detoxification enzyme activity were not identified in this strain.22 Subsequently, these two kdr mutations were linked with resistance to deltamethrin, and were found to be widely distributed across the United States.23,24 Four haplotypes have been classified in



Correspondence to: Stephen L Doggett, Department of Medical Entomology, Pathology West, Westmead Hospital, Locked Bag 9001, Westmead, NSW 2145, Australia. E-mail: [email protected]

a Department of Medical Entomology, Pathology West, Westmead Hospital, Westmead, NSW, Australia b Institute of Entomology, College of Life Sciences, Nankai University, Tianjin, China

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K Dang et al

Table 1. Susceptibility to d-allethrin and haplotypes of kdr mutations in Cimex lectularius in Australia Sample number

I II III IV V VI VII 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

State

NSW

QLD NSW

WA

VIC

QLD NT SA

Location

Collected year

Monheim strain Sydney strain Manly, SYDNEY Toongabbie, SYDNEY West Pennant Hills, SYDNEY Canley vale, SYDNEY Forster QLD Rainbow Beach Glebe, SYDNEY, St lves, SYDNEY Redfern I, SYDNEY Darlinghurst I, SYDNEY Auburn, SYDNEY Redfern II, SYDNEY Abbotsford, SYDNEY Darlinghurst II, SYDNEY North Parramatta, SYDNEY Northbridge, SYDNEY Byron Bay Beach, BYRON BAY Carlye St., BYRON BAY Maryland, NEWCASTLE Northbridge, PERTH Fremantle, PERTH Nedlands, PERTH Cottesloe, PERTH Southbank, MELBOURNE Ripponlea, MELBOURNE Moonee Ponds, MELBOURNE

Colony, late 1960s Colony, 2004 1994 1997 2002 2002 1998 1994 ∼2004 2007 2007 2007 2008 2012 Colony, 2011 Colony, 2011 Colony, 2011 Colony, 2012 Colony, 2013 2007 2007 Colony, 2013 ∼2007 ∼2007 ∼2007 Colony, 2013 2007 Colony, 2013 1 d2013  2 dColony, 2013  Colony, 2013 Colony, 2013 2007 Colony, 2013 Colony, 2013

South Yarra, MELBOURNE West Melbourne, MELBOURNE Narangba, BRISBANE Alice Springs Semaphore Park, ADELAIDE

Susceptibility Susceptible Resistant N/Aa N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Resistantc Resistant Resistant Resistantc Resistantc N/A N/A Resistantc N/A N/A N/A Resistant N/A Resistantc N/A Resistant Resistantc Resistant N/A Resistant Resistant

Haplotype A A/B Ab A A/Ab A Ab Ab B B B B B B B B B/C B B B B B B B/Bb B B B B C B/C B B B B Ab

a N/A, not available – these were all dead, preserved specimens. b With the novel mutation I936F. c The susceptibility was detected by Dang et al.16 d , 1  2 bed bugs from the same strain of Moonee Ponds, Melbourne:  1 tested samples directly collected from the field,  2 tested samples from the

established colony fed for several generations over one year; I–VII: from the reference collection of medically important arthropods deposited in the Department of Medical Entomology.

relation to these mutations: haplotype A (no target-site mutation), haplotype B (only L925I mutation), haplotype C (L925I and V419L mutations) and haplotype D (only V419L mutation). A quantitative sequencing (QS) protocol was established to monitor the resistant allele frequencies of C. lectularius from several locations within the US Army Garrison in Korea, confirming the roles of L925I and V419L mutations in pyrethroid resistance.25 In addition, various studies have shown the associations between kdr haplotypes and resistance to the pyrethroids.24,26 – 28 Consequently, the kdr-type resistance mechanism plays an important role in imparting resistance to pyrethroid insecticides in C. lectularius. In Australia, kdr-type resistance mechanisms have not yet been identified at the molecular level in C. lectularius, with only pyrethroid resistance profiling being undertaken on one laboratory strain, the ‘Sydney’ strain, and six field strains.13,14,16,29

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Therefore, the aim of the present study was to establish whether kdr-type resistance was present in Australian C. lectularius strains and the distribution of such mutations across the country. This information should assist pest managers in the selection of the most appropriate insecticides for the control of bed bugs.

2

MATERIALS AND METHODS

2.1 Bed bug strains Two reference laboratory colonies, an insecticide-susceptible ‘Monheim’ strain (Bayer, Germany) and an insecticide-resistant ‘Sydney’ strain, together with 13 field-collected strains that had recently been colonised, collected from Perth, Adelaide, Melbourne, Sydney, Newcastle and Alice Springs in six states (QLD, NSW, VIC, SA, WA, NT) across Australia, were maintained in the

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Pest Manag Sci (2014)

Detection of kdr mutations in Cimex lectularius

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Department as previously described.29 All strains were kept free from any insecticide exposure, and all field-collected strains were less than 3 years old, while the ‘Sydney’ strain originated from founder specimens collected in 2004. Bed bugs from a further 12 field infestations (Table 1) were also evaluated for kdr mutations. These bed bugs were collected after the bed bug resurgence from 2007 to 2013 and preserved in either 95% alcohol or dried and stored at −20 ∘ C. Furthermore, the Department of Medical Entomology maintains an historical reference collection of arthropods of medical importance, and the collection was examined for preserved C. lectularius samples. Eleven preserved (in 70% ethanol) specimens from seven separate collections that were obtained from various locations in Australia were tested to examine the temporal change in kdr mutations, which may give an indication on when the resistance alleles were introduced into the country. These specimens were collected over the years 1994 to 2004 (Table 1, I–VII). All bed bugs were identified according to Usinger prior to testing.30 2.2 Residual bioassay The knockdown susceptibility to pyrethroids in the colonised field strains was confirmed by the Dang mat assay.16 Briefly, ten mixed-sex adult bed bugs from each field strain were confined to a d-allethrin impregnated mat, together with identical numbers of unexposed controls, and knockdown was recorded at 10 min time intervals in the first hour, hourly to 6 h and then again at 24 h exposure. The ‘Monheim’ strain was used as a pyrethroid-susceptible reference. 2.3 The VGSC gene and kdr mutation Two regions of the VGSC gene, containing key putative kdr mutation sites associated with kdr-type resistance to pyrethroids found in a range of insect pests, were initially investigated. The first region (∼500 bp) included four putative kdr mutation sites, namely V410, V419, V421 and E435, encoding for the domain IS6 and part of the domain I–II linker region of the VGSC gene. The second region (∼800 bp) encompassed six putative kdr mutation sites, namely M918, L925, T929, L932, I936 and L1014, encoding for the domain IIS4–IIS6 region.21,23 2.4 DNA isolation DNA from individual bed bugs was extracted using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA), following the manufacturer’s protocol with a slight modification. Briefly, fresh bed bugs were homogenised individually in a 5 mL plastic tube (Labserv, Cat. No. LBSSP2006) containing five glass beads and 800 μL of phosphate buffered saline (PBS). The homogenate (180 μL) was treated with 20 μL of proteinase K at 55 ∘ C for 1 h and extracted as per the manufacturer’s protocol. DNA was eluted in 100 μL of Buffer AE provided in the Dneasy kit twice. The older (pre-2004) and dried specimens were macerated in a 2 mL microcentrifuge tube with a micropestle (Eppendoff, Germany) after freezing with liquid nitrogen. The grind was incubated at 55 ∘ C for 5 h or overnight with the addition of 180 μL of ATL buffer and 20 μL of proteinase K. The DNA was then extracted following the method mentioned above. 2.5 Detection of kdr mutations in the VGSC gene To monitor the putative kdr mutations mentioned above in the respective two regions of the VGSC gene, PCR and DNA Sanger sequencing (ABI BigDye Terminator Version 3.1) were used respectively. Fragment amplification was carried out using the primers Pest Manag Sci (2014)

BBparaF1 (5-AACCTGGATATACATGCCTTCAAGG-3), BBparaR1 (5-TG ATGGAGATTTTGCCACTGATG-3) for the first region (with the V419 mutation site) and BBparaF3 (5-GGAATTGAAGCTGCCATG AAGTTG-3), BBparaR3 (5-TGCCTATTCTGTCGAAAGCCTCAG-3) for the second region (with the L925 mutation site), designed by Zhu and colleagues.23 The PCR reaction contained 12.5 μL of MyTaq HS Mix (Bioline, London, UK), 0.5 μL of both forward and reverse primers (20 μM) and 2 μL of the DNA template in a total volume of 25 μL. PCR for both fragments was performed in a thermal cycler (Mastercycler gradient; Eppendorf, Germany) under the following conditions: 95 ∘ C for 2 min, followed by 40 cycles of 94 ∘ C for 20 s, 58 ∘ C for 30 s and 72 ∘ C for 40 s, followed by 72 ∘ C for 10 min. The amplified products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH) at 37 ∘ C for 30 min, followed by 80 ∘ C for 30 min, and then held at 4 ∘ C. The purified products were sequenced by the Australian Genome Research Facility Ltd (AGRF, Sydney, Australia). DNA sequencing was performed using primer BBparaR1 for the V419 mutation site and primer BBparaF3 for the L925 mutation site. The sequences were aligned by ClustalW and analysed by means of BioEdit, MEGA5 and Sequence scanner v.1.0. The sequenced VGSC genes of the test samples were compared against the ‘Monheim’ strain and C. lectularius (Genbank: FJ031996, FJ031997) prototypes to confirm the kdr mutations.

3

RESULTS

3.1 Susceptibility of C. lectularius field strains to d-allethrin Six strains from previous studies demonstrated high pyrethroid resistance when tested by the Dang mat assay (Table 1).16 In the present study, seven of the other field strains were mostly found to be highly resistant in the mat assay, with no significant knockdown (

Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia.

Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown res...
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