Insect Science (2011) 18, 30–39, DOI 10.1111/j.1744-7917.2010.01387.x

ORIGINAL ARTICLE

Insecticide resistance in Bemisia tabaci from Cyprus

Vassilis Vassiliou1, Maria Emmanouilidou1, Andreas Perrakis2,3, Evangelia Morou2, John Vontas2,Anastasia Tsagkarakou3 and Emmanouil Roditakis3

1Agricultural Research Institute, Nicosia, Cyprus, 2Faculty of Biotechnology and Applied Biology, Department of Biology, University of

Crete, Heraklion, Greece, 3National Agricultural Research Foundation (N.AG.RE.F.), Plant Protection Institute of Heraklion, Heraklion,

Greece

Abstract A comprehensive study on the Bemisia tabaci (biotype B) resistance to neon-icotinoid insecticides imidacloprid, acetamiprid and thiamethoxam, and pyrethroid bifen-thrin was conducted in Cyprus. The resistance level to eight field-collected B. tabacipopulations was investigated. The activities of enzymes involved in metabolic detoxifica-tion and the frequencies of pyrethroid and organophosphates target site resistance mutationswere determined. Moderate to high levels of resistance were detected for imidacloprid (re-sistance factor [RF] 77–392) and thiamethoxam (RF 50–164) while low resistance levelswere observed for acetamiprid (RF 7–12). Uniform responses by the Cypriot whitefliescould be observed against all neonicotinoid insecticides. No cross-resistance between theneonicotinoids was detected as well as no association with the activity of the P450 mi-crosomal oxidases. Only imidacloprid resistance correlated with carboxylesterase activity.Low to extremely high resistance was observed for insecticide bifenthrin (RF 49–1 243)which was associated with the frequency of the resistant allele in the sodium channel genebut not with the activity of the detoxification enzymes. Finally, the F331W mutation in theacetylcholinesterase enzyme ace1 gene was fixed in all B. tabaci populations from Cyprus.

Key words acetamiprid, B biotype, Bemisia tabaci, bifenthrin, Cyprus, imidacloprid,insecticide resistance, thiamethoxam

Introduction

The whitefly Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is one of the most important pests world-wide, causing damage to various crops via feeding ac-tivity, virus transmission and quality reduction (excreta)(Bedford et al., 1994; Denholm et al., 1998). Numer-ous biotypes with diverse capabilities have been identi-fied to date, by using genetic and physiological traits,while morphological differences have been detected insome cases (Costa & Brown, 1991; Rosell et al., 1997;

Correspondence: Emmanouil Roditakis, National Agricul-tural Research Foundation, Plant Protection Institute of Her-aklion, 71003 Heraklion, P.O. Box 2228, Greece. Tel: +30 2810302300; fax: +30 2810 245858; email: eroditakis@gmail.com

De Barro et al., 2000). B. tabaci is considered a cryp-tic species complex and there is a debate on the use ofthe term biotype (or race) (Brown et al., 1995; De Barroet al., 2005; De Barro et al., 2011). Here the term bio-type will be used for consistency with previous publishedliterature.

Biotypes B and Q in particular have been reported asthe most devastating and rapidly expanding biotypes incurrent agriculture. In several cases indigenous whiteflypopulations have been displaced by alien invasive biotypes(Liu et al., 2007). In Cyprus, both B and Q biotypeshave been found, however, the predominant biotype isB (Vassiliou et al., 2008; Papayiannis et al., 2009). B.tabaci is a major pest of vegetable and ornamental cropsof the island and its control is based mainly on chemicalinsecticides. In both greenhouse and open field crops,difficulties in controlling the pest have been reported,

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences

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B. tabaci insecticide resistance in Cyprus 31

raising substantial concerns on the efficacy of the appliedchemistries.

Bemisia tabaci has tremendous potential to develop re-sistance to different insecticides (Denholm et al., 1998;Horowitz et al., 2007). It is indicative that resistance to B.tabaci has been reported worldwide and to more than40 active ingredients (Arthropod Pesticide ResistanceDatabase, www.pesticideresistance.org). In conventionalcrop management systems, development of resistance re-sults in repeated applications of insecticides, increasingfurther the reliance upon chemicals, dramatically multi-plying crop production cost and causing concerns in thescientific community (Naranjo & Ellsworth, 2009).

This study is part of a project aiming to approach theissue of B. tabaci control in Cyprus under the perspec-tive of Integrated Pest Management (IPM). The particulartask involved the determination of B. tabaci resistance ata national level against organophosphate, pyrethroid andneonicotinoid insecticides, the major insecticide classesextensively used for pest management. In the present fun-damental study we combined toxicological, biochemicaland molecular tests to define the available tools in termsof chemical control, key components of IPM.

Material and methods

Whitefly strains

The susceptible reference strain SUD-S initially col-lected on cotton (Gossypium hirsutum L.) (Sudan, 1978)was obtained by IACR Rothamsted, UK, and it has beenmaintained in the absence of insecticides for the past30 years. SUD-S was maintained on cotton plants at25 ± 1◦C, 50%–60% RH and a photoperiod 16 : 8 hL : D.

Eight populations were collected during the survey fromgreenhouse and open-field crops as shown in Fig. 1. Mostof the populations were collected from July to October2007 (Table 1). Insects were collected from at least 10different sampling spots at each site and were transportedto the laboratory in a cool box within a few hours aftercollection. Whiteflies were reared on cotton plants for twoto three generations in large insect-proof cages coveredwith fine mess in a growth chamber at 25◦C (± 1◦C).

Insecticides

The following insecticides used were: the pyrethroidbifenthrin 250 g/L EC (Talstar; FMC, Philadelphia,PA, US) and the neonicotinoids imidacloprid 200 g/LSL (Confidor, Bayer AG, Leverkusen, Germany), thi-

Fig. 1 Whitefly collection sites during the 2007 resistance-monitoring survey in Cyprus.

amethoxam 250 g/L WG (Actara, Syngenta, Inofyta,Greece) and acetamiprid 200 g/L SP (Mospilan, NipponSoda Co, Tokyo, Japan).

Bioassays

Dose-response bioassays were based on a communalprocedure previously described (Roditakis et al., 2005).Briefly, a 39 mm diameter cotton leaf disc was immersedfor 5 sec in aqueous solution of insecticide containing0.2 g/L Triton X-100 (Merck, Darmstadt, Germany), al-lowed to dry and placed with the abraxial surface upper-most on a Petri dish embedded with thin sterile water agar(20 g/L). Twenty B. tabaci females were placed on eachleaf disc after a brief immobilization using carbon diox-ide. The dishes were inverted for the insects to orientatenormally and placed in a large controlled environmentroom (25 ± 1◦C, 50%–60% RH, 16 : 8 h L : D). Five con-centrations with five replications each (100 insects perconcentration) that gave between 0% to 100% mortalitywere tested. Final mortality was assessed after 24 h forbifenthrin and after 72 h for imidacloprid, thiamethoxamand acetamiprid.

Biotype diagnostics

A polymerase chain reaction – restriction fragmentlength polymorphism (PCR-RFLP) diagnostic assaybased on the mitochondrial DNA differences betweenbiotypes was used to determine the biotype of the pop-ulations. The procedure, which is described in detail inTsagkarakou et al. (2007) and in Vassiliou et al. (2008),implies the use of the AluI endonuclease to digest a mi-tochondrially encoded cytochrome c oxidase I (mtCOI)879 bp fragment that was PCR-amplified, yielding frag-ments of different size depending on the biotype. From

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32 V. Vassiliou et al.

Table 1 Whitefly collection sites, dates, host plants and cropping system. The application history is also displayed.

Applications prior to collectionCollection site Collection date Crop

Nic Pyr OPs/Carb IGRs

Agia Marina Pafos 20/9/2007 Cucumber open field 4 2 0 0Akaki Nicosia 26/7/2007 Hibiscus open field n/a n/a n/a n/aChlorakas Pafos 20/9/2007 Broccoli open field 3 2 5 0Chrysochou Pafos 22/10/2007 Rape open field n/a n/a n/a n/aFarmakas Nicosia 5/9/2007 Tomato open field 2 2 2 0Kiti Larnaca 12/7/2007 Bean open field 5 2 1 0

(under roof cover)Klirou Nicosia 24/9/2007 Squash open field 5 0 0 0Mandria Pafos 12/11/2007 Cucumber open field n/a n/a n/a n/aParalimni Ammochostos 4/10/2007 Squash open field 3 3 1 0Parekklisia 1 Lemesos 21/10/2006 Cucumber open field n/a n/a n/a n/aParekklisia 2 Lemesos 11/10/2007 Broccoli open field 6 0 0 1

Figures represent number of application with insecticides from the crop plantation grouped by mode of action (Nic = Neonicotinoids,Pyr = Pyrethroids, OPs = Organophosphates, Carb = Carbamates and IGRs = Insect Growth Regulators).

each population at least 15 individuals were included inthese assays.

Genomic DNA extractions from single females for bio-type and resistance diagnostics were performed as de-scribed in Tsagkarakou et al. (2007).

Resistance mutation diagnostic assays

PCR-RFLP assays were used to detect the resistancemutations as described in Tsagkarakou et al. (2009). Atotal of 136 and 168 females coming from eight popula-tions were analyzed for the kdr and ace1 resistance muta-tions, respectively. To ensure sequence conservation, PCRproducts from all diagnostic tests were also purified usingNucleoSpin

R©Extract (Macherey Nagel, Greece) and se-

quenced in both directions. Sequence data were analyzedusing BioEdit v. 7.0 software (Hall, 1999).

Biochemical assays

Mass homogenates of 50 adults were prepared usinga pestle in 0.25 mL ice cold sodium phosphate buffer(0.1 mol/L, pH 7.2), containing 0.2% Triton X-100. Thesupernatant was collected after a 10 min centrifugation at10 000 g at 4◦C. The protein concentration in the enzymesource was determined according to Bradford (1976) us-ing bovine serum albumin as a standard.

p-Nitrophenyl acetate esterase activity was measured at405 nm kinetically using 50 μL of the homogenate (10whitefly-equivalent) in the presence of 1 mmol/L paran-

itrophenol acetate. Total esterase activity was detectedusing 1- and 2-naphthyl acetate; 25 μL of the supernatant(equivalent to 5 whiteflies) was added to 200 μL sub-strate solution containing 2.25 mmol/L Fast Blue RR saltand 1 mmol/L 1- or 2-naphthyl acetate in sodium phos-phate buffer (0.1 mol/L, pH 7.2) as substrates. The activitywas measured continuously at 570 nm at 25◦C in a mi-croplate reader (Molecular Devices, Sunnyvale, CA, US)for 10 min in 4–6 replicates, utilising SOFTmax soft-ware (Molecular Devices) to fit kinetic plots by linearregression.

Glutathione S-transferase (GST) activity was deter-mined using 1-chloro-2,4-dinitrobenzene (CDNB) andreduced glutathione (GSH) as substrates, as previouslydescribed (Roditakis et al., 2006).

Cytochrome P450-dependent mono-oxygenase activitywas determined by O-deethylation of 7-ethoxycoumarinas previously described (Roditakis et al., 2006).

Data analysis

In dose-response bioassays, mortality data were anal-ysed by probit analysis based on Finney (Finney, 1964)using the Probit software 3.3 (Praxeme) (Raymond et al.,1993). This software tests the linearity of dose-mortalityresponse and provides the slope, the lethal concentrations(LC) and the 95% confidence limits (CL) of the LC foreach mortality line (Abbot, 1925).

Pair-wise comparisons were used to investigate for cor-relations between different attributes (enzyme activity,

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 30–39

B. tabaci insecticide resistance in Cyprus 33

LC50). A two-tailed test was applied to investigate thesignificance of Pearson Product Moment Correlation (2-tailed test). Analysis was conducted with SPSS 11 statis-tical programme (SPSS Inc. Chicago, IL, US).

Results

Insecticide resistance profile

The results of the probit analysis are presented inTable 2. The reference susceptible strain was always themost susceptible strain in this data set. The linearity wasrejected (P < 0.05) only in the case of bifenthrin.

Imidacloprid resistance status

For insecticide imidacloprid linearity in the dose-response mortality association was rejected in only onecase, suggesting homogeneity in the response of the pop-ulations. LC50 values ranged from 33.97 to 172 mg/L. TheCypriot populations exhibited high resistance factor lev-els (RF > 77). An extensive overlap of the 95% CL valuescould be observed. Populations from Farmakas and Kitiareas exhibited the highest resistance levels (RF = 281and 392 respectively).

Thiamethoxam resistance status

Linearity was rejected in six out of the eight cases tested.The Akaki and Kiti populations exhibited the highest het-erogeneity (P < 0.001). The population from Parekklisia2 exhibited resistance to thiamethoxam higher than the de-tection capacity of the bioassay experiment set up. LC50

values ranged from 230 to > 750 mg/L. The populationsexhibited high resistance levels (RF > 50). An extensiveoverlap of the 95% CL values could be observed. In ad-dition, only a four-fold difference in the responses to thi-amethoxam could be detected within the populations fromCyprus, suggesting a uniform status of thiamethoxam re-sistance all over the island.

Acetamiprid resistance status

Linearity was rejected in three out of the eight casestested. Populations Kiti and Paralimni exhibited the high-est heterogeneity (P < 0.001). LC50 values ranged from73.56 to 122.67 mg/L. The populations tested exhibitedlow resistance levels (RF < 12). A total overlap of the 95%CL values could be observed. The populations exhibitedstatistically uniform response to acetamiprid.

Bifenthrin resistance status

Linearity was rejected in five out of the eight casestested. Populations Akaki and Paralimni exhibited thehighest heterogeneity (P < 0.001). LC50 values rangedfrom 30.76 to 783 mg/L. The majority of the populationsexhibited high resistance levels (RF > 265). Populationsfrom Parekklisia 2 exhibited the lowest resistance levels(RF = 49) and from Paralimi the highest (RF = 1 243).An extensive overlap of the 95% CL values could also beobserved.

Cross-resistance and associations with applicationhistory records

In pair-wise comparisons absence of cross-resistancebetween neonicotinoid insecticides was detected(R2 < 0.1, P > 0.05). Moderate cross resistance be-tween neonicotinoid acetamiprid and pyrethroid bifen-thrin was observed (R2 = 0.630, P < 0.05). When com-paring responses to insecticides and application historyrecords of the respective populations, absence of signif-icant correlations was detected (R2 < 0.2 for neonicoti-noids and R2 = 0.406 for pyrethroids, P > 0.05 in allcases).

Detoxification enzyme activities

The results from the biochemical assays estimatingenzyme activities for the B. tabaci populations fromCyprus are presented in Table 3. High enzyme activitieswere detected in most cases when compared to SUD-S (up to a 6-fold increase). For GST activities, low orno increase has observed in relation to SUD-S. Com-parable levels of enzyme activities have been recentlydetermined by Roditakis et al. (2009). A moderate corre-lation was detected between imidacloprid resistance andcarboxylesterase (COE) activity (α- and β-naphthyl ac-etate, R2 = 0.51 and R2 = 0.65 respectively, P < 0.05,n = 7).

Biotype diagnostics

The biotype diagnostic tests showed that all B. tabacipopulations included in this study belong solely to bio-type B. This is in agreement with previous studies thatshowed that the B biotype is the predominant biotype ofB. tabaci in Cyprus (Vassiliou et al., 2008; Papayianniset al., 2009).

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 30–39

34 V. Vassiliou et al.

Table 2 Log-dose probit mortality results for B. tabaci populations tested with neonicotinoids imidacloprid, thiamethoxam andacetamiprid and the pyrethroid bifenthrin.

Insecticide Strain n† LC50 (mg/L) 95% CL Slope ± SE χ 2§ RF

Imidacloprid Sud-S 414 0.44 a‡ 0.38–0.51 3.86 ± 0.54 7.72 1GR176¶ 450 2.25 0.99–3.81 0.93 ± 0.14 1.10 5Klirou 568 33.97 bc 17.99–63.93 1.94 ± 0.55 25.99∗∗∗ 77Parekklisia 2 813 35.37 b 30.05–41.44 1.93 + 0.12 7.29 80Akaki 535 48.97 bc 40.42–58.34 2.64 ± 0.27 5.35 111Paralimni 530 67.43 c 50.38–89.41 1.17 ± 0.12 3.99 153Agia Marina 545 68.28 c 53.19–86.62 1.49 ± 0.14 3.63 155Chlorakas 512 71.23 c 51.06–97.66 1.11 ± 0.13 1.35 162Farmakas 559 123.51 d 97.87–155.95 1.56 ± 0.15 1.06 281Kiti 547 172.46 d 129.45–237.43 1.30 + 0.16 4.32 392

Thiamethoxam Sud-S 496 4.56 a 3.82–5.38 2.36 ± 0.31 2.08 1Klirou 555 230.02 b 175.81–300.22 2.79 ± 0.52 12.27∗∗ 50Akaki 573 362.26 bc 278.03–471.09 4.18 ± 1.13 19.05∗∗∗ 79Chlorakas 546 464.9 cd 356.81–608.70 2.48 ± 0.55 6.88∗ 102Kiti 472 556.98 bcde 89.83–3 453.42 3.79 ± 9.01 850.17∗∗∗ 122Agia Marina 554 568.56 de 478.06–710.52 2.15 ± 0.30 2.2 125Farmakas 579 589.99 cde 463.91–756.61 3.02 ± 0.76 9.47∗ 129Paralimni 544 622.63 cde 529.88–770.75 2.32 ± 0.29 0.2 137Parekklisia 2 520 750 1.26 + 0.41 8.67∗ 164

Acetamiprid Sud-S 557 10.08 a 8.68–11.81 2.47 ± 0.27 4.29 1Klirou 579 73.56 b 60.74–89.45 1.76 ± 0.14 6.98 7Parekklisia 2 459 67.1 bc 55.76–81.27 2.18 + 0.18 2.45 7Agia Marina 588 83.12 bc 67.09–103.51 1.66 ± 0.14 1.23 8Farmakas 547 85.67 bc 61.22–119.43 2.37 ± 0.40 8.23∗ 8Chlorakas 544 89.61 bc 73.51–108.74 2.29 ± 0.22 0.86 9Kiti 558 99.75 bc 53.86–184.99 1.41 ± 0.32 19.08∗∗∗ 10Akaki 554 110.87 b 88.11–140.41 1.57 ± 0.14 3.23 11Paralimni 610 122.67 bc 59.08–254.92 1.33 ± 0.36 23.58∗∗∗ 12

Bifenthrin Sud-S 554 0.63 a 0.39–1.01 2.28 ± 0.59 13.19∗∗ 1Parekklisia 2 380 30.76 b 20.76–45.25 1.93 + 0.36 9.02∗ 49Chlorakas 543 167.01 c 126.22–220.18 2.26 ± 0.34 9.21∗ 265Klirou 442 188.43 c 157.79–222.21 2.73 ± 0.29 6.9 299Farmakas 550 219.82 c 190.76–252.57 2.64 ± 0.22 0.09 349Akaki 545 239.37 cd 157.41–363.56 2.47 ± 0.57 21.17∗∗∗ 380Kiti 536 322.76 cd 205.12–507.37 1.67 ± 0.37 14.54∗∗ 512Agia Marina 550 335.13 d 277.76–403.13 1.86 ± 0.17 5.28 532Paralimni 547 782.98 d 314.03–1972.39 1.14 ± 0.43 24.66∗∗∗ 1243

†n, number of whiteflies tested. CL, confidence limits. RF, resistance factor.‡Different letters indicate non-overlap of confidence limits (P < 0.05).§Chi-square testing linearity of dose-mortality response:

∗P < 0.05;

∗ ∗P < 0.01;

∗ ∗ ∗P < 0.001.

¶Data for susceptible B. tabaci (Q biotype) originating form Crete are provided here for comparison – from Roditakis et al. (2009).

Resistance mutation diagnostic assays

Random sequencing of five individuals from differentcollection sites did not detect any polymorphism in theregions flanking the L925I and F331W mutations in the

para-type voltage-gated sodium channel gene and in theace 1 gene respectively.

PCR-RFLP diagnostic assays were used to analyzefield-collected females for the presence of the L925Imutation in the para-type sodium channel (pyrethroid

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 30–39

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resistance) and the F331W mutation in ace1 (organophos-phate [OP] resistance), in eight B. tabaci samples fromopen-field crops.

The F331W mutation in ace1 was fixed in the B. tabacipopulations as 166 of 167 females tested were homozy-gous for the resistance allele (Table 4). Only one individ-ual from Chrysochou area was found heterozygous, whileno individual was homozygous for the susceptible allele.

The frequency of resistant and susceptible alleles forthe sodium channel gene was determined in 136 individ-uals. The highest frequency of the susceptible alleles wasfound in two populations from Parekklisia 1 and Man-dria (0.29 and 0.27, respectively), but no bioassay data isavailable for the particular populations (Table 4). The pop-ulation from Parekklisia 2 that exhibited comparativelyhigh susceptible allele frequency (0.17) also exhibitedhigher susceptibility to bifenthrin (LC50 = 30.7 mg/L),while populations from Agia Marina and Paralimni withthe lowest frequency of the susceptible allele (0.05) dis-played the lowest susceptibility to the pyrethroid insecti-cide (LC50 = 335 and 783 mg/L, respectively). A strongpositive association was detected between the frequencyof resistant alleles and the resistance level to bifenthrin(R2 = 0.623, P < 0.05, n = 5).

Discussion

Insecticide resistance in B. tabaci has been extensivelystudied and was recently reviewed by Horowitz et al.(2007). Toxicological data from numerous countries andfor major insecticides have been congregated by theArthropod Pesticide Resistance Database and the Insec-ticide Resistance Action Committee (IRAC – www.irac-online.org). However, no information regarding B. tabaciinsecticide resistance status in Cyprus was available.

In the current study, a survey was conducted on B.tabaci resistance to major insecticide groups currentlyused for pest management in Cyprus. All tests were per-formed on biotype B B. tabaci populations, as indicatedby the biotype diagnostic assays. Moderate to high levelsof resistance were detected for neonicotinoids imidaclo-prid and thiamethoxam, while low resistance levels wereobserved for acetamiprid. When comparing the responsesof the different populations within Cyprus, uniform re-sponses could be observed, in general, for each neoni-cotinoid insecticide. In the case of acetamiprid, in partic-ular, no differences in the responses of the populationswere detected. The imidacloprid responses of the Cypriotpopulations were compared to a susceptible B. tabaci (Qbiotype) strain form Crete (Roditakis et al. 2009). Directcomparisons were possible only with the particular data

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 30–39

36 V. Vassiliou et al.

Table 4 Frequency of resistance alleles for the sodium channel and the Ace1 genes in different populations of Bemisia tabaci.

Sodium channel diagnostics Ace diagnostic

Population n Female genotypes Frequency of n Female genotypes Frequency of

r1r1 r1s ss r1 s rr rs ss Ace R Ace S

Agia Marina 23 22 1 0 0.95 0.05 20 20 0 0 1 0Chlorakas 19 17 2 0 0.89 0.11 19 19 0 0 1 0Chrysochou 14 13 1 0 0.95 0.05 19 18 1 0 0.95 0.05Klirou 13 11 2 0 0.84 0.16 20 20 0 0 1 0Mandria 15 11 4 0 0.73 0.27 23 23 0 0 1 0Paralimni 14 13 1 0 0.95 0.05 20 20 0 0 1 0Parekklisia 1 14 10 4 0 0.71 0.29 24 24 0 0 1 0Parekklisia 2 24 20 4 0 0.83 0.17 23 23 0 0 1 0

All field-collected samples were analyzed by polymerase chain reaction amplification of specific alleles (PASA) and polymerase chainreaction – restriction fragment length polymorphism. Naming of the sodium channel alleles I925 . . . T929 (r1) and L925 . . . T929 (s)are according to Alon et al. (2006).n, number of females.

set since the methodology used in both projects was iden-tical. The susceptible strain (GR176) exhibited a compa-rable resistance level to the reference strain SUD-S andsignificantly lower LC50 values (15- to 76-fold) comparedto the populations from Cyprus. The resistance levels toimidacloprid reported in this study are considered high;however, similar or higher resistance levels have been re-ported by Roditakis et al. (2009).

The resistance levels detected in this study may explainthe problematic performance of insecticide treatments inCyprus. Simulated field studies have shown a good asso-ciation between resistance factors and field performance(Cahill et al., 1996). However, the particular toxicologi-cal assays do not provide a direct determination of lethalconcentrations in the field since many additional bioticand abiotic factors (ecological and technical) are also in-volved at the farmer level of insecticide use (Cahill et al.,1996).

Resistance to neonicotinoids has been recorded in bothB and Q B. tabaci biotypes (Byrne et al., 2003; Nauen &Denholm, 2005; Wang et al., 2009; Schuster et al., 2010).and it has been associated with enhanced oxidative detox-ification by cytochrome P450 mono-oxygenases (Rauch& Nauen, 2003). However, we did not observe a strong as-sociation between resistance to neonicotinoids and meanCytP450 activity, possibly due to the small number of thepopulations and their heterogeneity.

Cross-resistance in the class of neonicotinoids has beenstudied extensively and is considered a contradictive is-sue. High cross-resistance among neonicotinoid insecti-cides was initially reported by Nauen et al. (2002). Re-

cent studies in China (Wang et al., 2009) and the US(Schuster et al., 2010) have also demonstrated cross-resistance among neonicotinoid insecticides. However,inconsistency in the neonicotinoid cross-resistance pat-tern has been reported by Prabhaker et al. (2005) andby Horowitz et al. (2004). Absence of cross-resistanceamong the neonicotinoids was detected in this study.

Over-expression of a single P450 gene, CYP6CM1, hasbeen closely related to imidacloprid resistance in both Band Q biotypes (Karunker et al., 2008). Recent studiesrevealed that recombinant BtCYP6CM1vQ, a functionalmono-oxygenase complex, has the potential to metabolizeimidacloprid (Karunker et al., 2009) but not acetamipridor thiamethoxam (Roditakis et al., 2010). Such findingsmay partially explain the variability reported on the neon-icotinoid cross-resistance issue.

High variability was observed in the susceptibility topyrethroid bifenthrin among the Cypriot whitefly popu-lations, where low to extremely high levels of resistancehave been detected. Resistance to pyrethroids has beenassociated with metabolic detoxification both by elevatedesterases and mono-oxygenases (Horowitz et al., 1988;Prabhaker et al., 1988; Dittrich et al., 1990; Roditakiset al., 2006). In addition, two independent target site mod-ifications, both associated with resistance to pyrethroids,were identified: the L925I and the T929V resistance mu-tations in the IIS4–5 linker of B. tabaci para-type voltagegated sodium channel gene (Morin et al., 2002; Alonet al., 2006; Roditakis et al., 2006). However, the T929Vmutation has not been reported in the B biotype (Alonet al., 2006).

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B. tabaci insecticide resistance in Cyprus 37

Recently, simple PCR–agarose gel visualization-basedassays were developed to reliably monitor pyrethroid andOPs resistance mutations. These assays were used to mon-itor the frequency of the resistance mutations in a largenumber of field-caught Q biotype B. tabaci from Crete(Greece) (Tsagkarakou et al., 2009). In this study the fre-quency of L925I mutation of the sodium channel gene wasinvestigated in the B biotype populations from Cyprus.It was shown that the frequency of the mutation variedamong the collected populations. An association was de-tected between the frequency of the resistant alleles andbifenthrin resistance. In contrast, no association was de-tected between the detoxifying enzyme activities and theresistance to the pyrethroid insecticide, suggesting that theparticular target site modification may be a key elementfor bifenthrin resistance in the Cypriot populations.

Resistance to OPs involves both metabolic (mono-oxygenases and carboxylesterases) and target site resis-tance mechanisms (Byrne & Devonshire, 1993; Byrneet al., 1994; Byrne & Devonshire, 1997). The over-expression of the carboxylesterase coe1 was associatedwith OP-resistant B and Q biotype B. tabaci populations(Alon et al., 2008; Morin et al., 2010). In addition theF331W mutation in the acetylcholinesterase enzyme ace1gene, conferring resistance to OPs, was identified in B.tabaci (Alon et al., 2008). This mutation was almost fixedin the B. tabaci populations from Cyprus. The same sit-uation was observed in a similar survey for Q biotypepopulations in Crete (Tsagkarakou et al., 2009) reflectingthe high OP selection pressure in both islands.

In conclusion, a comprehensive study on B. tabaci in-secticide resistance status was conducted in Cyprus. Im-portant elements, both on the susceptibility of B. tabacito chemistries as well as on the possible involvement ofknown resistance mechanisms were present. However, dueto the importance of the pest in crop production of thecountry, it is of critical importance to monitor the fluctua-tions in resistance level to the aforementioned chemistriesover time, as well as to investigate the baseline suscepti-bility to novel chemistries, which will play a key role infuture integrated pest management.

Acknowledgments

The project was funded by the Research Promotion Foun-dation and the Agricultural Research Institute of Cyprus,under the acronym “BETAREMO – AEIFO/0506/15”.The authors would like to thank Mr. Dimitris Kourriswho conducted the whitefly field collections and the tox-icological bioassays, and Mr. Andreas Hadjinikolis forhis valuable assistance in collecting Bemisia populations.

J. Vontas was supported by grants from Bayer Crop Sci-ence and Hellenic Secretariat General for Research andTechnology (Vioalevrioi).

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Accepted October 7, 2010

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 30–39