Effects of spirodiclofen on reproduction in a susceptible and resistant strain of Tetranychus urticae (Acari: Tetranychidae)

Exp Appl Acarol (2009) 47:301–309 DOI 10.1007/s10493-008-9226-y EVects of spirodiclofen on reproduction in a susceptible and resistant strain of Tetranychus urticae (Acari: Tetranychidae) Steven Van Pottelberge · Jahangir Khajehali · Thomas Van Leeuwen · Luc Tirry Received: 5 September 2008 / Accepted: 2 December 2008 / Published online: 20 December 2008  Springer Science+Business Media B.V. 2008 Abstract In this study the reproductive capacity of a laboratory-selected spirodiclofen resistant strain was investigated after treatment with spirodiclofen. Firstly, females were exposed to diVerent concentrations of spirodiclofen (200 and 1,000 mg/l) during 6, 12 or 24 h. In contrast to the susceptible parental strain, the fecundity and fertility of resistant mites was not aVected by treatment with these concentrations after any time of exposure tested. Secondly, pre-treatment of the resistant females with the synergists PBO or DEF could increase the inhibitory eVect of spirodiclofen on reproduction, demonstrating the possible involvement of monooxygenases and esterases in metabolic detoxiWcation of the acaricide. Because spirodiclofen interferes with lipid biosynthesis, total lipid content was measured in female adults. There were no signiWcant diVerences between treated and non-treated female adults, both in the susceptible and resistant strain. However, the total lipid content in the resistant females was signiWcantly higher than in susceptible females. Our data shows that the detection of spirodiclofen resistance should not be limited to mortality bioassays with eggs or larvae, but should be combined with inhibitory studies on female fertility and fecundity. Keywords Spirodiclofen resistance · Tetranychus urticae · Reproduction · DetoxiWcation · Lipid content Introduction Spirodiclofen, a tetronic acid derivative, is a selective, non-systemic acaricide with excellent eYcacy against all developmental stages of most economically important phytophagous mite species (Rauch and Nauen 2002). In contrast to the acute toxicity on eggs and immature stages, its activity against female adults is slower and less pronounced. However, spirodiclofen treatment strongly inXuences female fecundity in that the produced eggs S. Van Pottelberge · J. Khajehali · T. Van Leeuwen (&) · L. Tirry Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium e-mail: thomas.vanleeuwen@ugent.be 1C 302 Exp Appl Acarol (2009) 47:301–309 cannot be deposited. As a consequence, treated females die after several days due to the high number of eggs accumulated in their body. Spirodiclofen also aVects the fertility, i.e. eggs laid by females exposed to sublethal doses are not fertile (WachendorV et al. 2002; Nauen 2005; Marcic 2007). Considering that eggs and immatures account for around 90% of a population with a stable age distribution (Sabelis 1985), Marcic (2007) noted that a relatively low concentration of spirodiclofen can eliminate a considerable part of a Tetranychus urticae population. Furthermore, the decrease in fecundity and fertility in treated females results in signiWcantly reduced population growth rates, compared to control (Marcic 2007). Spirodiclofen diVers from other acaricides in its speciWc mode of action. The compound interferes with lipid biosynthesis, blocking the enzyme acetyl-CoA carboxylase that allows mites to form important fatty acids (Dekeyser 2005; Bretschneider et al. 2007). Biochemical studies showed that the lipid content in treated female adults of T. urticae was signiWcantly lower than in untreated females (WachendorV et al. 2002). No Weld resistance has yet been reported. However, laboratory-selected spirodiclofen resistant strains of T. urticae were characterised both biologically (cross-resistance pattern, genetics) and biochemically, emphasising the importance of metabolic detoxiWcation of the active compound (Rauch and Nauen 2002; Van Pottelberge et al. 2008). Since previous studies (Rauch and Nauen 2002; Van Pottelberge et al. 2008) quantiWed and investigated resistance mechanisms on the larval stages, the objective of the current study was to evaluate how this resistant genotype inXuences the reproductive capacity of female resistant mites after treatment with spirodiclofen. Because of the alleged interference in lipid biosynthesis, total lipid content in treated and non-treated female adults was measured. In addition, it was investigated how treatment with synergists inhibiting major detoxiWcation routes inXuenced the reproduction after treatment with spirodiclofen in susceptible and resistant strains. Materials and Methods Spider mites The reference laboratory susceptible strain (LS-VL) of T. urticae was originally collected in October 2000 from roses in a garden near Ghent (Belgium), where pesticides had not been used for at least 10 years (Van Leeuwen et al. 2004). The laboratory-selected spirodiclofen resistant strain (SR-VP) was obtained after successive applications of spirodiclofen as described previously (Van Pottelberge et al. 2008). Bio-assays on larvae showed a resistance ratio of 274. Mites of the LS-VL and SR-VP strain were reared on potted kidney bean (Phaseolus vulgaris L. cv. Prelude) plants in a climatically controlled room at 26(§0.5)°C, 60% relative humidity (RH) and a 16/8 h light/dark photoperiod. For the SR-VP strain, the bean plants were sprayed with 5,000 mg/l spirodiclofen containing 0.25 mg/l Citowet, a wetting agent. During all experiments, the same climatic conditions were used. Chemicals Spirodiclofen, commercial formulation Envidor® (suspension concentrate, 240 g a.i./l, Bayer Cropscience), was purchased from Fyto Vanhulle (Belgium). All other chemicals were of analytical grade and purchased from Sigma–Aldrich (Belgium). 1C Exp Appl Acarol (2009) 47:301–309 303 EVects of spirodiclofen on reproductive capacity In order to study the inXuence of spirodiclofen on fecundity and fertility, leaf discs (ca. 25 cm2) of kidney bean plants (Phaseolus vulgaris L. ‘Prelude’), placed on wet cotton wool in a Petri dish (9 cm diam), were sprayed with 200 mg/l (the maximum Weld-application rate) and 1,000 mg/l spirodiclofen by means of a Cornelis spray tower (1 bar pressure) (Van Laecke and Degheele 1993). This resulted in a homogenous spray coverage of 1.5 § 0.1 mg (mean § SE) aqueous acaricide deposit per cm2. Controls were sprayed with distilled water alone. Prior to the start of the experiment, a cohort of eggs that developed synchronously to adults was established for the strains LS-VL and SR-VP. After their preoviposition period, mated females from the synchronised cultures were transferred to the treated leaf discs after drying of the spray coverage. After a 6 12 or 24 h, respectively, all living mites were collected from the treated discs and placed on detached non-sprayed leaves. Immediately four replicates of 10 females were chosen randomly from these discs (with mites of the same age that were treated with the same spirodiclofen concentration during an identical pre-treatment period), placed on the upper side of 9 cm2 square-cut kidney bean leaf discs and allowed to lay eggs for 24 h. After this period, eggs were counted and surviving females were transferred to a new leaf disc. Dead females were replaced by individuals from the above mentioned detached leaves, in order to have 10 mites again on every leaf disc. Successive transferring of females was repeated from 3 to 4 times. Since mortality of the LS-VL females was very high after a 24 h contact of 1,000 mg/l spirodiclofen, the experiment for this pre-treatment period was stopped after 1 day. Eggs were left untouched on the original leaf disc for subsequent determination of the number of oVspring that survived until adulthood. The hatch rate of the eggs was calculated as the percentage of eggs that developed to the adult stage in proportion to the total number of eggs laid during 24 h (including shrivelled eggs). Data were analysed by ANOVA with means separated by Tukey (P < 0.05). Total lipid content in Tetranychus urticae For the determination of the total lipid content, procedures based on the method of Van Handel (1985) and described previously (WachendorV et al. 2002) were used. Two hours, 2 and 5 days after spirodiclofen treatment, groups of 10 living female adults (respectively 4, 6 and 9 days old) were randomly selected from (untreated) leaf discs with mites that had been exposed for 12 h to spirodiclofen (200, 1,000 mg/l) or distilled water (control), as described above. Additionally, groups of 100 adult females were sampled from the LS-VL and SR-VP strain and were pre-treated for 12 h with spirodiclofen. Surviving females were transferred to a non-sprayed leaf disc and lipid content was measured after 2 days. Data were analysed by ANOVA with means separated by Tukey (P < 0.05). If the assumption of normality or equality of variance was not met, a non-parametric Kruskal– Wallis test was used. Synergism studies In order to check the eVect of synergists on reproductive capacity, gravid adult female mites of the LS-VL and SR-VP strains were placed on leaf discs sprayed with PBO (piperonylbutoxide), DEF (S,S,S-tributyl-phosphorotrithioate) or DEM (diethylmaleate). PBO, DEF and DEM were used to inhibit cytochrome P450 monooxygenases, esterases and glutathione-S-transferases, respectively. After 24 h, living mites were collected and 1C 304 Exp Appl Acarol (2009) 47:301–309 transferred to leaf discs that had been sprayed with diVerent concentrations of spirodiclofen (ranging from 6.25 to 400 mg/l for LS-VL and from 312.5 to 20,000 mg/l for SR-VP) or with distilled water (control). After 24 h exposure, four replicates of 10 mites from each spirodiclofen concentration were transferred to unsprayed leaf discs for oviposition. The day after, the female mites were removed. The eggs were left to develop to adults, which were counted immediately after their Wnal moult. Based on preliminary tests, the doses of PBO, DEF and DEM were chosen as the highest dose that caused maximum 5% mortality and no inhibition of fecundity (2,000 mg/l PBO, 20 mg/l DEF, 2,000 mg/l DEM). For spraying, PBO (»90% purity), DEF (98% purity) and DEM (97% purity) were dissolved in a mixture of N,N-dimethylformamide and emulsiWer W (3:1 by weight) and subsequently diluted with distilled water (1,000-fold). Inhibition by spirodiclofen (the diVerence between the number of oVspring from mites from leaves treated with distilled water and those treated with spirodiclofen) was expressed as a percentage of the number of oVspring in mites treated with blank formulation. Spirodiclofen concentrations inhibiting 50% of the reproductivity (IC50-values) for LS-VL were calculated using the logistic equation of the Wtted curves. The concentration-inhibition data for SR-VP were Wtted with a second order regression curve. Results EVects of spirodiclofen on reproductive capacity The daily number of eggs reaching adulthood laid per 10 LS-VL or SR-VP females after a 6, 12 and 24 h treatment with 200 and 1,000 mg/l spirodiclofen is given in Table 1. At the start of the 12 h treatment, the synchronous cohort of mites was a few days older compared to other treatment times. A clear diVerence can be observed between the susceptible and resistant strain. Treatment of SR-VP with spirodiclofen most often did not aVect the egg-laying capacity of the females, even at the highest dose tested. The percentage of eggs that developed to adults Xuctuated between 87 and 100. The observed oviposition pattern showed an increase in egg laying from the second day of the adult stage onward to a maximum of 9–10 eggs/24 h/female, followed by a decrease. Fecundity and fertility of mites from the spirodiclofen resistant strain were obviously not aVected, even at 1,000 mg/l spirodiclofen. In contrast, when mites of the LS-VL strain came into contact with leaves sprayed with spirodiclofen, signiWcant diVerences in reproductive capacity could be observed. Spirodiclofen aVects female adults quite rapidly, even after a short exposure period of 6 h. The Wrst day a signiWcant decrease in female fecundity and fertility was found for both concentrations. Females still deposited an amount of eggs, but a lot of these eggs were shrivelled and not viable. However, mites treated with 200 and 1,000 mg/l recovered from the Wrst and fourth day onward, respectively. Pre-treatment of LS-VL females with 1,000 mg/l for 12 h, seemed to have a longer lasting eVect, especially on fecundity. The slight increase in number of eggs was caused by only a few females that started to lay viable eggs. The eVect of 200 mg/l during 12 h was similar to that of 6 h exposure. First a decrease and than a full recovery of the reproductivity was observed. Twenty-four hours of spirodiclofen exposure decreased the number of deposited eggs to almost zero. At 1,000 mg/l, none of the few eggs hatched. 1C Strain Time Concentration Female adult age (days) (h) (mg/l) 2–3 3–4 LS-VL 6 12 0 200 70.0 § 0.9 a (90) 75.3 § 1.6 a (82) 29.3 § 3.3 b (38) 85.3 § 4.4 a (85) 1,000 8.0 § 2.8 c (23) 46.0 § 3.0 b (80) 0 200 1,000 24a 0 200 1,000 SR-VP 6 12 – – – – 60.5 § 2.9 a (87) – 2.5 § 1.5 b (63) – 0.0 § 0.0 b (0) – 0 200 53.7 § 0.8 (87) 58.8 § 3.5 (90) 1,000 58.3 § 2.6 (91) 0 200 1,000 24 – – 4–5 5–6 6–7 105.5 § 1.2 a (98) 105.0 § 5.6 a (95) 101.0 § 7.9 a (91) 106.5 § 1.6 a (98) 73.5 § 1.6 b (91) 70.5 § 4.4 b (93) 110.3 § 2.5 a (98) 107.8 § 3.8 a (98) 21.5 § 6.5 b (41) 68.5 § 12.5 b (73) 2.5 § 1.7 c (40) – – – 78.0 § 2.3 a (90) 69.0 § 1.2 b (88) 90.8 § 0.9 (96) 87.0 § 2.3 (100) 16.0 § 10.1 c (76) – – – 97.3 § 4.8 (94) 88.5 § 3.4 (89) 7–8 8–9 100.0 § 6.1 (88) 107.0 § 3.3 (89) 96.8 § 7.3 (94) 104.0 § 6.0 (92) – – 83.8 § 9.3 (93) 97.7 § 5.5 (98) – 95.5 § 7.7 a (94) 90.0 § 3.9 a (93) 96.8 § 9.0 a (96) 80.8 § 7.1 a (98) 96.3 § 8.5 a (98) 71.8 § 8.0 a (96) 18.3 § 10.5 b (94) 22.5 § 9.0 b (98) 18.5 § 6.7 b (100) – – – 105.5 § 1.6 (97) 90.8 § 4.2 (89) – – – – – – Exp Appl Acarol (2009) 47:301–309 Table 1 Daily number of Tetranychus urticae eggs reaching adulthood laid per 10 LS-VL or SR-VP females after a 6, 12 and 24 h pre-treatment with 200 and 1,000 mg/l spirodiclofen 103.0 § 3.0 a (97) 91.5 § 2.8 b (93) 73.3 § 0.9 ab (91) 90.8 § 2.6 (96) 84.8 § 1.0 (88) 91.8 § 5.0 (93) – – – – 90.3 § 2.6 (98) 90.5 § 5.0 (97) 104.5 § 1.8 (98) 99.0 § 10.3 (91) 101.3 § 2.6 (95) 94.5 § 7.8 (95) 100.5 § 4.6 (97) 100.5 § 7.2 (96) 85.5 § 1.3 b (89) 88.0 § 1.4 (94) 84.0 § 4.8 (93) – – 88.8 § 2.3 (94) 102.8 § 5.5 (90) 101.3 § 2.7 (98) 101.5 § 4.5 (94) 85.0 § 5.6 (96) 0 200 62.0 § 4.4 (96) 54.8 § 2.5 (90) – – – – – – – – – – – – 1,000 63.8 § 2.1 (98) – – – – – – Means (§SEM, n = 4) within a column (and same treatment time) followed by diVerent letters are signiWcantly diVerent (Tukey, P < 0.05) The hatch rate of eggs (%) is written in brackets Since mortality of the LS-VL females was very high after a 24 h pre-treatment of 1,000 mg/l spirodiclofen, the experiment was stopped after 1 day 305 1C a 306 Exp Appl Acarol (2009) 47:301–309 Fig. 1 Inhibition of reproductivity of LS-VL Tetranychus urticae females after 24 h of spirodiclofen pretreatment without (䊉) or with prior application of synergists PBO (䊊), DEF (▼) or DEM (
). Data are mean values § SEM (n = 4) EVects of spirodiclofen after pre-treatment with synergists The eVect of spirodiclofen on reproduction after pre-treatment with PBO, DEF and DEM is presented in Figs. 1 and 2. Without synergists, the IC50 value for the LS-VL strain was 43.7 mg/l spirodiclofen. A similar value (42.2 mg/l) was obtained when the susceptible strain was pre-treated with DEM. Pre-treatment with PBO and DEF increased the inhibitory eVect of spirodiclofen on reproduction. The IC50 values decreased to 10.7 (DEF) and 16.9 mg/l (PBO). It can be noted that the slopes of these curves are less steep than that of the control, indicating a less homogeneous response. In SR-VP mites, spirodiclofen could not inhibit the reproductive capacity more than 20%, even at highest concentrations (Fig. 2), and the IC50 value could not be calculated. Pre-treatment with DEM even reduced the inhibitory eVect of spirodiclofen. However, as observed in LS-VL, the synergistic eVect of PBO and DEF treatment was clear. The estimated theoretical IC50 values obtained by extrapolation of the regression curves were 8 £ 103 (PBO) and 59 £ 103 mg/l (DEF). EVects of spirodiclofen on lipid content Mites of the LS-VL or the SR-VP strain that were exposed to diVerent concentrations of spirodiclofen did not show any signiWcant diVerence in total lipid content compared with the controls (Table 2). Also, no signiWcant diVerences could be observed between diVerent measurements in time. Only the lipid content in SR-VP mites treated with 1,000 mg/l measured after 5 days was signiWcantly diVerent from the lipid content 2 h after treatment (Table 2). When the lipid content of 100 mites (instead of 10) was measured, again no signiWcant diVerences were found between treatments within each strain (results not 1C Exp Appl Acarol (2009) 47:301–309 307 Fig. 2 Inhibition of reproductivity of SR-VP Tetranychus urticae females after 24 h of spirodiclofen pretreatment without (䊉) or with prior application of synergists PBO (䊊), DEF (▼) or DEM (
).Data are mean values § SEM (n = 4) Table 2 Total lipid content in Tetranychus urticae female adults of LS-VL and SR-VP strain (g/10 spider mites) after a 12 h pre-treatment with 200 and 1,000 mg/l spirodiclofen Strain LS-VL SR-VP Concentration (mg/l) 0 200 1,000 0 200 1,000 Female adult age (days) 4 6 9 21.7 § 2.3 21.4 § 0.2 19.0 § 0.9 25.9 § 1.4** 34.7 § 3.1 25.9 § 0.7 a 21.2 § 0.2 23.5 § 1.9 19.6 § 0.5 33.7 § 4.6 35.9 § 1.9 29.0 § 1.6 ab 25.1 § 2.1 24.5 § 2.4 21.4 § 0.8 34.1 § 5.2** 36.1 § 2.3 32.0 § 0.7 b Means (§SEM, n = 3) within a row followed by diVerent letters are signiWcantly diVerent (non-parametric test, Kruskal–Wallis, P > 0.05) Means within a column (and same strain) are not signiWcantly diVerent ** Not signiWcantly diVerent with LS-VL (Kruskal–Wallis, P > 0.05) shown). However, comparing both strains, the lipid content in SR-VP mites appeared to be signiWcantly higher than that in LS-VL mites, as can also be observed in most measurements in Table 2. Discussion Spirodiclofen strongly inhibits reproduction of the susceptible LS-VL strain in a dosedependent manner. WachendorV et al. (2002) and Marcic (2007) demonstrated similar inhibitory eVects. 1C 308 Exp Appl Acarol (2009) 47:301–309 Spirodiclofen eVects were most evident on the Wrst day after treatment, when not only fecundity, but also fertility of the eggs decreased, i.e. more non-viable eggs were laid. These eggs looked dim and were shrivelled. The symptoms of poisoning observed in the adult susceptible females were partly similar to those described by WachendorV et al. (2002). Some of the females treated with high spirodiclofen concentrations had a bigger size (due to an accumulation of the eggs in the genital tract), and, even more striking, they had egg remnants sticking to the ovipositor. These females were unable to lay eggs, often got stuck on the leaf due to this sticky appendage, and died within a few days. Another important observation, also reported by Marcic (2007), is the ability of females to recover from spirodiclofen intoxication after treatment with lower concentrations and/or shorter exposure periods. Surprisingly, the fecundity and fertility of SR-VP females was not at all aVected by spirodiclofen treatments. The hatching rate of the eggs remained very high, ranging from 87 to 100%. Remarkably, eggs of the SR-VP strain had a more brownish appearance, compared to the white eggs of the LS-VL strain. Even after 24 h exposure to the highest single dose sprayed in these bioassays (>10,000 mg/l), inhibition was lower than 20%. Due to this extreme resistance, it was not possible to calculate an IC50 value, but it’s clear it would be too high to be of any practical signiWcance in the Weld. Therefore, the resistance ratio based on the IC50 between resistant and susceptible mites must be much higher than the resistance ratio calculated with the LC50 values in larvae (=274) (Van Pottelberge et al. 2008). Reproduction in SR-VP is therefore less aVected by high spirodiclofen concentrations. Moreover, in the absence of spirodiclofen, the number of oVspring of the SR-VP strain was comparable to that of the susceptible strain. It seems that, in our experiments, selecting for high resistance did not aVect fecundity and fertility of the female adults. The possible involvement of monooxygenases and esterases in spirodiclofen detoxiWcation has already been reported in larval bioassays by Rauch and Nauen (2002) and Van Pottelberge et al. (2008), and is conWrmed in this study. Pre-treatment of resistant mites with PBO and DEF could partially restore the inhibition of the reproductive capacity caused by spirodiclofen. Although these synergists did not change the toxicity of spirodiclofen in larvae of the LS-VL strain (Van Pottelberge et al. 2008), they could synergize the reproductive eVect in females of this strain 2.3- and 4.1-fold, respectively. For both the resistant and susceptible strain, the eVect of DEM did not diVer from the control, underlining the minor role of glutathione-S-transferases in spirodiclofen detoxiWcation. Studies carried out by WachendorV et al. (2002) aiming at elucidating the exact molecular mode of action of spirodiclofen revealed complete inhibition of de novo lipid biosynthesis in treated female adults and thus, a signiWcant lower lipid content compared to untreated females 5 days after treatment with 200 mg/l spirodiclofen. These results could not be conWrmed in our experiments, where no signiWcant diVerences could be observed between treated and untreated mites. Based on this experiment it was not really possible to make conclusive remarks about de novo lipid biosynthesis. Noteworthy are the higher values of total lipid content in the SR-VP strain compared to the LS-VL strain, which could be an essential condition for spirodiclofen resistance. A more detailed analysis of the diVerent lipids may generate more relevant information to elucidate the mode of action of spirodiclofen and the role of speciWc lipids in this process. Based on our results, selection for spirodiclofen resistance can yield a strain with female adults that are still able to produce and lay fertile eggs after treatment with very high concentrations. It appears that studying spirodiclofen resistance based only on resistance ratios in larvae is not suYcient to estimate the total magnitude of resistance development. Determination of the inhibitory eVect on fecundity and fertility of resistant adult females should 1C Exp Appl Acarol (2009) 47:301–309 309 be equally addressed. The observation that spirodiclofen exposure no longer inXuences reproductivity in female resistant mites has important consequences for resistance management in the Weld. It is clear that adults surviving Weld concentrations (possibly still lethal to larvae), are able to migrate to untreated regions or newly formed leaves. In addition, since young females can lay eggs up to 30 days, they probably can survive on treated plants long enough to at least partially overcome the long residual spirodiclofen toxicity. These females carry the resistance traits and allow continuous selection in the larval stage by future treatments. If resistance development in the Weld would manifest itself Wrst as a lack of inhibition of reproduction, it is advisory to include the assessment of fecundity and fertility when monitoring resistance development in the Weld. Using spirodiclofen as an ovi- and larvicide, in combination with a diVerent mode of action group targeting adults, might be a good resistance management strategy. References Bretschneider T, Fischer R, Nauen R (2007) Inhibitors of lipid synthesis (acetyl-CoA-carboxylase inhibitors). In: Krämer W, Schirmer U (eds) Modern crop protection compounds. Wiley, Weinheim, pp 909–925 Dekeyser MA (2005) Acaricide mode of action. Pest Manag Sci 61:103–110. doi:10.1002/ps.994 Marcic D (2007) Sublethal eVects of spirodiclofen on life history and life-table parameters of two-spotted spider mite (Tetranychus urticae). Exp Appl Acarol 42:121–129. doi:10.1007/s10493-007-9082-1 Nauen R (2005) Spirodiclofen: mode of action and resistance risk assessment in tetranychid pest mites. J Pestic Sci 30:272–274 Rauch N, Nauen R (2002) Spirodiclofen resistance risk assessment in Tetranychus urticae (Acari: Tetranychidae): a biochemical approach. Pestic Biochem Physiol 74:91–101. doi:10.1016/S0048-3575(02) 00150-5 Sabelis MW (1985) Reproductive strategies. In: Helle W, Sabelis MW (eds) Spider mites, their biology, natural enemies and control, vol 1A. Elsevier, Amsterdam, pp 265–278 Van Handel E (1985) Rapid determination of total lipids in mosquitoes. J Am Mosq Control Assoc 1:302–304 Van Laecke K, Degheele D (1993) EVect of insecticide synergist combinations on the survival of Spodoptera exigua. Pestic Sci 37:283–288. doi:10.1002/ps.2780370308 Van Leeuwen T, Stillatus V, Tirry L (2004) Genetic analysis and cross-resistance spectrum of a laboratoryselected chlorfenapyr resistant strain of two-spotted spider mite (Acari: Tetranychidae). Exp Appl Acarol 32:249–261. doi:10.1023/B:APPA.0000023240.01937.6d Van Pottelberge S, Van Leeuwen T, Khajehali J, Tirry L (2008) Genetic and biochemical analysis of a laboratory-selected resistant strain of Tetranychus urticae Koch (Acari: Tetranychidae). Pest Manag Sci. doi:10.1002/ps.1698 WachendorV U, Nauen R, Schnorbach HJ, Rauch N, Elbert A (2002) The biological proWle of spirodiclofen (Envidor®)—a new selective tetronic acid acaricide. PXanzenschutz-Nachrichten Bayer 55:149–176 1C