Lethal and sublethal effects of cypermethrin and ...

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Lethal and sublethal effects of cypermethrin and methoxyfenozide on the larvae of Rachiplusia nu (Guenee) (Lepidoptera: Noctuidae) a

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Federico Rimoldi , Marilina N. Fogel , Marcela I. Schneider & Alicia E. Ronco

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Centro de Investigaciones del Medio Ambiente, Departamento de Química, Facultad de Ciencias Exactas, CONICET, Universidad Nacional de La Plata, La Plata, Argentina b

Centro de Estudios Parasitológicos y de Vectores (CCT La Plata CONICET-UNLP), Calle 2 No 584, (1900) La Plata, Argentina Available online: 1 January 2011

To cite this article: Federico Rimoldi, Marilina N. Fogel, Marcela I. Schneider & Alicia E. Ronco (2011): Lethal and sublethal effects of cypermethrin and methoxyfenozide on the larvae of Rachiplusia nu (Guenee) (Lepidoptera: Noctuidae), Invertebrate Reproduction & Development, DOI:10.1080/07924259.2011.591177 To link to this article: http://dx.doi.org/10.1080/07924259.2011.591177

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Invertebrate Reproduction & Development 2011, 1–9, iFirst

Lethal and sublethal effects of cypermethrin and methoxyfenozide on the larvae of Rachiplusia nu (Guenee) (Lepidoptera: Noctuidae) Federico Rimoldia, Marilina N. Fogela, Marcela I. Schneiderb* and Alicia E. Roncoa a

Centro de Investigaciones del Medio Ambiente, Departamento de Quı´mica, Facultad de Ciencias Exactas, CONICET, Universidad Nacional de La Plata, La Plata, Argentina; bCentro de Estudios Parasitolo´gicos y de Vectores (CCT La Plata CONICET-UNLP), Calle 2 No 584, (1900) La Plata, Argentina

Downloaded by [Marcela Schneider] at 11:58 27 June 2011

(Received 8 December 2010; final version received 23 May 2010) Rachiplusia nu is a defoliator lepidopteran species considered a potential pest in soybean and other crops. Cypermethrin is the main insecticide used in Argentina. Worldwide trends in pest control promote the use of insecticides with high specificity like methoxyfenozide; however, their usage in South America is still incipient. The effectiveness of cypermethrin and methoxyfenozide was studied on fifth larval instar of R. nu under chronic exposure in laboratory conditions. Four dilutions (between 10% and 80%) of the maximum field recommended concentration for each insecticide were assessed. Methoxyfenozide caused 100% larval mortality in all tested concentrations, while at tested cypermethrin concentrations, larval mortalities were between 75% and 98%. Cypermethrin treatments showed higher mean survival time values than methoxyfenozide. Both insecticides inhibited larval growth according to weight loss assessment. Cypermethrin also inhibited diet consumption during the first 24-h exposure. Keywords: effectiveness; Plusiinae species; development effects; pest control

Introduction Rachiplusia nu (Guenee) (Lepidoptera: Noctuidae) is a widely spread species on the South American continent, and is common in Argentina, Bolivia, Brazil, Chile, and Uruguay. Larvae of this defoliator can cause severe damage to crops including soybean (Glixine max L.), sunflower (Helianthus annuus L.), corn (Zea mays L.), wheat (Triticum sp L.), and alfalfa (Medicago sativa L.). Larval infestations can also be found in horticultural crops, such as beans (Phaseolus vulgaris L.), tomatoes (Lycopersicon esculentum L.), lettuce (Lactuca sativa L.), cucumbers (Cucumis sativus L.), squash (Cucurbita maxima D.), peas (Pisum sativum L.), cauliflower (Brassica oleracea L.), and meridian fennel (Carum carvi L.) (Aragoń et al. 1997). The early instar larvae of R. nu feed on leaf parenchyma without damaging the leaf veins, reaching their highest voracity during their fifth larval instar. Associated with the early instar larvae of R. nu is a rich community of parasitoids that help keep the population density of this defoliator under economic injury levels (Luna and Sanchez 1999). However, when these levels are surpassed, synthetic insecticides (mainly broad spectrum products) are commonly used as the primary tool for pest control. These compounds negatively impact on non-target organisms, such as

*Corresponding author. Email: [email protected] ISSN 0792–4259 print/ISSN 2157–0272 online ß 2011 Taylor & Francis DOI: 10.1080/07924259.2011.591177 http://www.informaworld.com

the pest’s natural enemies, plants, fish, and amphibians (Schneider et al. 2003, 2008; Rimoldi et al. 2008; Ronco et al. 2008). Integrated pest management (IPM) is a decision support system for the selection and use of pest control methods, singly or harmoniously coordinated into a management strategy, based on cost/benefit analyses that take into consideration the interests of, and impacts on, producers, society, and the environment (Kogan 1998). In this way, IPM promotes the use of more selective insecticides to reduce the impact on natural enemies, thereby promoting the natural control of pests by conservation of their natural enemies (predators, parasitoids, and entomopathogens), as well as other beneficial organisms (pollinators) present in the crops. Pyrethroids are currently among the major insecticides used against Lepidoptera and other pests (Pietrantonio et al. 2007) due to their broad spectrum and cost-effectiveness. Cypermethrin belongs to this insecticide group, which acts on the central nervous system, altering the axonic sodium channels and allowing excessive ion entrance, causing abnormal nervous activity and eventually paralysis (Stenersen 2004). Use of broad spectrum insecticides such as cypermethrin may cause resurgence of the key pests or induce outbreaks of secondary pests due to the


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suppression of their natural enemies or by pest resistance mechanisms (Pietrantonio et al. 2007). Indeed, previous studies have shown harmful effects of pyrethroids toward natural enemies (Usmani and Knowles 2001; Rimoldi et al. 2008). Insect growth regulator (IGR) insecticides are considered biorational pesticides that seem to be selective toward pests and show low or no toxicity to most natural enemies (Schneider et al. 2008; Pineda et al. 2011). Methoxyfenozide belongs to the molt accelerating compounds (MACs) group within the IGR insecticides. Its mode of action is to mimic the endogenous ecdysteroid hormone by binding to the natural hormone receptors and causing an anticipated lethal molt (Dhadialla et al. 1998). Although this compound has been marketed worldwide for more than 20 years, it was not registered in Argentina for soybean crops until 2006 (CASAFE 2007). Since 1996, with the introduction of the direct seeding technique and transgenic soybean crops, the cultivated area for this crop has now increased to near 18,000,000 ha in Argentina (SAGPyA 2009), leading to an increase in the use of broad spectrum insecticides (CASAFE 2007). Although the search for new compounds with low-environmental impact has been a matter of concern and a priority for IPM programs, their introduction into the productive systems of many developing countries has been delayed. Within this frame, the aim of this study was to assess the effectiveness of cypermethrin and methoxyfenozide on the fifth larval instar of R. nu by means of chronic laboratory toxicity bioassays, testing lethal and sublethal effects.

Materials and methods Insect rearing Rachiplusia nu used in toxicity tests were obtained from a colony (provided by the Institute of Microbiology and Animal Zoology [IMYZA] – Castelar, Buenos Aires) with no history of insecticide exposure. Larvae were reared on a semi-synthetic diet (Greene et al. 1976) in a controlled rearing chamber at 25 2 C, 75 5% R.H., and a photoperiod of 16/8 h (light/dark). Adults were fed with a 15% solution of honey. Dark cardboard was provided as an oviposition substratum and to ease the identification and extraction of eggs.

Insecticides The commercial products Glextrin25Õ (25% cypermethrin; Gleba S.A., Argentina) and IntrepidÕ (24% methoxyfenozide; Dow Agrosciences Argentina S.A., Argentina) were used in toxicity tests. Treatments of insecticides were expressed as concentrations of the

active ingredient (AI). Concentrations of cypermethrin used were 3.1, 6.2, 12.5, and 18.7 mg AI/kg diet (wet weight), corresponding to 12.5%, 25%, 50%, and 75% of the maximum field recommended concentration (MFRC) (25 mg AI/kg diet), respectively. Concentrations of methoxyfenozide used were 28.1, 57.6, 86.4, and 115.2 mg AI/kg diet (wet weight), corresponding to 20%, 40%, 60%, and 80% of the MFRC (144 mg AI/kg diet), respectively. The concentrations used in the experiments were selected taking into account that fifth instar larvae (L5) are found mainly in the upper strata of the crop, and the damage caused by these larvae is higher in phenological stages of the plant where coverage is not complete, so the penetration of the insecticide on the foliage is relatively high. Previous studies showed that between 10% and 90% of insecticide application doses reach the upper strata of the crops (Bird et al. 1996; Carlsen et al. 2006). Insecticide solutions were prepared immediately before toxicity testing and mixed with lepidopteran artificial diet according to Pineda et al. (2006). The artificial diet was chosen as way for the ingestion exposure due to its higher palatability than treated leaves.

Larval toxicity by ingestion The experimental design consisted of three to four replicates of 10 individuals each per treatment. Each newly molted fifth instar larva was individually placed in a plastic Petri dish (diameter, 9 cm; height, 1.4 cm). A cylinder (diameter, 0.7 cm; height, 1 cm) of the treated diet for each insecticide treatment was offered to the larva during the first 24-h to assess consumption. An equivalent quantity of untreated diet was offered to controls. After that, and during the rest of the experiment, exposed or non-exposed organisms were fed ad libitum with treated- or untreated-diet, respectively. After the first 24 h, the remaining diet was dried at 80 C and then weighed to calculate the proportion consumed. The weight of the surviving larvae and number of dead larvae, prepupae, and pupae were registered every 24 h until organisms reach the adult stage. Data were used to calculate the mortality percentages for each development stage (larvae, prepupae, and pupae), accumulated mortality, mean survival time, and growth inhibition. The following equation was used to calculate the growth inhibition: % growth inhibition ðweight control weight treatmentÞ 100: ¼ ðweight controlÞ


Invertebrate Reproduction & Development Chemical analysis

Larval toxicity by ingestion

Insecticide concentrations in the diet were verified by chemical analysis during the first 10 days, after extraction by sonication with acetonitrile (USEPA 2007). Cypermethrin and methoxyfenozide contents were determined by high-performance liquid chromatography mass detection. Separation was done in a C18 column of 15 cm 4.6 mm inner diameter using acetonitrile:water 80:20, at 0.8 mL/min, with the detection single ion monitor positive mode at 432 umas ([M þ NH4]þ) in an LC–MS Agilent 1100 LC system (Agilent Technologies Inc., USA) equipped with a binary pump and a diode array detector, and coupled with an MSD VL quadrupole (Agilent Technologies, USA) with an electrospray ionization interface set at 150 eV, using nitrogen to assist nebulization, at 300 C. A Rheodyne 7725i injector with a 20 mL loop was used. Data acquisition and analysis were performed using an LC/MSD Agilent ChemStation (Blasco et al. 2009).

Analysis of mortality

Data analysis Results are presented as the mean standard error (SE). Before data analysis, the Shapiro–Wilk test was used to assess data normality. One-way analysis of variance (ANOVA) or repeated measures ANOVA was used to observe differences between treatments. Data were transformed by arc-sine squareroot-transformed proportional data before analysis. A Mann–Whitney test was used for the set of data not reaching normality. After ANOVA, the means separation was done by multiple range test least significance difference ( p 0.05). Survival analysis was used to analyze the mean survival time at each concentration. Survival functions were estimated using the Kaplan–Meier method and log–rank test for treatment comparisons, using the Bonferroni correction for paired comparisons between treatments. The XLStat program (Addinsoft XLstat for Excel, Paris, France. 2009. http://xlstat.softonic.com) was used in analysis.

Results Chemical analysis According to the results of chemical analysis, on the 10th day, cypermethrin and methoxyfenozide concentrations of the first two test dilutions remained within 85% of the starting nominal dilutions, and the highest dilution dropped to almost 50%. Due to the decrease in the concentrations of insecticides with exposure time, in the rest of the results we will refer to nominal rather than actual concentrations.

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The tested insecticides were highly toxic to fifth instar R. nu under chronic exposure by ingestion, leading to a significant increase in the exposed organisms’ mortality. All of the tested concentrations of methoxyfenozide as well as treatments with cypermethrin concentrations of 6.2, 12.5, and 18.7 mg AI/kg diet induced between 97.5% and 100% mortality at the end of testing. Likewise, treatment with cypermethrin at 3.1 mg AI/kg diet caused high- and significant-mortality with respect to controls, though mortality did not reach 100% (Table 1). Treatments with methoxyfenozide induced the presence of dead larvae with double cephalic capsule formation. The fifth instar larvae of R. nu were the most affected by these insecticides (Table 1). The statistical assessment of the prepupal and pupal mortality at the lowest tested concentration of cypermethrin did not show differences from the control group (Table 1). Assessment of prepupal and pupal mortality for the remaining treatments was not performed because only a few organisms survived in each treatment.

Mean survival time The mean survival time assessment of a target organism is relevant to know the speed of action of insecticides. This endpoint contributes to defining their efficacy in the field. All tested cypermethrin concentrations induced longer mean survival times than tested methoxyfenozide concentrations. Within the range of the tested cypermethrin concentrations, a decrease in mean survival time with an increase in insecticide concentration was observed. The mean survival times in concentrations of 3.1 and 6.2 mg AI/ kg diet of cypermethrin were 11.07 0.91 and 8.70 0.66 days, respectively. In the concentrations of 12.5 and 18.7 mg AI/kg diet of this compound, the mean survival time values were 6.01 0.36 and 6.19 0.35 days, respectively. No differences in mean survival time values for methoxyfenozide were observed among all concentrations (log/ rank ¼ 265.44; p50.0001). The survival time values were 3.10 0.19, 3.70 0.16, 3.57 0.20, and 3.37 0.20 days to methoxyfenozide concentrations of 28.1, 57.6, 86.4, and 115.2 mg/kg diet, respectively. Figure 1 shows percentages of accumulated larval mortality during exposure time for each treatment. These time-response curves illustrate that in cypermethrin treatments, the times to reach final mortality are longer than in methoxyfenozide treatments. Also, it is possible to observe that the two lower concentrations of cypermethrin induced lower responses than the rest of the tested concentrations of this insecticide (Figure 1a). Moreover, in the case of the time-response

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Table 1. Effects of cypermethrin and methoxyfenozide on the mortality of R. nu.

Treatments Control Methoxyfenozide

Cypermethrin

Concentrations (mg AI/kg)

Larval mortality (%)a

Prepupal mortality (%)b

Pupal mortality (%)b

Accumulated mortality (%)a

0 28.1 57.6 86.4 115.2 3.1 6.2 12.5 18.7

17.14 (3.50)a 100.00 (0.00)c 100.00 (0.00)c 100.00 (0.00)c 100.00 (0.00)c 75.00 (6.45)b 95.00 (2.88)c 95.00 (2.88)c 97.50 (2.50)c F ¼ 56.457 p50.0001 df ¼ 8.29

4.80 (1.98)a – – – – 8.33 (8.33)a * * * U ¼ 21.000 p ¼ 0.909 n ¼ 40

3.17 (2.42)a – – – – 0.00 (0.00)a * * * U ¼ 24.000 p ¼ 0.989 n ¼ 40

24.29 (3.49)a 100.00 (0.00)c 100.00 (0.00)c 100.00 (0.00)c 100.00 (0.00)c 77.50 (6.29)b 97.50 (2.50)c 100.00 (0.00)c 100.00 (0.00)c F ¼ 118.530 p50.0001 df ¼ 8.29


50.05

Notes: The data correspond to mean values (SE). Means within the column followed by different letters are significantly different. Three or four replications of 10 larvae each were used for each treatment, and 10 replications were used for control. a One-way ANOVA. b Mann–Whitney test. *Treatment was not considered in the statistical analysis due to the few surviving organisms.

curves of methoxyfenozide, no differences were observed between them (Figure 1b). In this frame, methoxyfenozide tested concentrations were more effective than cypermethrin ones, not only due to the mortality observed, but also due to the mean survival time.

Effect on larval weight and growth Both insecticides significantly inhibited the growth of exposed larvae at all tested concentrations, and a direct relationship was observed between the growth inhibition and the exposure time. Significant inhibition with respect to the control was detected from the first- and second-day of exposure for all concentrations of cypermethrin and methoxyfenozide, respectively. No significant differences between concentrations higher than 6.2 mg AI/kg diet of cypermethrin were observed during 7-day exposure. Although the lowest cypermethrin concentration tested, significantly inhibited larval growth with respect to the control, this effect was significantly lower than that observed with 18.7 mg AI/ kg diet during the exposure time (Figure 2a). No significant differences between methoxyfenozide concentrations of 28.1 and 57.6 mg AI/kg diet were observed. However, these concentrations induced significantly lower larval growth inhibition than 86.4 and 115.2 mg/kg diet methoxyfenozide treatments during exposure (Figure 2b) ( ¼ 0.05; F ¼ 14.150; p50.0001). The growth inhibition assessment was only possible until the 7th and 4th days of exposure for cypermethrin and methoxyfenozide, respectively, due to the low number of organisms remaining after that time.

Proportion of consumed diet The organisms exposed to cypermethrin did not consume, or consumed very little, diet during the first 24 h of testing. By contrast, the larvae exposed to methoxyfenozide consumed a similar amount of diet as the control organisms, or even more (organisms in methoxyfenozide treatment with 86.4 mg/kg diet exhibited significantly higher percentages of consumption) (Figure 3) ( ¼ 0.05; F ¼ 22.426; p50.0001).

Discussion According to the concepts considered within the IPM paradigm, studying the biological effects of pesticides on target organisms is very relevant for the minimization of treatment thresholds and the assessment of new pesticide efficiency. Moreover, in addition to mortality, the sublethal effects could also contribute to reducing pest population levels. The selection of highly efficient and selective compounds has usually been associated with the study of the worst possible scenarios by means of laboratory toxicity tests. The same strategy is undertaken with beneficial organism by the IOBC (Hassan 1994). Hence, these tests have become useful tools in the first stages of pesticide assessment decisionmaking steps. Regarding the observed variation in insecticide concentrations in the diet along the exposure time in this study, results are consistent with the half-life times reported in the literature in different types of matrices. Although no reported data were found for the type of diet used in this study, other authors have registered cypermethrin half-life times in cotton leaves of 0.71

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Invertebrate Reproduction & Development

Figure 1. Insecticide effects on mortality of R. nu fifth larval instar throughout the exposure time: (a) cypermethrin concentrations and (b) methoxyfenozide concentrations. The Kaplan–Meier method was used in survival function estimations and the log-rank test for treatment comparisons, using Bonferroni’s correction for pair comparisons between treatments.

days for doses of 42.5 mg/kg, and 0.69 days for doses double of that (Battu et al. 2009). For the case of methoxyfenozide, the FAO (2003) has published halflife times of 14–20 days in pears. According our results, both insecticides exhibit a higher loss at higher concentrations compared to lower doses. The half-life of a compound not only depends on its intrinsic chemical proprieties, but also on the type of containment matrix and the concentration. Actual exposure depends on whether the organism is in direct contact with it or ingests it in the diet.

The high effectiveness of cypermethrin and methoxyfenozide for the control of lepidopteran pests has been well-documented (Abdullah et al. 2001; Usmani and Knowles 2001; Pineda et al. 2007). This study also showed high effectiveness of both insecticides in the control of R. nu, even at concentrations below the MFRCs. Methoxyfenozide was more effective than cypermethrin on this pest, because treatments at equivalent dilutions showed that cypermethrin did not induce higher mortality than those detected in methoxyfenozide treatments. Although survivors were


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Figure 2. Effects of cypermethrin and methoxyfenozide on growth inhibition of R. nu fifth larval instar throughout the exposure times: (a) cypermethrin concentrations and (b) methoxyfenozide concentrations. The results correspond to mean values (SEM). Repeated measures ANOVA was used for treatment comparisons ( ¼ 0.05; F ¼ 14.150; p50.0001). Treatments with different letters are significantly different.

Figure 3. Effects of cypermethrin and methoxyfenozide on the larvae’s diet consumption at 24 h of exposure. The results correspond to mean values (SEM). Bars with different letters are significantly different. One-way ANOVA was used for comparison among treatments ( ¼ 0.05; F ¼ 22.426; p50.0001).


Invertebrate Reproduction & Development observed at the lowest tested concentrations of cypermethrin in this study, mortality rates were high (over 75%), and it may be desirable to keep the number of individuals of the pest population below the economical injury level according to control pests strategies. The high rate of mortality with cypermethrin in the tests agrees with those reported by Usmani and Knowles (2001). These authors detected high toxicity with the pyrethroids cypermethrin and permethrin on larvae of Helicoverpa zea (Boddie), Spodoptera frugiperda (Smith), and Agrotis ipsilon (Hufn) (Lepidoptera: Noctuidae) by topical exposure. Similarly, Zhang et al. (2008) registered high mortalities of Bombyx mori (L.) (Lepidoptera: Bombycidae) exposed to several pyrethroids. Furthermore, they detected additive effects when these were mixed with organophosphate compounds. Pineda et al. (2007) detected high mortality in larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) exposed to methoxyfenozide. This observation was also detected at concentrations 50-fold lower than the ones tested in this study. Likewise, Gobbi et al. (2000) reported high mortality of S. littoralis, Mytimna unipuncta (Haworth) (Lepidoptera: Noctuidae), and Spodoptera exigua (Hu¨bner) after exposure to another MAC, tebufenozide. Moreover, Budia et al. (1994) observed effects on the survival of fifth larval instar of S. exigua at 10 mg AI/kg tebufenozide. Despite the fact that the accumulated and partial mortalities per stage induced by an insecticide are relevant in pest management, it is very important to consider its action speed. The present results show that although the accumulated percentage of mortalities was similar for all cypermethrin concentrations above 3.1 mg/kg diet, larvae exposed to cypermethrin took longer to die than those exposed to methoxyfenozide. The reduction of larval weight is an important sublethal effect to be considered since this could lead to alterations in the population dynamics of lepidopteran pests. Both insecticides reduced the individual weight of organisms. In the case of cypermethrin, the reduction of larval weight could be observed from the first day of testing. This effect could be attributable to different causes. Several studies have reported pyrethroid repellence. For example, Longley and Jepson (1996) detected lower percentages of parasitism of Aphidius rhopalosiphi (De Stefani-Perez) (Hymenoptera: Aphidiidae) to aphids previously treated with deltamethrin. In this study, organisms exposed to cypermethrin consumed very little of the treated diet during the first 24 h of testing. Likewise, anti-feeding effects were observed by Baumler and Potter (2007) to different pyrethroids on Popillia japonica (Newman) (Coleoptera: Scarabaeidae). In this way, the reduction of larval weight could be

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attributable to the reduction of the body fat because larvae consume their reserves (mainly lipids, proteins, and carbohydrates) and do not feed because of repellence (anti-feeding effect) and/or disorders at the level of the central nervous system induced by the toxicant modifying the feeding behavior. By contrast, taking into account the mode of action of pyrethroids, cypermetrin provokes paralysis in the exposed organisms and consequently may induce behavioral alterations, such as cessation of feeding. Haynes (1988) mentioned several effects on behavior of insects exposed to pyrethroids that impact its feeding. In the case of the treatments with methoxyfenozide, significant effects on larval weight were detected after 2 days of exposure. All dead larvae that had been treated with this insecticide showed the presence of a double cephalic capsule. Before the molting process, the organisms stop feeding as a result of the molting signaling hormone, 20-hydroxyecdysone (Shaaya and Levenbook 1982). Since the methoxyfenozide mode of action mimics natural ecdysone, the reduction of organism weight could be due to a drop in feeding as a consequence of the activity of the insecticide and the presence of the double cephalic capsule, which interferes with larval feeding. Inhibition of this endpoint was also detected for this and other IGRs on Diatrea grandiosella (Dyar) (Lepidoptera: Crambidae), S. littoralis, and M. unipuncta (Smagghe and Degheele 1998; Gobbi et al. 2000; Pineda et al. 2007). The efficacy of an insecticide is given by the percentage of mortality induced on the key pest and its speed of action, because if there is a delay in the mortality, the pest could continue feeding. However, repellence associated with an insecticide (like the behavior observed for cypermethrin in this study) makes the time factor irrelevant, because during this period organisms do not feed. Given the present results and taking into account the information in the literature on the low toxicity of methoxyfenozide on non-target organisms, this MAC insecticide should be considered for the control of R. nu larvae within IPM programs. Although toxicity testing under controlled laboratory conditions is usually considered a worst-case scenario compared with field assessments of pesticide action on target and non-target organisms according to IOBC guidelines, data from these studies provides relevant preliminary information for decision-making steps within the frame of IPM. In relation to the magnitude of the effects and the short-time response at dilutions equivalent to the MFRCs, methoxyfenozide appears to be more effective than cypermethrin for the control of this lepidopteran pest in controlled laboratory conditions. Both insecticides induce high mortality and important sublethal effects at concentrations below their MFRCs. Therefore, future studies should include the assessment

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of these concentrations in semi-field and field conditions toward diminishing recommended application doses. These results could be useful in the development of environmentally friendly pest control strategies to replace conventional insecticides, most of which are not compatible with IPM programs. This study is the first report on the toxicity assessment of methoxyfenozide and cypermethrin on a Plusiinae species such as R. nu, a potential key pest in soybean crops.


Acknowledgements We thank D. Marino for help with chemical analysis, C. Knipp for editorial and English style corrections, G. Bulus Rossini for statistical contributions, and DowAgrosicence SA and Gleba SA for providing the tested insecticides. We are grateful to two anonymous reviewers for valuable suggestions that greatly improved this manuscript. We acknowledge the support of Dr Guy Smagghe (Ghent University, Ghent, Belgium) for his suggestions on an earlier version of this manuscript that helped improve it. This research was supported by a PICT 38350 BID 1728 OC-AR project from the Argentinean National Agency for the Promotion of Science and Technology (ANPCyT). F. Rimoldi and M. N. Fogel are fellows of Consejo Nacional de Investigaciones Cientı´ ficas y Tećnicas (CONICET).

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