Geraldo Andrade Carvalho¹, Maurício Sekiguch i Godoy², Douglas Silva Parreira³ and Denise Tourino Rezende⁴

¹ Prof. Dr. in Entomology, Selectivity of pesticides for natural enemies. Universidade Federal de Lavras (UFLA), Departamento de Entomologia, Lavras, Minas Gerais, Brazil, C.P. 3037, CEP: 37200-000. E mail: gacarval@den.ufla.br. Corresponding Author

² Pós Dr. in Entomology, Selectivity of pesticides for natural enemies. Universidade Federal de Lavras (UFLA), Departamento de Entomologia, Lavras, Minas Gerais, Brazil, C.P. 3037, CEP: 37200-000.

³ Ms. Sc. in Entomology, Selectivity of pesticides for natural enemies. Universidade Federal de Lavras (UFLA), Departamento de Entomologia, Lavras, Minas Gerais, Brazil, C.P. 3037, CEP: 37200-000.

⁴ Graduate student of Agronomy, Selectivity of pesticides for natural enemies. Universidade Federal de Lavras (UFLA), Departamento de Entomologia, Lavras, Minas Gerais, Brazil, C.P. 3037, CEP: 37200-000.

Recibido: 10-jun-2009 - Aceptado: 1-may-2010

Tomato crops are affected by a large number of pests and diseases (Santini 2001). In Brazil, tomato pests are controlled mainly by the indiscriminate use of chemical pesticides, which generally lead to biological imbalances and environmental pollution. A safer alternative for controlling some of the tomato pests is biological control utilizing parasitoids belonging to the genus Trichogramma (Hymenoptera: Trichogrammatidae). According to Pratissoli and Parra (2001) and Haji et al. (2002), trichogrammatids have significantly contributed to reducing the populations of lepidopteran pests and the number of insecticide applications on tomato crops.

According to Haji et al. (1998), Trichogramma pretiosum (Riley, 1879) (Hymenoptera: Trichogrammatidae) has been used in the control of the tomato leaf caterpillar Manduca diffissa (Butler, 1871) (Lepidoptera: Sphingidae), tomato moth Tuta absoluta (Meyrick, 1917) (Scrobipalpuloides) (Lepidoptera: Gelechiidae), and tomato fruit borer Neoleucinodes elegantalis Guenée, 1854 (Lepidoptera: Crambidae), which demonstrates the potential of this species in pest control. However, one of the major obstacles to the use of this and other parasitoids in tomato crops is the continued use of large quantities of chemical pesticides. Another limiting factor is that there are few studies about the toxic effects of the new molecules on natural enemies of pests in tomato crops (Degrande et al. 2002; Moura et al. 2005; Moura and Rocha 2006). Thus, the aim of the present work was to evaluate the toxicity of some new insecticides used in tomato crops applied during the immature developmental stages of T. pretiosum on F₁ generation adults, and also to evaluate the sublethal effects on the F₂ generation adults.

The studies were carried out with parasitoids of the species T. pretiosum obtained from parasitized Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) eggs, collected from a maize crop in the municipality of Piracicaba, São Paulo State, Brazil. The parasitoids were reared in the laboratory under controlled conditions (24±2 °C, 70 ± 10% RH, and 12h photoperiod) on eggs of the factitious host Anagasta kuehniella (Zeller, 1879) (Lepidoptera: Pyralidae). The insecticides used in the tests with T. pretiosum, together with their technical and trade names, dosages, formulations, and chemical groups, are presented in Table 1. They are the latest products registered for control of tomato crop pests in Brazil. Distilled water was used as a control.

To evaluate the effects of the insecticides on immature stages of T. pretiosum, twenty fertile T. pretiosum females, aged about 24 h, were placed in individual glass tubes (8 cm x 2.5 cm) and fed with honey. The tubes were closed with polyvinyl chloride (PVC) film. About 125 UV-killed eggs of A. kuehniella were glued onto 5 x 0.5-cm paper cards and exposed to the females for parasitization for 24 h (Parra 1997). The supposedly parasitized eggs were kept in an acclimatized chamber at 24±2 °C, 70±10% RH and exposed to a 12-h photoperiod until the parasitoids reached the desired developmental stage for the application of the insecticides.

A total of 20 paper cards per treatment with parasitized A. kuehniella eggs (parasitoids in the egg-larval or pre-pupal or pupal stages; 0-24 h, 72-96 h, and 168-192 h after parasitism, respectively) were dipped into the insecticide solutions or into water for 5 s, dried in the shade for about 30 min and then placed in glass tubes. The tubes were kept in the acclimatized chamber as previously described. The effects of the insecticides applied during the immature stages on T. pretiosum adults emerging from the treated host eggs were also studied. The same number of females, card size and number of host eggs as previously described were employed; however, untreated 0-24-h old A. kuehniella eggs were presented to T. pretiosum F₁ and F₂ generation females, and biological parameters like adult emergence, sex ratio, and parasitization capacity were studied.

The effects of the insecticides on each immature stage of the parasitoid were measured by evaluating percent emergence [(number of eggs with parasitoid emergence holes/total number of parasitized eggs) x 100], sex ratio (number of females/number of females + males) according to the equation proposed by Pereira et al. (2004), and parasitism ratio (number of parasitized eggs/female/24 h) of F₁ and F₂ generations females. Each treatment was replicated five times, with the experimental plot represented by four paper cards with parasitized host eggs. A completely randomized three x seven factorial experimental design with three periods of parasitoid development vs. seven substances, totaling 21 treatments were used. The obtained data were submitted to analysis of variance and the means were compared by the Scott-Knott grouping test at 5% significance (Scott and Knott 1974).

The insecticides were also classified using the International Organization for Biological Control (IOBC) protocols (Sterk et al.1999), considering the percent reduction of emergence and parasitism capacity of F₁ and F₂ generation females due to insecticide application at each immature stage in relation to the control treatment, as follows: class 1 = harmless (<30% reduction), 2 = slightly harmful (30-79% reduction), 3 = moderately harmful (80-99% reduction), and 4 = harmful (>99% reduction). The mean percent reduction of emergence capacity and number of eggs of the parasitoid were calculated with the equation: % reduction = 100 – [(% general mean of emergence or number of eggs in the treatment with the insecticide/% general mean of emergence or number of eggs of control) x 100].

Emergence and sex ratio of F₁ generation parasitoids. Only pyriproxifen affected the emergence of T. pretiosum (F₁ generation) negatively, 28.4% emergence, when applied to host eggs with the parasitoid in the egg-larval stage (Table 2). This compound also produced a 24-h delay in the emergence of F₁ adults. The observed effects are thought to be related to the mechanism of action of the compound, since some insect growth regulators are agonists or antagonists of the juvenile hormone, which is responsible for the growth and/or the development of the immature forms of the insects. Pyriproxifen mimics the action of the juvenile hormones in a number of physiological processes and is a potent inhibitor of embryogenesis, metamorphosis, and adult formation (Glancey et al. 1990; Ishaaya and Horowitz 1992; Miyamoto et al. 1993).

None of the evaluated insecticides reduced adult emergence in the F₁ generation when applied on the host eggs containing parasitoids in the pre-pupal stage. However, imidacloprid and triflumuron treatments during the parasitoid prepupal stage reduced adult emergence in the F₁ generation by 16.2 and 18.7%, respectively, as compared to the emergence recorded when applied during the egg-larval period. The applications of imidacloprid and lufenuron on host eggs with parasitoids in the pupal stage led to a significant reduction of emergence of F₁ generation adults of T. pretiosum in comparison to the control and other treatments (Table 2). Carvalho et al. (2003) presented similar results for imidacloprid and lufenuron products, with only 70.3% and 72.8% adult emergence, respectively, when pupae in eggs of A. kuehniella were contaminated. Similar results were reported by Pratissoli et al. (2004), who observed that the number of T. pretiosum adults that emerged from lufenuron-treated S. frugiperda host eggs fell sharply.

A cetamiprid and novaluron were harmless, as they did not cause any reduction in adult emergence in the F₁ generation, regardless of the developmental stage of the parasitoid subjected to insecticide treatment. Different results were reported by Moura et al. (2005, 2006), who found that acetamiprid significantly decreased percent adult emergence in all stages tested. These differences in biological responses may be associated with distinct populations of T. pretiosum, as well as their geographical origins, as also discussed by Bleicher and Parra (1990) and Brunner et al. (2001).

When pyriproxifen was applied in the egg-larval stage, it was slightly harmful (class 2) due to 30-70% reduction in adult emergence in the F₁ generation. Acetamiprid, imidacloprid lufenuron, triflumuron, and novaluron were classified as harmless (class 1) when they were applied during the pre-pupal and pupal stages of T. pretiosum, giving an adult emergence of >70% (Table 2). Similar results were obtained by Moura et al. (2005) for acetamiprid, which was also categorized as class 1. However, imidacloprid has been classified as slightly harmful (class 2), differing from the result obtained in this work. This difference is possibly associated with the dose used by other authors, which was eight times higher than that of the present study.

The sex ratio of males and females of the F₁ generation of T. pretiosum that emerged from treated host eggs during the egg-larval period and the pre-pupal and pupal stages was not affected by any of the evaluated insecticides (P > 0.05), and there was no statistical difference between treatments, which presented average ratios of 0.5-0.6.

Parasitism capacity of F₁ generation parasitoids. Only lufenuron did not significantly reduce the parasitism capacity of F₁ females of T. pretiosum treated during the egg-larval period. However, this insecticide significantly reduced (14.7 and 13.2% parasitized eggs, respectively) the parasitism capacity of F₁ females treated during the pre-pupal and pupal stages, when compared with those treated in the egg-larval stage (Table 3). Carvalho et al. (2003) also observed negative effects for 0.28 g a.i.L-1 and 0.4 g a.i.L-1 imidacloprid and lufenuron, respectively, pulverized onto eggs of A. kuehniella with T. pretiosum parasitoids in the pupal stage.

Treatments with acetamiprid and pyriproxifen reduced the parasitism capacity of females from treated host eggs with parasitoids in the egg-larval period, as compared to other developmental stages of treatment with the insecticides. The parasitism capacity of F₁ generation females that emerged from parasitized host eggs treated during the pre-pupal and pupal stages was not negatively affected by the insecticides (Table 3). In a study of acetamiprid sprayed through a Potter tower, Moura and Rocha (2006) observed significant reduction in the capacity of parasitism (57.3%) of F₁ generation females when eggs of A. kuehniella with T. pretiosum were treated in the pupal stage. These conflicting results obtained in the treatment of the pupal stage are possibly associated with the different techniques used to treat host eggs.

IOBC classification (F₁ generation). According to the IOBC toxicity indices, acetamiprid, imidacloprid, and pyriproxifen are slightly harmful (class 2), reducing parasitism by 30- 79%, but only when applied to host eggs with parasitoids in the egg-larval stage. Lufenuron, triflumuron, and novaluron were harmless (class 1) when applied to all immature stages of T. pretiosum, which reduced parasitism by less than 30% in comparison to the control (Table 3). Rocha and Carvalho (2004) reported that lufenuron and triflumuron were classified as slightly harmful (class 2) and imidacloprid as moderately harmful (class 3), differing from the present study. The possible discrepancy of toxicological classification is associated with the different methodology used, since the studies were carried out by spraying insecticides directly onto adults and not onto immature forms of the parasitoid.

Emergence and Sex ratio (F₂ generation). The percent emergence of F₂ generation parasitoids was reduced by all insecticides, except lufenuron, when A. kuehniella eggs with parasitoids in the egg-larval stage were treated. However, treatment with lufenuron in the pre-pupal and pupal stages of the parasitoid reduced the percent emergence of F₂ generation parasitoids, as compared to treatment during the egg-larval stage (Table 4). Carvalho et al. (2003) reported different results, in which lufenuron showed significant reduction in relation to the control only when eggs of A. kuehniella containing parasitoids in the pupal stage were infected, which did not happen in egg-larval or pre-pupal stages. These differences in biological responses may be associated with distinct populations of T. pretiosum studied, as well as their geographical origins, as previously mentioned.

Considering the percent reduction in the emergence of F₂ generation specimens, pyriproxifen was slightly harmful (class 2) to parasitoids in the egg-larval period, and lufenuron was slightly harmful only to parasitoids in the pupal stage. All the other compounds were harmless (class 1) to all immature stages (Table 4).

The sex ratio of F₂ generation parasitoids submitted to acetamiprid, imidacloprid, and triflumuron treatments was not adversely affected. Lufenuron treatment in the egg-larval and the pre-pupal stages reduced the proportion of females in the F₂ generation by from 0.2 to 0.4 on average, respectively. Novaluron application in the pre-pupal stage adversely affected the sex ratio of F₂ generation adults. Pyriproxifen treatment in pre-pupal stage was less harmful with regard to sex ratio of F₂ generation adults as compared to its treatment during the other developmental stages (Table 5). Parasitism capacity of F₂ generation parasitoids. Insecticide application during the egg-larval stage of T. pretiosum did not significantly reduce the parasitism capacity of F₂ generation females. Treatments with acetamiprid, imidacloprid, triflumuron, and novaluron applied in the pre-pupal stage and acetamiprid and triflumuron applied in the pupal stage of T. pretiosum reduced the parasitism capacity of F₂ generation females (Table 6). The capacity of parasitism of the different stages of the F₂ generation of parasitoids exposed to lufenuron, triflumuron, and novaluron did not present any negative effect from the pesticides. Imidacloprid affected parasitism capacity, except in the egg-larval stage. Acetamiprid and pyriproxifen applied in the pupal stage of the parasitoid adversely affected the parasitism capacity of F₂ generation females. IOBC classification (F₂ generation). Acetamiprid, when used during the pupal stage of the parasitoid, was slightly harmful (class 2) with respect to parasitism capacity of F₂ generation females, while the other insecticides were harmless (class 1) when applied during all immature stages (Table 6). The compounds that were harmful to T. pretiosum in the present study may be more or less toxic in field conditions due to the effect of various abiotic factors. Thus, there is a need for field validation of the laboratory results. Furthermore, the experiments must also be carried out using the natural host to evaluate the impact of the pesticides on this parasitoid species so that safer pesticides can be associated with the release of parasitoids in integrated pest control programs.

When applied to T. pretiosum in the egg-larval period, acetamiprid, imidacloprid, and pyriproxifen were slightly harmful to the parasitism capacity of F₁ generation females, and so was pyriproxifen to the emergence rates of F₁ and F₂ generation adults. When applied to T. pretiosum in the pupal stage, acetamiprid was slightly harmful to the parasitism capacity of F₂ generation females, as well as lufenuron to the percent emergence of F₂ generation adults. In general, acetamiprid, imidacloprid, lufenuron, and pyriproxifen were toxic to T. pretiosum when applied in different immature stages. Greenhouse and field studies are required to validate the laboratory trial results. Regardless of the immature stage of T. pretiosum at the time of pesticide application, triflumuron and novaluron were harmless to F₁ and F₂ generation parasitoids, thus being recommended as safe insecticides for integration with parasitoid use in pest control programs.

We acknowledge research grants from the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.

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