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Interactions between Bacillus thuringiensis proteins, Spodoptera frugiperda (Lepidoptera: Noctuidae) and Campoletis flavicincta (Hymenoptera: Ichneumonidae) | S.M.D. | Bt Research

Interactions between Bacillus thuringiensis proteins, Spodoptera frugiperda (Lepidoptera: Noctuidae) and Campoletis flavicincta (Hymenoptera: Ichneumonidae)  

Salles S.M.D. , Pinto L.M.N. , Pavani F. , Machado V. , Fiuza L.M.
Programa de Pós-Graduação em Biologia, Laboratório de Microbiologia e Toxicologia, Ciências da Saúde, Universidade do Vale do Rio dos Sinos (UNISINOS), CEP: 93001-970 Caixa Postal 275. São Leopoldo, RS, Brasil
Author    Correspondence author
Bt Research, 2015, Vol. 6, No. 5   doi: 10.5376/bt.2015.06.0005
Received: 21 May, 2015    Accepted: 23 Jun., 2015    Published: 16 Aug., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Salles et al., 2015, Interactions between Bacillus thuringiensis proteins, Spodoptera frugiperda (Lepidoptera: Noctuidae) and Campoletis flavicincta (Hymenoptera: Ichneumonidae), Bt Research, Vol.6, No.5 1-10 (doi: 10.5376/bt.2015.06.0005)

Abstract

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene hás reduced the damage created by insect pests in several economically important cultures. The commercialization of Bt plants for insect management has revolutionized agriculture and become a major tool for integrated pest management (IPM) programs. Among the parasitoids of the pest species Spodoptera frugiperda Campoletis flavicincta (Ashmead) is a major parasitoid of larvae. In this context, this study aimed to investigate the interactions of the Bt proteins (from strains of B. thuringiensis, rice and Bt corn) on the parasitoid C. flavicincta and the armyworm S. frugiperda.  To evaluate this interaction, S. frugiperda larvae were exposed to four different assays: (T1) without parasitism or exposure to the Bt proteins (control); (T2) exposure only to Bt proteins (Bt plants or monogenic strains); (T3) exposure only to parasitism by C. flavicincta; and (T4) exposure to both parasitism and Bt proteins (Bt plants or monogenic strains). In each treatment, 30 larvae and three replications were included, for a total of 360 larvae. In general, the treatments T4 (Bt proteins and C. flavicincta) were more effective in controlling S. frugiperda thus indicating a potential to be used for the pest control in an integrated pest management system.The biological characteristics of C. flavicincta that developed into larvae under the action of Bt thuringiensis differed from parasitoids that emerged from healthy larvae (control). Therefore, the results in this laboratory system may significantly differ from what would be seen in the Field.

Keywords
Parasitoids; Biotech crops; Cry proteins; Integrated pest management; Rice; Corn

Background
Some methods, solely or together, get the satisfactory control of Spodoptera frugiperda (J.E Smith). Among the entomopathogenic agents used in biological control of lepidopterous pests the Bacillus thuringiensis Berliner bacterium (Bt) has gained special attention as an alternative method (Höfte and Whiteley, 1978; Alves et al., 1998).

The cultivars transformed with cry genes, present several advantages over Bacillus thuringiensis formulations, as they do not require foliar spraying for insect control because the toxin is expressed by the plant itself. The commercialization of Bt plants for insect management has revolutionized agriculture and become a major tool for integrated pest management (IPM) programs (Shelton et al., 2002; Romeis et al., 2008).

The responses of resistant and susceptible insects to Bt toxins maybe useful for understanding, monitoring, and managing evolution of pest resistance to Bt crops (Shikano and Cory, 2014ª) Several Bt rice genotypes showing high resistance against lepidopteran pests have been developed since 1993 (Cohen et al., 2008). While rice is the most important human food in China, corn is the essential grain for farm animals. More than 35 million acres of corn are grown in China (James, 2014).

However, a major concern related to Bt plants is their potential effects on non-target organisms, especially on natural enemies that help control pest populations, in addition to the ability of pest insects to develop resistance to Bt proteins (Gould, 1998; Onstad, 2008; Tabashnik et al., 2013; Shikano and Cory, 2014 ).

The inclusion of non-Bt refuges among plants may be an alternative for suppressing the evolution of resistance, through maintaining the Bt protein-susceptible alleles in the population (Tabashnik et al., 2009; Huang et al., 2012). The maintenance of natural enemies helping to control pest populations in Bt crop areas is another possibility for delaying the evolution of resistance (Wolfenbarger et al., 2008; Desneus et al., 2010).

The maintenance of natural enemies is critical to prevent phytophagous insect populations from reaching levels that are capable of causing economic damage (Berti and Ciociola, 2002). Studies show that parasitoids belonging to the order Hymenoptera can promote the natural control of pests (mainly from Lepidoptera and Hemiptera) in rice and maize cultivation areas (Martins et al., 2004).

These parasitoids are very sensitive to changes in their hosts after the ingestion of toxins, as they usually complete their development in a single host. When susceptible hosts are treated with Bt toxins, there is a greater possibility of both predators and parasitoids being affected because generalists often feed on different prey (Salama et al., 1983). Godfray (1994) suggests that the quality of the host may affect the survival, size and development time of the parasitoid. If the host is not able to survive, the parasitoid will also not complete its development (Godfray, 1994).

One of the most destructive and economically important pest insects found in Brazil is S. frugiperda, which is a polyphagous species native in tropical regions and is widely distributed because of its wide range of host plants species (Carvalho et al., 2013). Among the parasitoids of S. frugiperda, C. flavicincta is a major parasitoid of the larvae of this species (Lucchini and Almeida, 1980). These micro-hymenopterans are approximately 15 mm in size and perform oviposition inside the S. frugiperda larvae during their early instars, where the C. flavicincta larvae complete their entire cycle feeding on the inner content of the host, reaching the pupal stage at the end of the cycle and replacing the S. frugiperda adult with an adult parasitoid (Cruz et al., 1995).

In this context, the consequences of the effects of Bt plants on parasitoids have received attention, and studies have revealed both positive and negative impacts (Johnson and Gould, 1992; Baur et al., 2003; Sanders et al., 2007; Dhillon and Sharma, 2010). Although important, there are few studies on the interactions between Bt plants and parasitoids of insect pests (Rasmann and Turlings, 2008; Lundgren et al., 2009; Bruck, 2010; Tian et al., 2013; Liu et al., 2014; Kumar et al., 2014).

The identification of the effects of a control agent, in this case Bt plants, on parasitoids can lead to the development of control practices that aim to emphasize the strengths of each of these agents. This study aimed to investigate the interactions of the Bt proteins (from strains of B. thuringiensis, rice and Bt corn) on the parasitoid C. flavicincta and the armyworm S. frugiperda.

1 Results
1.1 Mortality bioassays of Spodoptera frugiperda parasitized by Campoletis flavicincta and exposed to genetically modified plants of Bt rice (Cry1B) or Bt maize (Cry1Ab).
The results of these bioassays are shown in Figures 1 and 2. The mortality observed in all treatments at ten days after the installation of the bioassay differed significantly from the control (F= 5,0. df= 3,8 p< 0,05). The mortality recorded in larvae fed with Bt rice (Cry1B) for ten days, was 84.3%, while that in larvae that were only parasitized by C. flavicincta was 52%. However, when the larvae were both parasitized and fed with Bt rice, mortality reached 95.5%. The results observed in the last two treatments also differed significantly from those in larvae exposed only to the parasite (F= 22.3. df= 3,8 p= 0.000). The mortality of the larvae that were both exposed to parasitism and fed with Bt rice was also higher than that observed in the larvae that were either fed Bt rice or parasitized. 

 
Figure 1 Schematic representation of the methodology followed for the mortality bioassays with Spodoptera frugiperda parasitized by Campoletis flavicincta and treated with Bt plants (rice or maize) or Bt bacterial suspensions (Bt thuringiensis 4412 or Bt kurstaki HD1) 

 
Figure 2 Mortality of Spodoptera frugiperda subjected to different treatments for intervals of 3 to 10 days: (A) larvae exposed to parasitism by Campoletis flavicincta and fed with Bt rice for 10 days; (B) larvae exposed to parasitism and fed Bt rice for three days (the same letters indicate no significant difference by the Tukey test at a 5% probability) 


In the assays for assessing the mortality of larvae that were both exposed to parasitism and fed with Bt rice (Cry1B) for three days after the installation of the bioassay (Figure 2B), all of the treatments were significantly effective compared with the control (F= 48,8. df= 3,8 p= 0.000). The highest mortality was observed on plants where the larvae were simultaneously exposed to parasitism and fed with Bt rice (T2, 73.3%), followed by the treatment with only Bt rice plants (T3, 63.3%), and that where larvae were only exposed to parasitism (T3, 61%). The efficiency of Bt decreased compared with the previous assay (Figure 2A).

In these experiments, which aimed to evaluate the mortality of larvae exposed to parasitism and fed with Bt maize plants (Cry1Ab), treatment T4 (Bt maize and C. flavicincta) was the most efficient (F=53,0, gl= 3,8, p= 0.000, Figure 3A), causing 90% mortality of the larvae, followed by the treatments where larvae were either fed with Bt maize (T2, 87.6%) or parasitized by C. flavicincta (T3,72%). In the treatments where the larvae were both parasitized and fed with Bt maize for only three days (Figure 2B), the results indicated significant effectiveness in controlling S. frugiperda compared with the control (F= 10,2 gl= 3,8, p= 0.04). Again treatment T4 was the most effective (71%). In the groups where the larvae were either fed with Bt maize (T2) or parasitized by C. flavicincta (T3), the average mortalities were 67.7 and 63.3%, respectively. The data did not differ statistically.

 
Figure 3 Mortality of Spodoptera frugiperda subjected to different treatments for intervals of 3 to 10 days: (A) larvae exposed to parasitism by Campoletis flavicincta and fed with Bt maize for ten days; (B) larvae exposed to parasitism and fed with Bt maize for three days (the same letters indicate no significant difference by the Tukey test at a 5% probability) 


1.2 Mortality bioassays of Spodoptera frugiperda parasitized by Campoletis flavicincta and infected by the strains Bt thuringiensis 4412 (Cry1B) and Bt. kurstaki HD1 (Cry1Ab)

In trials for assessing the mortality of larvae exposed to Bt thuringiensis strain 4412 (Cry1B) and the parasitoid C. flavicincta (Figure 4A), all treatments were found to be effective, differing significantly from the control (F= 42,5 df= 3,8 p= 0.000).

 
Figure 4 Mortality of Spodoptera frugiperda larvae subjected to (A) the parasitoid and the Bt thuringiensis 4412 strain (cry1B); or (B) the parasitoid and the Bt kurstaki HD1 strain (cry1Ab). (the same letters indicate no significant difference by the Tukey test at 5% probability) 


In the evaluation of the mortality of larvae exposed to Bt kurstaki strain HD1 (Cry1Ab) and the parasitoid C. flavicincta (Figure 4B), treatment T4 (Bt kurstaki and C. flavicincta) caused 48.8% mortality of S. frugiperda and was found to be significantly more efficient compared with either treatment with Bt kurstaki strain HD1 (33.3%) or exposure to C. flavicincta parasitism, where the average mortality was 42.2% (F= 11,3, df= 3,8, p= 0.003).

1.3 Biology of C. flavicincta descendants emerging from Spodoptera frugiperda that were exposed or not exposed to the monogenic strain Bt thuringiensis 4412
Data on the biology of C. flavicincta were evaluated through the analysis of parasitoid off spring from couples that emerged from larvae that were infected or non-infected by Bt thuringiensis 4412. The development period for each phase of C. flavicincta is show in Table 1.

 
Table 1 Average duration of the developmental stages (days) of C. flavicincta from pairs obtained from S. frugiperda larvae that were either infected or non-infected by B. thuringiensis 4412 


The parasitoids obtained from infected or non-infected larvae showed significantly different average egg-larvae durations, of 11.1 and 9.9 days, respectively (t = 5.6, df = 36.9, p <0.05). Regarding the pupal period, the parasitoids that emerged from the infected larvae exhibited an average pupal period of 8.9 days, versus 8.5 days for those obtained from uninfected larvae (t= 1.48, df = 16.8, p> 0.05).

The average longevity of the adults that emerged from infected or uninfected larvae was 15.3 days versus 16.4 days, respectively (t = 5.6, df = 17.3, p> 0.05). The sex ratios of the parasitoids that emerged from couples obtained from infected (n = 61) and uninfected (n = 111) larvae were 1 ♀: 5.1 ♂ and 1 ♀: ♂ 12.75, respectively. The egg-larva and pupal periods and the longevity of adult insects were also evaluated, which ranged from 7-25 days and peaked at 14 days (Figure 5), when the parasitoids that emerged from infected (n = 61) and non-infected (n = 111) larvae showed percentages of 22.9 and 15.3%, respectively.

 
Figure 5 Percentage distribution of Campoletis flavicincta individuals between different life cycle periods and descendants of couples from Spodoptera frugiperda thatwere either infected or non-infected with Bacillus thuringiensis 4412. (a) egg-larva phase, (b) pupal phase 


2 Discussion
Studying potential impacts of insect-resistant genetically- engineered plants on beneficial non-target arthropods is an important component of the environmental risk assessment. Our findings demonstrated that the T4 treatment (exposure to both Cry proteins and C. flavicincta) was more effective in controlling S. frugiperda compared with the treatments that assessed either only the toxicity of the Cry proteins (T2) (Bt plants or strains) and or only parasitism (T3). Indicating a potentiating in mortality of larvae, when used both control methods (Cry proteins and parasitoid).

A similar result was obtained by Dequech et al. (2005), who observed that the use of C. flavicincta combined with B. thuringiensis aizawai resulted in increased mortality of S. frugiperda. Additionally, (Ahmad et al., 1978) reported that the mortality of Lymantria dispar was greater in the presence of both Bt. thuringiensis and the parasitoid Apanteles melanoscelus compared with A. melanoscelus alone.

The average percentage of larvae mortality in all bioassays that were only exposed to parasitism by C. flavicincta was 60%, indicating a high potential for the control of micro-hymenopteran larvae of S. frugiperda. A similar result was obtained by (Dequech et al., 2005), who found that larvae of S. frugiperda that were only exposed to parasitism by C. flavicincta showed a high average mortality, of 78.4%.

In the present work, the offspring of the parasitoids that developed in larvae treated with Bt thuringiensis 4412 exhibited altered biological characteristics, because these toxins affect the survival of the host, which ranged from only 5-6 days, while the parasitoid C. flavicincta requires 8-11 days to complete larval development. These effects were largely indirect, related to the sensitivity of lepidopteran larvae to B. thuringiensis.

Herbivores that have consumed tissues from Bt crops, when used as prey or hosts for a natural enemy, provide a realistic exposure pathway. However, Bt proteins will affect Bt susceptible herbivores and consequently affect their quality as a resource for natural enemies. Such ‘host/prey-quality mediated effects’ have been observed in numerous tri-trophic feeding studies with Bt crops (Flexner et al., 1986; Vinson, 1990; Sharma et al., 2008; Meissle et al., 2004; Prutz and Dettner, 2004; Dhillon and Sharma, 2010).

The toxic action of Bt in S. frugiperda most likely prevents the host larvae from supplying sufficient nutrients for healthy development of the parasitoid larvae. The Bt toxin is known to modify amino acids and ions within the hemolymph composition of herbivores such as S. frugiperda. Thus, the existence of direct effects cannot be excluded, although they are quite unlikely because the Cry proteins act by binding to specific receptors in the insect gut epithelium, and has shown been to be specific for Lepidoptera (Salama et al., 1983).

Hence, the level of injury occurring in a parasitoid in its host also depends on the biology of the parasitoid. In contrast to Campoletis sonorensis, which consumes the body of its host (Ridgway and Wilson, 1975), other parasitoids such as Cotesia marginiventris feed only a portion of the inner body. The decreased levels of some essential amino acids may be one of the mechanisms through which larvae of Helicoverpa armigera infected with Bt toxins impact the parasitoid Campoletis chlorideae (Yazlovetzky, 2001).

Dhillon and Sharma (2010) evaluated the developmental period of the first generation of Campoletis chlorideae, and their results revealed a longer period of pupal formation and a reduced adult emergence period in Helicoverpa armigera treated with Bt (Biolep) compared with untreated individuals (control); however, in the second generation, there were no significant effects in any of the examined cases.

Dequech et al. (2005) found no significant difference when evaluating the biology of the descendants of Campoletis flavicincta parasitoids that had emerged from Spodoptera frugiperda that were infected or non-infected with Bacillus thuringiensis aizawai.

It is likely that our results are related to the poor quality of prey exposed to the protein Cry1B. The proteins Cry affected survival, development times and growth rates larvae of S. frugiperda and consequently affected the development of the parasitoid C. flavicincta. For S. frugiperda, (Mendes et al., 2011) reported a 20-fold reduction in the weight of larvae fed with Cry1Ab maize compared to those fed with the same non-Bt hybrid. Studies using Bt-resistant lepidopteran larvae, the hosts for parasitic wasps, support such indirect adverse effects (Schuler et al., 2004; Chen et al., 2008).

The preservation of natural enemies is critical because they help control primary and secondary pests not controlled by the Bt crop. Furthermore, recent modeling work (Onstad et al., 2013) has suggested natural enemies can also delay the evolution of resistance to the Bt plants by the targeted pest. Additional important issues to be analyzed in this work include whether parasitoids of insect pests can be used to increase the efficiency of pest control in the areas of refuges (no Bt plants) . Also, the results of this study suggested that proteins Cry1B may have a direct effect on C. flavicincta. The occurrence of direct effects of Cry proteins on a hymenopteran parasitoid, such as C. flavicincta, merits further research because of the importance of these parasitoids as natural enemies in agroecosystems.

3 Materials and Methods
3.1. Insects
3.1.2
Spodoptera frugiperda
S. frugiperda larvae were obtained from rearing conditions established in a controlled room at the UNISINOS Laboratory of Microbiology and Toxicology, with a temperature of 25ºC, a relative humidity (RH) of ± 65%, and photoperiod of 12 hours. Adults obtained from material collected in rice fields were placed in cages with a substrate for oviposition and glucose-based feeding. The eggs collected daily, were transferred to Petri dishes lined with moistened filter paper. After hatching, the larvae were individually placed in PVC cups containing an artificial diet (Poitout et al., 1974) developed specifically for the rearing of noctuids, until the pupae had formed, when they were separated by sex and placed in glass jars. The adults were maintained in cages under controlled conditions as described above.

3.1.3 Rearing of Campoletis flavicincta in the laboratory
The laboratory culture of C. flavicincta parasitoids was initiated from larvae parasitized by S. frugiperda collected in the field. The larvae were individually placed in 50 mL polypropylene vials containing an artificial diet (Poitout et al., 1974), until the emergence of adults or parasitoid.

The emerged parasitoids were multiplied through the exposure of 20 larvae to a pair of parasitoids in glass jars (11 cm high and 7 cm diameter) for approximately 24 hours. The larvae were fed with the artificial diet, and the parasitoids were fed with a 10% glucose solution. The larvae were subsequently individually placed in 50 mL plastic pots containing the artificial diet and evaluated until the formation of pupae or the emergence of the parasitoids. Both larvae and parasitoids were kept in B.O.D. chambers, adjusted to a temperature of 25°C, a 12 h photoperiod, and ± 65% RH.

3.2 Bt plants
The genetically modified rice plants (Bt rice) used in this study belong to the IRGA 424 cultivar, which was transformed using Agrobacterium tumefaciens carrying the cry1B gene of B. thuringiensis with a constitutive ubi (ubiquitin) promoter, as described by Pinto et al (2013). The vegetative growth of Bt rice was maintained at the Laboratory of Microbiology and Toxicology in Biochemical Oxygen Demand (B.O.D) chambers under a temperature of 25°C, a RH of 65% and a photoperiod of 12 hours.

The Bt maize hybrids used in this study, which are also resistant to S. frugiperda, belong to the commercial cultivar DKB-350 (YieldGard® Corn Borer-YGCB), which was transformed with the cry1Ab gene of B. thuringiensis (recommended for planting in Brazil) and the near isogenic negative checks (recommended for planting in midwestern Brazil). The Bt maize was grown under the same environmental conditions as the rice in PVC pots 15 cm diameter containing 3seeds per pot). In these experiments, seeding was performed weekly, and the plants were used between 2 and 6 weeks of vegetative growth. In these treatments, no fertilizers or chemical treatments were used.

3.3 Mortality bioassays of Spodoptera frugiperda parasitized by Campoletis flavicincta and fed with genetically modified plants (Bt maize or rice)
In these experiments, adapted from (Dequech et al., 2005), S. frugiperda larvae were exposed to the following treatments for three or ten days: (a results obtained by Ramirez-Romero et al. (2007) suggested that Cry1Ab is not acutely lethal, and mortality depends on level (dosage) and length of exposure) (T1) without parasitism or Bt plants (control); (T2) exposed to Bt plants (Bt rice or maize); (T3) only exposed to parasitism by C. flavicincta; and (T4) exposed to both Bt plants (Bt rice or maize) and parasitism (Figure 1). For each treatment, 30 larvae from the 2nd instar were used, and three repetitions performed, for a total of 360 assessed larvae.

The period of exposure was 3-10 days to compare the mortality rates of the larvae. To obtain the insects to be used for treatments T3 and T4, 2nd instar larvae of S. frugiperda were exposed to parasitoids in acrylic cages for 24 hours. After this period, both T4 and T2 larvae, at four days of age, were fed with Bt plants (maize or rice). Exposure of S. frugiperda larvae to the genetically modified plants (rice or maize) was performed in acrylic plates (35 mm diameter) whose bottom was covered with soaked filter paper discs and plant leaves. The same procedure was adopted in treatments T1 and T3, but with the transgenic plants being replaced with the non-transformed cultivar IRGA 424 or conventional Dekalb® corn.

The S. frugiperda larvae from all treatments remained exposed to the transgenic or non-transgenic plants, and the moisture in the filter paper was regularly monitored. Replacement of the leaves was performed daily, and the number of dead and living larvae and the timing of parasitoid emergence were recorded. The acrylic plates (35 mm diameter) containing the larvae (S. frugiperda) were maintained in a B.O.D. chamber, set at a temperature of 25°C, a 12 hour photoperiod and an RH of 65%.

3.4 Mortality bioassay of Spodoptera frugiperda parasitized by Campoletis flavicincta and exposed to bacterial suspensions of the monogenic strains Bt thuringiensis 4412 (Cry1B) and Bt kurstaki HD1 (Cry1Ab)
These assays were performed according to the method described above in section 3.5 (Figure 1), except that the plants were replaced with suspensions of the monogenic strains Bt kurstaki HD1 (Cry1Ab) and Bt thuringiensis 4412 (Cry1B), which were provided by the Institute Pasteur (Paris). For the assays, the strains were grown in Glucosed Usual Medium (Barjac, 1976) at 28°C and 180 rpm for 48 hours.

Then, the suspensions were centrifuged at 5,000 rpm for 15 min, after which the supernatant was discarded, and the pellet was diluted in sterile distilled water. The concentration was determined and standardized to 8.6 x 106 cells/mL for Bt thuringiensis 4412 and to 1 x 109 cells/mL for Bt kurstaki HD1 (Polanczyk et al., 2000) using a Neubauer chamber at optical microscopy. A 100µL aliquot of the suspension was applied to the surface of the artificial diet (Poitout et al., 1974), previously conditioned in mini-acrylic plates (35 mm diameter).

After the evaporation of the excess of moisture, a total of 30 larvae of S. frugiperda from 2nd instar were individually introduced per treatment. In the control, sterile distilled water was applied at a volume equivalent to that in the treatment groups. Three repetitions were performed, totalizing 360 larvae assessed in this bioassay. The experiment was conducted in a B.O.D., set at a temperature of 25°C, a ± 65% RH, and 12 hours of photoperiod.

3.5 Biology of Campoletis flavicincta descendants emerging from Spodoptera frugiperda that were exposed or not exposed to the monogenic strain Bt subesp. thuringiensis strain 4412 (Cry1B)

In the evaluation of the descendants of C. flavicincta, parasitized larvae of S. frugiperda were exposed to Bt thuringiensis 4412 as described above. As a control, parasitized larvae that were not exposed to Cry proteins were used. For each treatment, 100 larvae were assayed to ensure the emergence of the required number of parasitoids. From these insects, the parasitoids obtained from larvae that were either exposed to the Cry proteins or not exposed were divided into two pairs (two days old) for each treatment.

Each pairs was maintained for 24 h in a glass jar (11 cm high and 7 cm diameter). After this period, 50 larvae from the 2nd instar were exposed to each pair of parasitoids for 24 h. The larvae were fed with the artificial diet, and the parasitoids were fed with 10% glucose. This procedure was repeated three times, and the larvae were then isolated according to the experimental procedure described above. Each track was monitored daily, evaluating: (a) the date of formation of the parasitoid pupa, (b) the date of emergence, (c) sex, and (d) adult parasitoid longevity.

Figure 1 Schematic representation of the methodology followed for the mortality bioassays with Spodoptera frugiperda parasitized by Campoletis flavicincta and treated with Bt plants (rice or maize) or Bt bacterial suspensions (Bt thuringiensis 4412 or Bt kurstaki HD1).

3.6 Statistical analysis
Using the statistical program Systat, the log10-transformed mortality data from each treatment were subjected to one-way analysis of variance (ANOVA), followed by the Tukey test at a 5% probability. To assess the frequency of parasitoid emergence from infected and uninfected larvae and the average life cycle of these parasitoids, the data were subjected to the 2-square and t-test at a 5% significance level.

4 Conclusions
The treatments T4 (Bt proteins and C. flavicincta) were more effective in controlling S. frugiperda, thus indicating a potential of proteins Cry and C. flavicincta to be used in the control of pest an integrated pest management system. The offspring of the parasitoids that developed in larvae treated with Bt thuringiensis 4412 exhibited altered biological characteristic when compares to the control. These effects were largely indirect, related to the sensitivity of lepidopteran larvae to B. thuringiensis. . However, the existence of direct effects cannot be excluded.

Authors'Contributions:

LP oversaw the development of bioassays and insect rearing, helped in the analysis of results and preparation of the manuscript. FP produced some bioassays and helped in the creation of insects.VM helped the statistical analysis and drafting of the manuscript, LF oversaw the development of the tests, helped in data analysis and preparation of the manuscript.

Acknowledgements:
We thank the researcher Jaime Vargas de Oliveira, from the Rice Experimental Station (IRGA). We also thank the researcher Ivan Cruz, from Embrapa Maize and Sorghum, for providing the parasitoid pupae of Campoletis flavicincta used in this work. We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for granting a doctoral scholarship to the senior author.

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