Development of Dichelops melacanthus and its egg parasitoid Telenomus podisi reared on Bt-soybean MON 87701 x MON 89788 and its near conventional isoline under different temperatures

An Acad Bras Cienc aabc Anais da Academia Brasileira de Ciências An. Acad. Bras. Ciênc. 0001-3765 1678-2690 Academia Brasileira de Ciências Dichelops melacanthus foi avaliado em condições con troladas (60 ± 10% UR e fotoperíodo de 14/10 h L/E) em três temperaturas constantes (19, 25, e 31 ± 2 °C). Vagens frescas da soja MON 87701 x MON 89788 e de sua isolinha não-Bt (A5547) foram oferecidas a ninfas e adul tos. A biologia de T. podisi foi estudada nessas mesmas condições controladas de UR, mas apenas na temperatura padrão de 25 ± 2 °C. Em geral, o desenvolvimento de D. mela canthus foi melhor nas temperaturas mais altas, as quais aceleraram o desenvolvimento do percevejo, mas sem afetar os parâmetros biológicos da fase adulta. Não houve influência da soja-Bt na biologia da praga em nenhuma das temperaturas estudadas, o que mostra que D. melacanthus não é afetado pela soja transgênica. O parasitoide de ovos T. podisi também não foi impactado quando parasitou ovos da praga alimentada com soja MON 87701 x MON 89788, com resultados semelhantes àqueles obtidos na soja isogênica não-Bt. Portanto, este estudo comprova que D. melacanthus é favorecido por temperaturas mais altas (31 ± 2 °C), e que soja-Bt MON 87701 x MON 89788 não afeta o desenvolvimento desta praga assim como de seu parasitoide T. podisi. INTRODUCTION The soybean [Glycine max (L.) Merril] is one of the most important crops worldwide, with an average annual production of around 260 million tons in an area of about 103 million ha (Faostat 2013). However, the occurrence of pests has become one of the main factors that has hindered obtaining good yields of this grain, mainly defoliating insects (Panizzi and Corrêa-Ferreira 1997, Bueno et al. 2011) and sucking insects (Panizzi 1997, Smith et al. 2009, Musser et al. 2009). Currently there is evident population growth of the green belly stink bug Dichelops melacanthus (Dallas) (Hemiptera: Pentatomidae), in soybean growing areas of Brazil, especially after the adoption of a no-tillage system in most producing regions of the country (Chocorosqui and Panizzi 2004). This species attacks the soybean crop, but now also occurs in winter crops such as wheat (Manfredi-Coimbra et al. 2005). The influence of temperature on the life cycle of insects has been demonstrated in several studies (Hochachka and Sommero 1984, Praslicka and Huszár 2004, Silva et al. 2011). In this regard, Chocorosqui and Panizzi (2002) conducted a preliminary study on the impact of temperature on the nymphal stage of D. melacanthus, and found that the nymphs did quite poorly in low temperatures. However, the literature lacks detailed data on the impact of temperature on the life cycle of this stink bug, so this is crucial information that would make it practical to determine the regions and times when the pest could benefit from climate changes. Within the context of integrated pest manage ment, one of the tools that have been long adopted by producers is the cultivation of plants resistant to insects. The process of gene insertion with the bacterium Bacillus thuringiensis Berlinier (Bt) in the plant has been developed primarily for the control of lepidopterans, as in the case of Bt cotton and Bt corn. This bacterium expresses an insecticidal protein (Cry) that contacts the gut of susceptible insects, debilitating them immediately after ingestion (Santos et al. 2009). MON 87701 × MON 89788 soybeans express the insecticidal cry1Ac insecticide gene, which is resistant to the main crop defoliators, Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Eribidae) and the soybean looper Chrysodeixis (= Pseudoplusia) includens (Walker, 1857) (Lepidoptera: Noctuidae) (Bernardi et al. 2012). Among the advantages of the cultivation of Bt plants are the reduction in insecticide use and lower environmental impact compared to the use of insecticides (Kouser and Qaim 2011, Lu et al. 2012). However, it is still unclear what impact this technology can have on non-target pests, since the information in the literature is scarce and conflicting. For example, Kim et al. (2012) reported in a laboratory study that the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) has higher fecundity when developed on Bt corn. This benefit for the biology of the pest can change the scenario of reduced insecticide use on crops, because of the need to use pesticides to control pests that were formerly secondary on the crop (Li et al. 2011). In Asia, after the large-scale adoption of Bt cotton farming, the population growth of the mirid Apolygus locurum (Meyer-Dur) (Hemiptera: Miridae) has been observed. These studies indicate that the expansion of Bt plant cultivation can benefit non-target pests through changes in the physico-chemical composition of plants (Latham et al. 2006), where the main beneficiaries may be sucking insects sucking, such as D. melacanthus. Among the important natural enemies of this bug are the egg parasitoid Telenomus podisi (Ashmed) (Hymenoptera: Platygastridae) (Corrêa-Ferreira and Moscardi 1994, Laumann et al. 2010). This parasitoid is distributed in almost all Brazilian agroecosystems, but its efficiency can be affected by many factors. Thus, some studies have shown that the cultivation of Bt plants can alter the nutritional status of the host and consequently reduce the performance of natural enemies (Zhang et al. 2006, Sanders et al. 2007), while other studies have not demonstrated such a relationship (Manachini and Lozzia 2002, Fernandes et al. 2007), suggesting that different results may be obtained depending on the taxon studied. Thus, the aim of this study was to assess the biology of D. melacanthus at different temperatures when fed MON 87701 × MON 89788 soybean pods and possible impacts of this technology on the egg parasitoid T. podisi. MATERIALS AND METHODS Rearing And Multiplication of D. melacanthus and T. podisi Eggs of D. melacanthus for the study came from the rearing laboratory of the Agronomic Institute of Paraná (located in Londrina, Paraná), where it has been established for 19 years. The parasitoids were derived from the rearing of the Embrapa Soybean Laboratory, where it has been reared for over 27 generations in the laboratory. Both populations were raised under controlled laboratory conditions (25 ± 2 °C, 60 ± 10% RH, and a photoperiod of 14:10 h [L:D]). Soybean Cultivation and Preparation of the Material Used in Bioassays The soybean cultivar was developed and made available by the company Monsanto Brazil Ltda. The near isoline A5547 (non-Bt soybean, maturation group 5.5) and MON 87701 × MON 89788 expressing the Cry1Ac protein (Bt soybean) were used. The material was sown in sterile soil in plastic pots (8 L) maintained in a greenhouse. Five seeds per pot were distributed in a total of 25 pots per breeding line. In terms of cultural practices, there was weekly control of powdery mildew with the application of a product based on sulfur (S) (Kumulus(r)) at a dose of 0.5 g L-1. At the V2 phenological stage of soybeans (Fehr and Caviness 1977), plants were fertilized with a chemical fertilizer with the formulation 0-20-20 (N-P-K), according to the technical recommendations of the region. The humidity of the vessels was monitored daily and drip irrigation of plants occurred whenever necessary. The pods were collected from the R6 phenological stage (Fehr and Caviness 1977) when they were already full. Before being offered to the stink bugs, the pods went through a process of cleaning, in which they were immersed for 15 min in a solution of water and 5% sodium hypochlorite. After this period, the pods were removed from the solution, rinsed, and dried for 2 h before the material was offered to insects. Biology of D. melacanthus FED non-Bt and Bt Soybean Pods at Different Temperatures The testing was conducted in a completely rando mized design in a 3 × 2 factorial [three temperatures vs. two soybean isolines (Bt and non-Bt)] with seven replicates of 10 nymphs per treatment. The temperatures to which the specimens were submitted were 19, 25, and 31 ± 2 °C under controlled relative humidity (60 ± 10%) and 14/10 h L/D photoperiod conditions. The sources of food offered were the soybean MON 87701 × MON 89788, which expresses the Cry1Ac protein (Bt soybean), and its non-Bt isoline A5547 (non-Bt soybean). The nymphs were placed in Petri(r) dishes (Ø = 6 cm), lined with filter paper. The individualization occurred from the second instar, as in the first, the nymphs had herd behavior and did not feed. Each individual was offered a soybean pod (each two days) and a plastic Eppendorf(r) tube containing water and capped with cotton. Plates were moistened daily to aid in maintaining the relative humidity (RH%) of the plates. Every day we reviewed the nymphal stage and recorded dead individuals in all treatments. Within 24 h after the last molt, the adult stink bugs were weighed (g) with the aid of an analytical balance and separated by sex. Later, stink bugs were put into Gerbox(r) boxes that had perforated lids to allow air to enter and were lined with filter paper. Additionally, within each box a cap with cotton soaked in water was made available for adults. There were two females and one male per Gerbox(r), and every 48 h their filter paper and food were changed. The biological parameters evaluated were the weight (g), sex ratio, fecundity, and egg viability (%). To assess the viability of the eggs, these were taken from each Gerbox(r), packaged in petri dishes (Ø = 6 cm), and kept under the same conditions of temperature, humidity, and photoperiod as adults. As the nymphs emerged, they were counted to calculate viability. Biology of T. podisi Developed in Eggs of D. melacanthus Reared With Bt and non-Bt Soybean Pods The eggs of D. melacanthus used to study the bio logy of T. podisi were obtained from rearing with the same methodology used in the study of the biology of the stink bugs, except for the use of large plastic boxes of 25 × 30 × 30 cm. The eggs collected from the boxes were stored in liquid nitrogen at -196 °C, which does not affect the quality of these eggs for a period of 360 d (Corrêa-Ferreira and Oliveira 1998). A mean of 25 eggs from each treatment were separated and glued to non-toxic white cardboard glue mats (0.8 cm × 5 cm). The experiment was carried out in a completely randomized design with 35 replications. In each repetition, we used a newly emerged T. podisi female (between 24 h and 48 h) that had previously mated and individualized in Duran tubes (6 cm × 1 cm Ø) containing a droplet of honey to feed the parasitoid. The cards were individually introduced into the tubes and immediately sealed with plastic wrap. Parasitism was allowed for 24 h, after which time the cards were removed and transferred to separate flat-bottom glass tubes (8 cm × 2 cm Ø) until adult emergence. Females also remained acclimatized under the same conditions in which they developed in the chambers. We evaluated the longevity of parental females (days), the egg-to-adult period (days), parasitism, parasitism viability (% emergence), and sex ratio. Statistical Analysis Data were analyzed for normality (Shapiro and Wilk 1965) and homogeneity of variance of treatments (Burr and Foster 1972), and if necessary transformed to perform ANOVA. The treatment means were then compared by a Tukey test at the 5% probability level (SAS Institute 2001). RESULTS Biology of D. melacanthus Fed non-Bt and Bt Soybean Pods at Different Temperatures The results demonstrated that the increase in temperature accelerated the nymphal development of D. melacanthus at all instars of the nymphal stage of the species (Table I). At 31 °C, the nymphal stage of D. melacanthus took 16.73 d, while at 25 °C it was 27.50 d (F = 195.8, DF = 1, P = <0.01). When the insects were subjected to a temperature of 19 °C, development was highly degraded, and only one individual reached the adult stage. Overall, the highest mortality rate was always observed in the second instar of the stink bug, demonstrating that the food is not very appropriate for D. melacanthus development (Table II). TABLE I - Influence of temperature on the nymphal longevity of Dichelops melacanthus developed on Bt and non Bt isogenic soybean pods. Variables Nymphal logevity (d) of Dichelops melacanthus 2° instar 3° instar 4° instar 5° instar Total Temperature (oC) 19 12.84 ± 0.35 a 15.28 ± 1.45 a - - - 25 4.55 ± 0.25 b 7.18 ± 0.52 b 7.26 ± 0.29 a 9.58 ± 0.18 a 27.50 ± 0.58 a 31 3.76 ± 0.12 c 3.68 ± 0.18 c 3.99 ± 0.22 b 5.44 ± 0.12 b 16.73 ± 0.52 b Soybean Bt 6.97 ± 0.82ns 6.70 ± 1.08ns 5.34 ± 0.44ns 7.36 ± 0.58ns 22.85 ± 1.56ns Non-Bt 7.14 ± 1.04 8.44 ± 1.22 5.91 ± 0.58 7.66 ± 0.59 21.38 ± 1.60 CV (%) 8.63 34.25 17.33 3.97 9.21 DFtemp. 2 2 1 1 1 DFsoybean 1 1 1 1 1 DFtemp. x soybean 2 2 1 1 1 Ftemp. 259.46 46.35 74.55 364.13 195.8 Fsoybean 0.55 1.04 2.28 2.41 3.62 Ftemp. x soybean 2.96 0.09 1.91 0.74 0.01 Ptemp. < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Psoybean 0.46 0.31 0.14 0.14 0.07 Ptemp. x soybean 0.07 0.91 0.17 0.40 0.90 Means ± SEM followed by the same letter in the column for each factor (temperature or soybean) do not differ by Tukey test (P>0.05). nsANOVA not significant. - Parameter nonexistent. TABLE II - Influence of temperature on the nymphal viability (%) of Dichelops melacanthus developed on Bt and non-Bt isogenic soybean pods. Variables Dichelops melacanthus Nymphal stage viability (%) 2° instar 3° instar 4° instar 5° instar Total Temperature (oC) 19 29.90 ± 3.04ns 41.76 ± 10.39 a 0.00 ± 0.00 b 0.00 ± 0.00 b 0.42 ± 0.22 a 25 28.57 ± 3.76 85.83 ± 4.93 b 98.07 ± 1.92 a 96.15 ± 2.60 a 17.85 ± 1.55 b 31 38.57 ± 3.29 87.62 ± 4.48 c 97.14 ± 1.94 a 100.00 ± 0.00 a 29.28 ± 2.21 c Soybean Bt 30.47 ± 2.97ns 77.38 ± 6.30ns 70.26 ± 10.06ns 71.05 ± 10.16ns 16.85 ± 3.29ns Non-Bt 33.80 ± 2.80 66.10 ± 8.25 68.42 ± 10.92 66.66 ± 11.43 14.85 ± 2.52 CV (%) 26.14 36.42 7.92 16.02 17 DFtemp. 2 2 2 2 2 DFsoybean 1 1 1 1 1 DFtemp. x soybean 2 2 2 2 2 Ftemp. 2.31 13.84 1156.37 293.11 294.74 Fsoybean 0.7 1.96 3.43 0.03 0.51 Ftemp. x soybean 0.01 1.08 1 1.65 2.19 Ptemp. 0.12 < 0.01 < 0.01 < 0.01 < 0.01 Psoybean 0.41 0.17 0.07 0.85 0.48 Ptemp. x soybean 0.98 0.35 0.38 0.21 0.13 Means ± SEM followed by the same letter in the column for each factor (temperature or soybean) do not differ by Tukey test (P>0.05). nsANOVA not significant. - Parameter nonexistent. Although nymphal viability was relatively low at both temperatures, it was superior when the insects were subjected to a temperature of 31 °C (approximately 30%), while that at a 25 °C average was 17.85% (F = 294.74, DF= 1, P = <0.01). On the other hand, MON 87701 × MON 89788 soybeans did not affect the nymphal biology at any temperature, thus indicating that they were not detrimental to the development of D. melacanthus (Tables I and II). TABLE III - Biological parameters of Telenomus podisi developed in Dichelops melacanthus eggs fed on Bt and non Bt soybean. Soybean Longevity of parental female (d)1 Period Parasitism (%)1,2 egg - adult (d)1 Bt 12.24 ± 1.56ns 12.88 ± 0.10ns 85.16 ± 2.26ns Non-Bt 11.80 ± 1.33 12.68 ± 0.13 83.32 ± 3.42 CV (%) 27 2.08 7.71 DFresidue 8 8 8 F 0.05 1.39 0.20 P 0.83 0.27 0.66 Soybean Viability of parasitism (%)1 Sexual rate1 Bt 76.79 ± 4.03ns 0.80 ± 0.04ns Non-Bt 79.29 ± 4.38 0.82 ± 0.03 CV (%) 12.07 11.25 DFresidue 8 8 F 0.18 0.06 P 0.68 0.81 1Means ± SEM followed by the same letter in the column do not differ by test t (P>0.05). nsANOVA not significant. 2Data transformed (). Due to the high mortality of nymphs at 19 °C, no data on the biological parameters of adults were obtained from that treatment. Overall, the adults that emerged at 31 °C showed a greater weight than those at 25 °C, but the other biological characteristics, such as sex ratio, fecundity, and egg viability, were similar at the two temperatures (Fig. 1). As observed for nymphal stage D. melacanthus, the adults fed on MON 87701 × MON 89788 soy beans had biological parameters similar to those fed on non-Bt soybeans (Fig. 2), confirming that the transgenic soybean does not affect the biology of the pest. Figure 1 - Biological characteristics of the adult phase Dichelops melacanthus developed with soybean pods submit ted at different constant temperatures. Means were compared by Tukey test (* = P ≤ 0.05). (PS. At the temperature of 19 °C hatched only 1 adult individual, not allowing to perform statistical analysis). Figure 2 - Biological characteristics of the adult of Dichelops melacanthus developted on Bt and non-Bt soybean pods. Means were compared by Tukey test. Biology of T. podisi Developed in Eggs of D. melacanthus Reared With Bt and non-Bt Soybean Pods The development of the parasitoid T. podisi was not harmed when it parasitized eggs of D. melacanthus reared on MON 87701 × MON 89788 soybeans (Table III). Parental females lived about 12 d in both treatments, and had a parasitism rate exceeding 80%, indicating good nutritional quality of the host. Confirming this statement, the viability parameters of parasitism and sex ratio were also similar between treatments (Table III). DISCUSSION Our study proves that the stink bug D. melacanthus develops better in higher temperatures, explaining the higher occurrence of this species in the warmer regions of Brazil. Even when the insects were subjected to 31 °C, the biological parameters of adult females were not affected. Among nymphs that developed at 19 °C, almost all individuals died in the third instar without performing ecdysis, confirming that the species is better adapted to higher temperatures. These results corroborate Chocorosqui and Panizzi (2002), where the authors found that at low temperatures (15-20 °C) there was a high mortality of the pest, while the best performance was at 25 °C. However, the maximum temperature studied by the authors was 25 °C, and the study was limited only to the impact on the nymphal stage of the stink bug. In our study examining the different stages of D. melacanthus, temperatures up to 31 °C shortened the juvenile phase of the stink bug, but did not affect the biological parameters of adults, indicating that in areas or times of higher temperatures, this species can cause major damage to crops, due to a possible increase in the number of generations of the pest. The fecundity of females that emerged at 31 °C was 104.63 eggs, with a viability of 65.22%. These results did not differ for females from 25 °C, which had fecundity and viability of 86.33 eggs and 60.18%, respectively. These results are underpinned by Chocorosqui and Panizzi (2008), who claim that fertility can vary from 74.1 to 131.4 eggs/female with viability between 55.8 and 74.9%. The importance of the influence of temperature on the biology and biodynamics of pests has been increasingly studied (Nondillo et al. 2008, Silva et al. 2011, Kendrick and Benstead 2013). In Brazil, some insects have recently gained greater importance, and it is believed that the occurrence of higher temperatures has favored some insect pests. For example, currently, the brown stink bug E. heros is the main problem in areas cultivated with soybeans, and this species has become highly adapted to high temperature conditions (Cividanes and Parra 1994). According to Cividanes and Parra (1994), the highest oviposition of this species is in the range of 26 to 28 °C, but it also has good growth at 30 °C. Currently, the soybean looper C. includens is reported as a key pest of soybeans, and its occurrence is often associated with periods of higher temperatures and lower rainfall. According to Mason and Mack (1984), the maximum fecundity of moths of this species occurs between 26 and 32 °C, which explain the greater abundance of this pest in the warmer regions of Brazil. Despite the climatic significance of the dynamics of insects, the nutritional quality of the host plant is crucial for the establishment of a pest. In this sense, our results showed that soybean pods are probably not suitable for the development of green belly stink bugs, due to the high mortality rate observed in general. Similar studies have also indicated a high mortality of D. melacanthus when only fed with soybean pods (Panizzi et al. 2007, Chocorosqui and Panizzi 2008), achieving mortality rates of around 63% (Chocorosqui and Panizzi 2008). This high mortality is observed even when the insect feeds on other food sources, such as an artificial diet (Panizzi et al. 2007) or seeds of wheat or maize seedlings (Chocorosqui and Panizzi 2008). Therefore, the survival of D. melacanthus in nature is not guaranteed by only one food source, but probably by a diversity of host plants (Silva et al. 2013), which may explain the current status of this pest, which is still of secondary importance in soybeans. The nymphal stage of D. melacanthus at 25 °C took about 25 d to complete, independent of the genetic material of soybeans. These values ​​are similar to those obtained by Chocorosqui and Panizzi (2008), where this stink bug took between 25 and 28 d to reach the adult stage while feeding on non-Bt soybean pods. When the insects reached the adult stage, the parameters of sex ratio, fecundity, and egg viability did not differ between the treatments, thus proving the ineffectiveness of MON 87701 × MON 89788 soybeans on the biology of D. melacanthus. Thus, these observations demonstrate that MON 87701 × MON 89788 soybeans have no im pact on D. melacanthus. However, although the sucking insects do not come in direct contact with the insecticidal protein, the safety of Bt plants on sucking insects cannot be generalized because there are other factors that can alter the biology of non-target pests. Accordingly, Kim et al. (2012) reported that the development of the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) on Bt maize surprisingly increased its fecundity. The authors also found a high concentration of insecticidal protein (Cry1F) in insects, proving the ingestion of Bt. While the conducting vessels of plants do not carry the insecticidal protein, insects can accidentally ingest protein when they are inserting their stylus on the leaf area of the host. Therefore, it is possible that D. melacanthus has ingested the Bt protein Cry1Ac from soybeans, but is unlikely that buildup occurred in its tissues, since the biological parameters of the pest were not affected. Although under laboratory conditions the stink bug has not shown differences in their biology, in field conditions there are many contradictory results, where the non-target pest may or may not be influenced by Bt (Pons et al. 2005, Mann et al. 2010, Li et al. 2011). This was evidenced in the work of Dhillon and Shama (2013), in which the authors reported a higher incidence of the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) on Bt cotton, while the population of Amrasca biguttula (Ishida) (Hemiptera: Cicadellidae) was negatively influenced by the transgenic plant. In another study, Fernandes et al. (2012) observed the opposite behavior, with a lower incidence of aphids in Bt cotton. Therefore, these divergent results demonstrate that this relationship is very specific, depending on the plant and insect species studied. The gene cry1Ac expressed in Bt soybeans is also present in some Bt cotton (Bollgard I(r)), which is nowadays cultivated on a large-scale worldwide. Some research has been performed with this transgenic cotton, finding no influence of this technology on non-target insects (Torres and Ruberson 2006, Sujii et al. 2013), similar to the results of our study. However, for the cultivation of Bt maize, a few different results have been observed, including reported population increases in the leafhoppers Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) (Virla et al. 2010) and Zyginidia scutellaris (Herrich - Schäffer) (Pons et al. 2005). One hypothesis raised by the authors is that the pest may have benefited from the high quality of the uninjured leaves of the plant, due to the lower occurrence of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) (Virla et al. 2010). In the case of Bt soybean crops, the expected reduction in leaf injury caused by A. gemmatalis and C. includens, particularly, may also favor the higher abundance of other pests such as sucking pests like stink bugs. However, this hypothesis needs to be investigated under field conditions. In the field, the quality of the host plant pest may interfere with the rate of parasitism (Koppel et al. 2009), probably due to being able to change the nutritional quality of host eggs. In our study, this was not observed, and the biology of T. podisi was not affected when developed in eggs of D. melacanthus fed MON 87701 × MON 89788 soybeans. This indicates that the nutritional quality of the eggs of the pest does not affect the development of its natural enemy. This was confirmed by the high parasitism rate (above 80% in both treatments), resembling that on eggs of E. heros, its preferred host (Pacheco and Corrêa-Ferreira 1998). These results indicate that this parasitoid can be used to mitigate the impact of stink bugs on MON 87701 × MON 89788 soybeans, since the efficiency of this natural enemy will not be impaired. The innocuous effect of Bt on natural ene mies has been shown in other research. Chen et al. (2008) showed that the parasitoid Diadegma insu laris (Cresson) (Hymenoptera: Braconidae) is not affected by the Bt protein, regardless of the form in which it is offered to the natural enemy. Similarly, egg parasitoids of Lepidoptera belonging to the genus Trichogramma are also not affected when fed with pollen from Bt corn and cotton in their diet (Geng et al. 2006, Wang et al. 2007). However, other studies have found adverse results on natural ene mies. For example, Gao et al. (2010) found that the development time of the first generations of Anagrus nilaparvatae (Pang and Wang) (Hyme noptera: Mymaridae) in eggs of the non-target pest Nilapavarta lugens (Stal) (Hemiptera: Delphacidae) were affected when the pest was fed Bt rice. In a review by Lovei and Arpaia (2005), 40% of studies found some negative impact on natural enemy biology caused directly or indirectly by Bt plants. Nevertheless, the authors stress that there is still little research investigating this interaction, mainly because most of the studies were performed with a small group of natural enemies. Finally, our work demonstrated that the stink bug D. melacanthus has better development at elevated temperatures, and in this condition the pest may cause further damage to cultivated plants. Thus, it is necessary to monitor the temperature over the years and in different regions with soybean crops to determine whether the pest will not be affected by warmer temperatures at these sites. Additionally, we can clearly observe that our study has an important contribution in proving that D. melacanthus and its parasitoid T. podisi are not affected by soybean MON 87701 × MON 89788 technology. Further field studies are needed where it is possible to study the bioecology of the pest, especially considering that there is less competition for food because of the population reduction of the target lepidopteran with this technology, thus changing the biodynamics of pests in crops. ACKNOWLEDGMENTS Authors wish to thank Embrapa Soja, the sponsor agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support. Bernardi O, Malvestiti GS, Dourado PM, Oliveira WS, Martinelli S, Berger GU, Head GP and Omoto C. 2012. Assessment of the high -dose concept and level of control provided by MON 87701 x MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil. Pest Manag Sci 68: 1083-1091. Bernardi O Malvestiti GS Dourado PM Oliveira WS Martinelli S Berger GU Head GP Omoto C 2012 Assessment of the high -dose concept and level of control provided by MON 87701 x MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil Pest Manag Sci 68 1083 1091 Bueno Rcof, Bueno AF, Moscardi F, Parra Jrp and Hoffmann-Campo CB. 2011. Lepidopteran larva consumption of soybean foliage: basis for developing multiple- species economic thresholds for pest management decisions. Pest Manag Sci 67: 170-174. Bueno Rcof Bueno AF Moscardi F Parra Jrp Hoffmann-Campo CB 2011 Lepidopteran larva consumption of soybean foliage: basis for developing multiple- species economic thresholds for pest management decisions Pest Manag Sci 67 170 174 Burr IW and Foster LA. 1972. A test for equality of vari ances. Mimeo Series No. 282. University of Purdue, West Lafayette, 26 p. Burr IW Foster LA 1972 A test for equality of vari ances University of Purdue West Lafayette 26 p Chen M, Zhao JZ, Collins HL, Earle ED, Cao J and Shelton AM. 2008. A critical assessment of the effects of Bt transgenic plants on parasitoids. Plos One 3: e2284. Chen M Zhao JZ Collins HL Earle ED Cao J Shelton AM 2008 A critical assessment of the effects of Bt transgenic plants on parasitoids Plos One 3 e2284 e2284 Chocorosqui VR and Panizzi AR. 2002. Influência da temperatura na biologia de ninfas de Dichelops melacanthus (Dallas, 1851) (Heteroptera: pentatomidae). Semina 23: 217-220. Chocorosqui VR Panizzi AR 2002 Influência da temperatura na biologia de ninfas de Dichelops melacanthus (Dallas, 1851) (Heteroptera: pentatomidae) Semina 23 217 220 Chocorosqui VR and Panizzi AR. 2004. Impact of cultivation systems on Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) populations and damage and its chemical control on wheat. Neotrop Entomol 33: 487-492. Chocorosqui VR Panizzi AR 2004 Impact of cultivation systems on Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) populations and damage and its chemical control on wheat Neotrop Entomol 33 487 492 Chocorosqui VR and Panizzi AR. 2008. Nymph and adult biology of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) feeding on cultivated and non-cultivated host plants. Neotrop Entomol 37: 356-360. Chocorosqui VR Panizzi AR 2008 Nymph and adult biology of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) feeding on cultivated and non-cultivated host plants Neotrop Entomol 37 356 360 Cividanes FJ and Parra Jrp. 1994. Biologia em diferentes temperaturas e exigências térmicas de percevejos pragas da soja: II. Euschistus heros (Fabr.) (Heteroptera: Pentatomidae). Pesq Agropec Bras 29: 1841-1846. Cividanes FJ Parra Jrp 1994 Biologia em diferentes temperaturas e exigências térmicas de percevejos pragas da soja: II. Euschistus heros (Fabr.) (Heteroptera: Pentatomidae) Pesq Agropec Bras 29 1841 1846 Corrêa-Ferreira BS and Moscardi F. 1994. Seasonal occurrence and host spectrum of egg parasitoids associated with soybean stink bugs. Biol Control 5: 196-202. Corrêa-Ferreira BS Moscardi F 1994 Seasonal occurrence and host spectrum of egg parasitoids associated with soybean stink bugs Biol Control 5 196 202 Corrêa-Ferreira BS and Oliveira Mcn. 1998. Viability of Nezara viridula (L.) eggs for parasitism by Trissolcus basalis (Woll.), under different storage techiniques in liquid Nitrogen. An Soc Entomol Brasil 27: 101-107. Corrêa-Ferreira BS Oliveira Mcn 1998 Viability of Nezara viridula (L.) eggs for parasitism by Trissolcus basalis (Woll.), under different storage techiniques in liquid Nitrogen An Soc Entomol Brasil 27 101 107 Dhillon MK and Sharma HC. 2013. Comparative studies on the effects of Bt-transgenic and non-transgenic cotton on arthropod diversity, seed cotton yield and bollworms control. J Environ Biol 34: 67-73. Dhillon MK Sharma HC 2013 Comparative studies on the effects of Bt-transgenic and non-transgenic cotton on arthropod diversity, seed cotton yield and bollworms control J Environ Biol 34 67 73 Faostat - Food and Agriculture Organization of the United Nations. 2013. http://faostat3.fao.org/home/index.html. Faostat Food and Agriculture Organization of the United Nations 2013 http://faostat3.fao.org/home/index.html Fehr WR and Caviness CE. 1977. Stages of soybean de velopment. Ames: University of Sciense and Technology. Special Report 80: 11. Fehr WR Caviness CE 1977 Stages of soybean de velopment Ames: University of Sciense and Technology. Special Report 80 11 11 Fernandes FS, Ramalho FS, Malaquias JB, Nascimento Junior JL, Correia ET and Zanuncio JC. 2012. Within-plant distribution of cotton aphid (Hemip tera: Aphididae) in cotton cultivars with colored fibers. An Acad Bras Cien 84: 707-719. Fernandes FS Ramalho FS Malaquias JB Nascimento Junior JL Correia ET Zanuncio JC 2012 Within-plant distribution of cotton aphid (Hemip tera: Aphididae) in cotton cultivars with colored fibers An Acad Bras Cien 84 707 719 Fernandes OA, Faria M, Martinelli S, Schmidt F, Carvalho VF and Moro G. 2007. Short-term assessment of Bt maize on non-target arthropods in Brazil. Sci Agric 64: 249-255. Fernandes OA Faria M Martinelli S Schmidt F Carvalho VF Moro G 2007 Short-term assessment of Bt maize on non-target arthropods in Brazil Sci Agric 64 249-255 Gao MQ, Hou SP, Pu DQ, Shi M, Ye GY and Chen XX. 2010. Multi generation effects of Bt rice on Anagrus nilaparvatae a parasitoid of the nontarget pest Nilapavarta lugens. Environ Entomol 39: 2039-2044. Gao MQ Hou SP Pu DQ Shi M Ye GY Chen XX 2010 Multi generation effects of Bt rice on Anagrus nilaparvatae a parasitoid of the nontarget pest Nilapavarta lugens Environ Entomol 39 2039 2044 Geng JH, Shen ZR, Song K and Zheng L. 2006. Effect of pollen of regular cotton and transgenic Bt-CpTI cotton on the survival and reproduction of the parasitoid wasp Trichogramma chilonis (Hymenoptera: Trichogrammatidae) in the laboratory. Environ Entomol 35: 1661-1668. Geng JH Shen ZR Song K Zheng L 2006 Effect of pollen of regular cotton and transgenic Bt-CpTI cotton on the survival and reproduction of the parasitoid wasp Trichogramma chilonis (Hymenoptera: Trichogrammatidae) in the laboratory Environ Entomol 35 1661 1668 Hochachka PW and Sommero GN. 1984. Temperature adaptation. In: Hochachka PW and Sommero GN (Eds), Biochemical Adaptation. Princeton University Press, Princeton, NJ, p. 355-449. Hochachka PW Sommero GN 1984 Temperature adaptation Hochachka PW Sommero GN Biochemical Adaptation Princeton University Press Princeton, NJ 355 449 Kendrick MR and Benstead JP. 2013. Temperature and nutrient availability interact to mediate growth and body stoichiometry in a detritivorous stream insect. Freshwater Biol 58: 1820-1830. Kendrick MR Benstead JP 2013 Temperature and nutrient availability interact to mediate growth and body stoichiometry in a detritivorous stream insect Freshwater Biol 58 1820 1830 Kim YH, Hwang CE, Kim TS, Lee JH and Lee S. 2012. Assessment of potential impacts due to unintentionally released Bt maize plants on non-target aphid Rhopa losiphum padi (Hemiptera: Aphididae). J Asia Pac Entomol 15: 443-446. Kim YH Hwang CE Kim TS Lee JH Lee S 2012 Assessment of potential impacts due to unintentionally released Bt maize plants on non-target aphid Rhopa losiphum padi (Hemiptera: Aphididae) J Asia Pac Entomol 15 443 446 Koppel AL, Herbert DA, Kuhar TP and Kamminga K. 2009. Survey of stink bug (Hemiptera: Pentatomidae) egg parasitoids in wheat, soybean, and vegetable crops in Southeast Virginia. Environ Entomol 38: 375-379. Koppel AL Herbert DA Kuhar TP Kamminga K 2009 Survey of stink bug (Hemiptera: Pentatomidae) egg parasitoids in wheat, soybean, and vegetable crops in Southeast Virginia Environ Entomol 38 375 379 Kouser S and Qaim M. 2011. Impact of Bt cotton on pesti cide poisoning in smallholder agriculture: A panel data analysis. Ecol Econ 70: 2105-2113. Kouser S Qaim M 2011 Impact of Bt cotton on pesti cide poisoning in smallholder agriculture: A panel data analysis Ecol Econ 70 2105 2113 Latham JR, Wilson AK and Steinbrecher RA. 2006. The Mutational Consequences of Plant Transformation. J Biomed Biotechnol 2006: 1-7. Latham JR Wilson AK Steinbrecher RA 2006 The Mutational Consequences of Plant Transformation J Biomed Biotechnol 2006 1 7 Laumann RA, Moraes Mcb, Silva JP, Vieira Amc, Silveira S and Borges M. 2010. Vespas parasitoides de ovos como inimigos naturais do percevejo neotropical Dichelops melacanthus. Pesq Agropec Bras 45: 442-449. Laumann RA Moraes Mcb Silva JP Vieira Amc Silveira S Borges M 2010 Vespas parasitoides de ovos como inimigos naturais do percevejo neotropical Dichelops melacanthus Pesq Agropec Bras 45 442 449 Li G, Feng H, McNeil JN, Liu B, Chen P and Qiu F. 2011. Impacts of transgenic Bt cotton on a non-target pest, Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae), in northern China. Crop Protect 30: 1573-1578. Li G Feng H McNeil JN Liu B Chen P Qiu F 2011 Impacts of transgenic Bt cotton on a non-target pest, Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae), in northern China Crop Protect 30 1573 1578 Lovei GL and Arpaia S. 2005. The impact of transgenic plants on natural enemies: a critical review of laboratory studies. Entomol Exp Appl 144(1): 1-14. Lovei GL Arpaia S 2005 The impact of transgenic plants on natural enemies: a critical review of laboratory studies Entomol Exp Appl 144 1 1 14 Lu Y, Wu K, Jiang Y, Guo Y and Desneux L. 2012. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nat 487: 362-367. Lu Y Wu K Jiang Y Guo Y Desneux L 2012 Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services Nat 487 362 367 Manachini B and Lozzia GC. 2002. First results into the effects of Bt corn crop on nematofauna. Boll Zool Agric Bachic 34: 85-96. Manachini B Lozzia GC 2002 First results into the effects of Bt corn crop on nematofauna Boll Zool Agric Bachic 34 85 96 Manfredi-Coimbra S, Silva JJ, Chocorosqui VR and Panizzi AR. 2005. Danos do percevejo barriga-verde Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) em trigo. Cienc Rural 35: 1243-1247. Manfredi-Coimbra S Silva JJ Chocorosqui VR Panizzi AR 2005 Danos do percevejo barriga-verde Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) em trigo Cienc Rural 35 1243 1247 Mann RS, Gill RS, Dhawan AK and Shera PS. 2010. Relative abundance and damage by target and non-target insects on Bollgard and Bollgard II cotton cultivars. Crop Prot 29: 793-801. Mann RS Gill RS Dhawan AK Shera PS 2010 Relative abundance and damage by target and non-target insects on Bollgard and Bollgard II cotton cultivars Crop Prot 29 793 801 Mason LJ and Mack TP. 1984. Influence of temperature on oviposition and adult female longevity for the soybean looper, Pseudoplusia includens (Walker) (Lepidoptera Noctuidae). Environ Entomol 13: 379-383. Mason LJ Mack TP 1984 Influence of temperature on oviposition and adult female longevity for the soybean looper, Pseudoplusia includens (Walker) (Lepidoptera Noctuidae) Environ Entomol 13 379 383 Musser FR, Stewart SD and Catchot Jr AL. 2009. 2008 soybean insect losses for Mississippi and Tennessee. Midsouth Entomol 2: 42-46. Musser FR Stewart SD Catchot Jr AL 2009 2008 soybean insect losses for Mississippi and Tennessee Midsouth Entomol 2 42 46 Nondillo A, Redaelli LR, Botton M, Pinent Smj and Gitz R. 2008. Exigências térmicas e estimativa do número de gerações anuais de Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) em morangueiro. Neotrop Entomol 37: 646-650. Nondillo A Redaelli LR Botton M Pinent Smj Gitz R 2008 Exigências térmicas e estimativa do número de gerações anuais de Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) em morangueiro Neotrop Entomol 37 646 650 Pacheco Djp and Côrrea-Ferreira BS. 1998. Potencial reprodutivo e longevidade do parasitóide Telenomus podisi Ashmead, em ovos de diferentes espécies de percevejos. An Soc Entomol Brasil 27: 585-591. Pacheco Djp Côrrea-Ferreira BS 1998 Potencial reprodutivo e longevidade do parasitóide Telenomus podisi Ashmead, em ovos de diferentes espécies de percevejos An Soc Entomol Brasil 27 585 591 Panizzi AR. 1997. Wild hosts of pentatomids: ecological and role in their pest status on crops. Annu Rev Entomol 42: 99-122. Panizzi AR 1997 Wild hosts of pentatomids: ecological and role in their pest status on crops Annu Rev Entomol 42 99 122 Panizzi AR and Corrêa-Ferreira BS. 1997. Dynamics in the insect fauna adaptation to soybean in the tropics. Trends Entomol 1: 71-88. Panizzi AR Corrêa-Ferreira BS 1997 Dynamics in the insect fauna adaptation to soybean in the tropics Trends Entomol 1 71 88 Panizzi AR, Duo Ljs, Bortolato NM and Siqueira F. 2007. Nymph developmental time and survivorship, adult longevity, reproduction and body weight of Dichelops melacanthus (Dallas) feeding on natural and artificial diets. Rev Bras Entomol 51: 484-488. Panizzi AR Duo Ljs Bortolato NM Siqueira F 2007 Nymph developmental time and survivorship, adult longevity, reproduction and body weight of Dichelops melacanthus (Dallas) feeding on natural and artificial diets Rev Bras Entomol 51 484 488 Pons X, Lumbierres B, López C and Albajes R. 2005. Abundance of non-target pests in transgenic Bt-maize: A farm scale study. Eur J Entomol 102: 73-79. Pons X Lumbierres B López C Albajes R 2005 Abundance of non-target pests in transgenic Bt-maize: A farm scale study Eur J Entomol 102 73 79 Praslicka J and Huszár J. 2004. Influence of temperature and host plants on the development and fecundity of the spider mite Tetranychus urticae (Acarina: Tetranychidae). Plant Prot Science 40: 141-144. Praslicka J Huszár J 2004 Influence of temperature and host plants on the development and fecundity of the spider mite Tetranychus urticae (Acarina: Tetranychidae) Plant Prot Science 40 141 144 Sanders CJ, Pell JK, Poppy GM, Raybould A, Garcia-Alonso M and Schuler TH. 2007. Host-plant mediated effects of transgenic maize on the insect parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae). Biol Control 40: 362-369. Sanders CJ Pell JK Poppy GM Raybould A Garcia-Alonso M Schuler TH 2007 Host-plant mediated effects of transgenic maize on the insect parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) Biol Control 40 362 369 Santos K, Neves P, Meneguim AM, Santos RB and Santos WJ. 2009. Selection and characterization of the Bacillus thunringiensis strains toxic to Spodoptera eridania (Cramer), Spodoptera cosmioides (Walker) and Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Biol Control 50: 157-163. Santos K Neves P Meneguim AM Santos RB Santos WJ 2009 Selection and characterization of the Bacillus thunringiensis strains toxic to Spodoptera eridania (Cramer), Spodoptera cosmioides (Walker) and Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) Biol Control 50 157 163 Sas Institute. 2001. User's Guide: Statistics, Version 6e. Cary, 2001.201p. Sas Institute 2001 User's Guide: Statistics, Version 6e. Cary, 2001 201p Shapiro SS and Wilk MB. 1965. An analysis of variance test for normality. Biometrika 52: 591-611. Shapiro SS Wilk MB 1965 An analysis of variance test for normality Biometrika 52 591 611 Silva DM, Hoffmann-Campo CB, Bueno AF, Bueno Rcof, De Oliveira Mcn and Moscardi F. 2011. Biological characteristics of Anticarsia gemmatalis (Lepidoptera: Noctuidae) for three consecutive generations under different temperatures: understanding the possible impact of global warming on a soybean pest. Bull Entomol Res 102: 285-292. Silva DM Hoffmann-Campo CB Bueno AF Bueno Rcof De Oliveira Mcn Moscardi F 2011 Biological characteristics of Anticarsia gemmatalis (Lepidoptera: Noctuidae) for three consecutive generations under different temperatures: understanding the possible impact of global warming on a soybean pest Bull Entomol Res 102 285 292 Silva JJ, Ventura MU, Silva Fac and Panizzi AR. 2013. Population Dynamics of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) on Host Plants. Neotrop Entomol 42: 141-145. Silva JJ Ventura MU Silva Fac Panizzi AR 2013 Population Dynamics of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) on Host Plants Neotrop Entomol 42 141 145 Smith JF, Luttrell RG and Greene JK. 2009. Seasonal abundance, species composition, and population dynamics of stink bugs in production fields of early and late soybean in south Arkansas. J Econ Entomol 102: 229-236. Smith JF Luttrell RG Greene JK 2009 Seasonal abundance, species composition, and population dynamics of stink bugs in production fields of early and late soybean in south Arkansas J Econ Entomol 102 229 236 Sujii ER, Togn Phb, Ribeiro PA, Bernardes TA, Milane Pvgn, Paula DP, Pires Css and Fontes Emg. 2013. Field Evaluation of Bt Cotton Crop Impact on Nontarget Pests: Cotton Aphid and Boll Weevil. Neotrop Entomol 42: 102-111. Sujii ER Togn Phb Ribeiro PA Bernardes TA Milane Pvgn Paula DP Pires Css Fontes Emg 2013 Field Evaluation of Bt Cotton Crop Impact on Nontarget Pests: Cotton Aphid and Boll Weevil Neotrop Entomol 42 102 111 Torres JB and Ruberson JR. 2006. Interactions of Bt-cotton and the omnivorous big-eyed bug Geocoris puncti pes (Say), a key predator in cotton Welds. Biol Control 39: 47-57. Torres JB Ruberson JR 2006 Interactions of Bt-cotton and the omnivorous big-eyed bug Geocoris puncti pes (Say), a key predator in cotton Welds Biol Control 39 47 57 Virla EG, Casuso M and Frias EA. 2010. A preliminary study on the effects of a transgenic corn event on the non-target pest Dalbulus maidis (Hemiptera: Cicadellidae). Crop Prot 29: 635-638. Virla EG Casuso M Frias EA 2010 A preliminary study on the effects of a transgenic corn event on the non-target pest Dalbulus maidis (Hemiptera: Cicadellidae) Crop Prot 29 635 638 Wang ZY, Wu Y, He KL and Bai SX. 2007. Effects of transgenic Bt maize pollen on longevity and fecundity of Trichogramma ostriniae in laboratory conditions. Bull Insectol 60: 49-55. Wang ZY Wu Y He KL Bai SX 2007 Effects of transgenic Bt maize pollen on longevity and fecundity of Trichogramma ostriniae in laboratory conditions Bull Insectol 60 49 55 Zhang SY, Fie BY, Cui J and Li DM. 2006. Biology of Campoletis chlorideae (Uchida) (Hym., Ichneumonidae) developing in Bt-treated, Bt-resistant Helicoverpa armigera (Hübner) (Lep., Noctuidae) larvae. J Appl Entomol 130: 268-274. Zhang SY Fie BY Cui J Li DM 2006 Biology of Campoletis chlorideae (Uchida) (Hym., Ichneumonidae) developing in Bt-treated, Bt-resistant Helicoverpa armigera (Hübner) (Lep., Noctuidae) larvae J Appl Entomol 130 268 274