Introduction
Cropping of plants that express Cry proteins from Bacillus thuringiensis Berliner is increasing worldwide (James Reference James2013) not only due to their high pest control efficacy but also because of their ease of cultivation. These genetically modified plants express proteins from the cry genes targeting Lepidoptera and Coleoptera pests (Yu et al., Reference Yu, Yun and Kong2011; Head et al., Reference Head, Carrol, Clark, Galvan, Huckaba, Price, Samuel and Storer2014). Bt maize and cotton are widely cultivated worldwide, frequently in combination with herbicide tolerance (James, Reference James2013). A Bt soybean was recently developed by Monsanto by combining the transformation events MON 87701 (expressing Cry1Ac protein) and MON 89788 (glyphosate tolerance). This product was commercially released in Brazil in the 2013–14 season and will be commercialized in Argentina in the near future.
Based on the experience with Bt maize and cotton, Bt soybean technology is expected to control major Lepidoptera pests. Considering the ease of cultivation of Bt soybean in addition to the increasing caterpillar problems faced by Brazilian growers, it is conceivable that this new technology will be highly adopted. The Bt soybean MON 87701×MON 89788, efficiently targets a range of species including the velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae) and the soybean looper, Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) (Bernardi et al., Reference Bernardi, Malvestiti, Dourado, Oliveira, Martinelli, Berger, Head and Omoto2012) but it is not efficient against Spodoptera spp. (Bernardi et al., Reference Bernardi, Sorgatto, Barbosa, Domingues, Dourado, Carvalho, Martinelli, Head and Omoto2014).
Even though Bt soybean, as a new strategy in soybean integrated pest management, has shown to be efficient against important Lepidoptera pests, it is still of theoretical and practical interest to keep studying its effects on a larger diversity of insects, including pests and their biological control agents. Among the non-target organisms which are increasingly becoming economically important soybean pests, the southern armyworm Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) stands out. Spodoptera eridania feeds on both soybean leaves and pods (Santos et al., Reference Santos, Neves and Meneguim2005; Bueno et al., Reference Bueno, Carneiro, Bueno, Pratissolli, Fernandes and Vieira2010a , Reference Bueno, Carneiro, Pratissolli, Bueno and Fernandes b ). This species is already recognized as a key pest in some Brazilian soybean production areas (Bueno et al., Reference Bueno, Bueno, Moscardi, Parra and Hoffmann-Campo2011), with a high tolerance toward the Cry1Ac protein expressed in Bt soybean (Bernardi et al., Reference Bernardi, Sorgatto, Barbosa, Domingues, Dourado, Carvalho, Martinelli, Head and Omoto2014). Furthermore, S. eridania is polyphagous (Michereff-Filho et al., Reference Michereff-Filho, Torres, Andrade and Nunes2008; Delaney, Reference Delaney2012), a property that may contribute to its rapid adaptation to different agroecosystems and its modifications, illustrating the importance of this species to soybean integrated pest management.
Some studies have already reported that the adoption of Bt crops leads to a reduction in insecticide use (Sisterson et al., Reference Sisterson, Biggs, Manhardt, Carrière, Dennehy and Tabashnik2007; Hutchison et al., Reference Hutchison, Burkness, Mitchell, Moon, Leslie, Fleischer, Abrahamson, Hamilton, Steffey, Gray, Hellmich, Kaster, Hunt, Wright, Pecinovsky, Rabaey, Flood and Saun2010; Kouser & Qaim, Reference Kouser and Qaim2011; Lu et al., Reference Lu, Wu, Jiang, Guo and Desneux2012) possibly favoring certain non-target pest outbreaks (Zhao et al., Reference Zhao, Ho and Azadi2011). Therefore, it is important to understand the direct and indirect impact of Bt plants on both non-target pest species and their natural enemies. Although risks and benefits of insect management strategies with the use of Bt plants are not fully explored, risks seem to be lower than with chemical technologies and benefits seem to be greater (Wolfenbarger et al., Reference Wolfenbarger, Naranjo, Lundgren, Bitzer and Watrud2008; Naranjo, Reference Naranjo2009).
Among the biological control agents of pests belonging to the genus Spodoptera, the egg parasitoid Telenomus remus Nixon (Hymenoptera: Platygastridae) stands out as highly efficient (Bueno et al., Reference Bueno, Carneiro, Bueno, Pratissolli, Fernandes and Vieira2010a ; Pomari et al., Reference Pomari, Bueno, Bueno and Menezes2012). The high levels of parasitoid control recorded for this species may be due to its efficient parasitism in multiple layers of egg mass. Moreover, each T. remus female can parasitize as many as 270 Spodoptera eggs (Morales et al., Reference Morales, Gallardo, Vásquez and Rios2000). Because the Bt soybean MON 87701×MON 89788 does not control pests of the genus Spodoptera, the use of T. remus may be a feasible strategy to replace chemical control, and at the same time benefit from the decreased insecticide use associated with Bt crops. Therefore, it is of theoretical and practical interest to understand the impact of Bt soybean on this egg parasitoid. Our study aimed to evaluate the impact of Bt soybean on the biological characteristics of S. eridania, as well as on the development of the egg parasitoid T. remus.
Material and methods
All trials were carried out in the laboratory under controlled environmental conditions (25±2 °C, RH of 70±10%, photoperiod of 14:10 [L:D] h). The first trial (Bioassay 1) compared the performance of the caterpillar S. eridania on Bt soybean, MON 87701×MON 89788, and on its non-Bt isoline. The second trial (Bioassay 2) compared leaf consumption of S. eridania on both soybeans. The third trial (Bioassay 3) studied the comparative performance of the parasitoid T. remus on S. eridania eggs from Bt soybean, MON 87701×MON 89788, and its non-Bt near isoline.
Colonies of S. eridania and T. remus
Spodoptera eridania as well as the parasitoid T. remus were reared in the laboratory according to the methods described by Pomari et al. (Reference Pomari, Bueno, Bueno and Menezes2012) for approximately 34 generations under controlled environmental conditions (25±2 °C, RH of 70±10%, photoperiod of 14:10 [L:D] h), using eggs of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) as the parasitoid host.
Soybean plants
Both soybean cultivars used in the study, MON 87701×MON 89788, which express the Cry1Ac protein, and its non-Bt near isoline, were provided by Monsanto do Brasil Ltda. Plants were grown in 8 liters plastic pots in a greenhouse. Powdery mildew (Microsphaera diffusa Cooke & Peck) was controlled with a sulfur-based fungicide (Kumulus® 0.5 g l−1) applied weekly. No other pesticide was used.
Comparative S. eridania performance on Bt and non-Bt soybeans (Bioassay 1)
The trial was carried out in a completely randomized experimental design with two treatments (Bt and non-Bt soybeans) and five replicates, with each replicate containing 20 individualized larvae of S. eridania. Newly hatched larvae (up to 24 h old) were individualized in transparent plastic pots (150 ml). The pot lids had small holes to ensure airflow to both caterpillars and leaves. Water was provided by placing small cotton balls on the stem of each leaf to avoid excessive dehydration.
To feed the caterpillars, soybean leaves were excised daily from plants at the V7 developmental stage (Fehr & Caviness, Reference Fehr and Caviness1977) and cleaned for approximately 15 min in a 5% sodium hypochlorite solution, and then dried for 2 h before being fed to S. eridania larvae. In all cases, the second expanded leaf (from top to bottom part of the plant) was used. Several soybean seeds were sown on a daily basis to ensure the availability of leaves at the appropriate developmental stage required for this trial.
Spodoptera eridania instar, lifespan, survival of larvae, adult longevity, fecundity and egg viability were recorded daily. During the 24 h interval after the caterpillars had pupated, each individual was sexed and weighed with 0.001 g precision. To assess the parameters of the adult stage, moths less than 24 h old were paired. After mating, female moths were placed in enclosures made of plastic pipes (10 cm in diameter×21.5 cm tall), lined with white paper on their inner surface to allow oviposition. A cotton wad soaked with 10% honey solution was placed inside each enclosure to feed the moths.
Comparative S. eridania leaf-feeding on Bt and non-Bt soybeans (Bioassay 2)
The trial was carried out in a completely randomized experimental design with two treatments (Bt and non-Bt soybeans) and five replicates, each with 20 larvae of S. eridania. As in Bioassay 1, individual leaflets were collected daily from plants at the V7 developmental stage (Fehr & Caviness, Reference Fehr and Caviness1977) and washed in 5% sodium hypochlorite solution prior to being fed to lepidopteran larvae. Leaflets were kept fresh in closed plastic bags and used within 2 h of excision. Single leaflets were placed on double sheets of moist filter paper in 2.5×11 cm diameter plastic Petri dishes. Leaflets were replaced daily to avoid excessive dehydration. The insects were kept in an environmental chamber (25±2 °C temperature, RH of 70±10%, and photoperiod of 14:10 [L:D]) until pupation. The soybean foliage area (cm2) was determined using a leaf area meter (Model LI-3100, Li-Cor, Lincoln, NE) before and after larval feeding. The daily foliage consumption by each specimen was then calculated by subtraction of the final (defoliated) from the initial (offered) leaf area. A control leaflet was used for each genotype to estimate leaf dehydration and consequent reduction in leaflet size. This was performed during the entire period of evaluation, by measuring the leaf area (in cm2) of the control leaflets, and then using these changes to adjust the results for daily larvae consumption. Total consumption by individual larvae was recorded for each specimen, and average consumption obtained from each replicate was used for analysis (Bueno et al., Reference Bueno, Bueno, Moscardi, Parra and Hoffmann-Campo2011).
Comparative T. remus performance in S. eridania eggs from Bt and non-Bt soybeans (Bioassay 3)
The trial was carried out in a completely randomized experimental design with two treatments (Bt and non-Bt soybean) and five replicates. Telenomus remus females (with a maximum age of 24 h) were individualized in Duran® glass vials (1 cm×6 cm) containing a droplet of pure honey as food source. Each replicate contained five parasitoid females totaling 25 parasitoids per treatment. A single egg mass of S. eridania with approximately 150 eggs was exposed to the parasitoid (one T. remus female) for 24 h. Spodoptera eridania egg masses were not older than 24 h and were laid by females reared on either Bt or non-Bt soybean as larvae. For both treatments, the egg masses offered to parasitoids were fixed on a rectangular card of white Bristol board (0.8 cm×5 cm) using non-toxic Tenaz® glue, and placed into glass tubes containing parasitoid females for 24 h. After exposure to the parasitoids, the cards were transferred to larger glass tubes (8 cm tall×2 cm in diameter) and kept in climatic chambers at a temperature of 25±2 °C, 70±10% RH and a photoperiod of 14/10 h L/D until the emergence of T. remus adults. Telenomus remus parental females were also kept in climate chambers for daily observation and longevity recorded under the same experimental conditions. The biological parameters assessed were: longevity of parental females (days), egg-to-adult period (days), number of S. eridania parasitized eggs, parasitoid viability (%) and sex ratio.
Statistical analysis
Results of bioassays were subjected to exploratory analyses to assess the assumptions of normality of residuals (Shapiro & Wilk, Reference Shapiro and Wilk1965), homogeneity of variance of treatments and additivity of the model (Burr & Foster Reference Burr and Foster1972). Means were then compared by Student's t test (N=5) (P≤0.05) (SAS Institute, 2001).
Results
Spodoptera eridania leaf-feeding and performance on Bt and non-Bt soybeans (Bioassays 1 and 2)
Biological characteristics of S. eridania were similar when fed with Bt or non-Bt soybeans, with larval survival higher than 80% (table 1), confirming that Bt soybean MON 87701×MON 89788 consumption did not control S. eridania. Also, it did not affect foliar consumption of S. eridania, although larval development was reduced by approximately 2 days (table 1). Despite this change in larval development time, S. eridania pupae weight and duration, sex ratio, adult fecundity and longevity of female moths, as well as S. eridania egg viability did not differ between Bt and non-Bt soybeans (table 1). In contrast, Bt plants impacted the lifetime of adults. Male lifetime increased by approximately 3 days after caterpillars had fed on Bt soybeans (table 1).
Table 1. Spodoptera eridania biology on the Bt soybean, MON 87701×MON 89788, and its non-Bt isoline (25 ± 2 °C; 60 ± 10% RH; and photoperiod of 14/10 h) (N=5).
Within each row, means ± SEM followed by the same letter do not statistically differ from each other (t test, P≤0.05).
1
Statistic performed on data transformed into arcsine
$\sqrt {X/100} $
.
Comparative performance of T. remus on S. eridania eggs from Bt and non-Bt soybeans (Bioassay 3)
The average number of S. eridania eggs did not differ between Bt (155.6 eggs) and non-Bt plants (137.5 eggs) (table 2), nor did the number of S. eridania eggs parasitized by T. remus on Bt (87.3 parasitized eggs) and non-Bt (102.3 parasitized eggs) soybean treatments (table 2). Similarly, there was no difference between treatments for the other biological parameters evaluated for T. remus (table 2: parental females longevity (days), egg-adult period (days), parasitism viability (%) and sex ratio).
Table 2. Telenomus remus biology in eggs of S. eridania originating from caterpillars reared on the Bt soybean, MON 87701×MON 89788, and its non-Bt isoline under controlled climatic conditions (25±2 °C, RH 60±10% and photoperiod of 14/10 L:D) (N=5).
Within each row, means±SEM followed by the same letter do not statistically differ from each other (t test, P≤0.05).
1
Statistic performed on data transformed into arcsine
$\sqrt {X/100} $
.
Discussion
Although the pest control of the Cry1Ac protein, expressed in Bt soybean MON 87701×MON 89788, is specific to lepidopterans (CTNBio, 2010), it did not control S. eridania. The Cry1Ac protein's low level of activity against S. eridania was confirmed by the similar results of caterpillar leaf-feeding on Bt and non-Bt soybeans as well as by the observed minor influence of this protein on the pest's biological characteristics during larval and adult stages. Tolerance of caterpillars of the genus Spodoptera to Cry1Ac has been reported previously (Luttrell et al., Reference Luttrell, Wan and Knighten 1999 ; Adamczyk et al., Reference Adamczyk, Greenberg, Armstrong, Mullins, Braxton, Lassiter and Siebert2008; Santos et al., Reference Santos, Neves, Meneguim, Santos, Santos, Villas Boas, Dumas, Martins, Praça, Queiroz, Colin Berry and Monnerat2009; Greenberg et al., Reference Greenberg, Li and Liu2010; Bernardi et al., Reference Bernardi, Sorgatto, Barbosa, Domingues, Dourado, Carvalho, Martinelli, Head and Omoto2014) and may be related to inactivation of the insecticidal proteins by proteases produced by these insects (Miranda et al., Reference Miranda, Zamudio and Bravo2001; Rahman et al., Reference Rahman, Abdullah, Ambati, Taylor and Adang2012). Plant developmental stage also may play an important role in the efficacy of the Cry protein. For example, Bt soybean plants did not affect larval survival of Spodoptera exigua Hübner (Lepidoptera: Noctuidae) throughout the growing season, but larval weight was reduced when larvae fed on leaves from two Bt soybean varieties before the anthesis stage, when Cry1Ac concentrations were at a maximum (Yu et al., Reference Yu, Li, Li, Romeis and Wu2013).
The reported difference in the duration of larval development and parental male longevity of S. eridania reared on Bt and non-Bt is less than 3 days. It therefore has almost no effect on the mating synchrony of adults, which is an important aspect for refuge area management. Also, larval development was shorter on Bt than on non-Bt soybean. Similarly, male adult longevity was greater in Bt soybean. These results clearly indicate that the observed effects are due to changes in Bt plant components that might be beneficial to some insect species but not to others. It is known that the impact of transgenic plants on entomophagous insects can result not only directly from toxins but also indirectly via unintended changes in plant characteristics caused by the transgene insertion (Faria et al., Reference Faria, Wackers, Pritchard, Barrett and Turlings2007).
It can happen because the composition of a transgenic Bt plant and the corresponding non-transformed near-isoline are likely to differ to some extent due to genetic differences between them (Motavalli et al., Reference Motavalli, Kremer, Fang and Means2004). Although near-isolines show the highest genetic similarity to the Bt counterpart, they are always separated by several steps of conventional breeding (crosses, back-crosses, etc.). It occurs because the Bt trait has to be introduced into the conventional isoline after transformation. This requires several steps of selection and breeding, resulting in genetic differences in the range of those obtained by conventional breeding (Zurbrugg et al., Reference Zurbrugg, Honemann, Meissle, Romeis and Nentwig2010). As a consequence, composition assessments of transgenic soybeans compared to their near isolines showed that plant components can differ among them. MON 8701 and MON 89788 had higher levels of carbohydrates and lower levels of proteins. Even though natural variation (due to geographical factors) contributes more than biotechnology-driven genetic modifications to this variability in composition (Berman et al., Reference Berman, Harrigan, Riordan, Nemet, Hanson, Smith and Sorbet2010), changes in plant components are more likely to explain the differences in larval duration (days) or male adult longevity found in our work. Plant components such as phenolics, rutin or chlorogenic acid, are considered models for the study of plant antiherbivore defenses. Aglycone quercetin and rutin, one of its glycosides, increased the mortality and elongated the larval period of the caterpillar A. gemmatalis, although nutritional and post-ingestive effects of such flavonols needs further investigation (Hoffmann-Campo et al., Reference Hoffmann-Campo, Ramos Netro, Oliveira and Oliveira2006). Whether biotechnology-driven genetic modification may change any of these mentioned components are still unclear. Very few studies have looked specifically at the impact of other plant characteristics that may have been unintentionally altered, as a result of genetic modification (Faria et al., Reference Faria, Wackers, Pritchard, Barrett and Turlings2007). Reports suggest that aphids actually perform better on Bt maize lines than on their near isogenic counterparts (Pons et al., Reference Pons, Lumbierres, Lopez and Albajes2005), but the generality and cause of these differences remain unknown (Faria et al., Reference Faria, Wackers, Pritchard, Barrett and Turlings2007).
Susceptibility to B. thuringiensis varies greatly among different species (Schnepf et al., Reference Schnepf, Crickmor, Van Rie, Lereclus, Baum, Feitelson, Zeigler and Dean1998). Many examples of Bt plants that impair the development of non-target lepidopteran pests have been documented. For example, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and Sesamia calamistis (Sugi) (Lepidoptera: Noctuidae) are susceptible to Bt corn with the Cry1Ab protein developed to control Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) (Vojtech et al., Reference Vojtech, Meissle and Poppy2005; Van den Berg & Van Wyk, Reference Van den Berg and Van Wyk2006). Likewise, the mortality of Mamestra brassicae larvae (Linnaeus) (Lepidoptera: Noctuidae) was high when fed on the Cry1Ac protein containing Chinese Bt cabbage that is resistant to Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) (Kim et al., Reference Kim, Roh, Kang, Wang, Shim, Li, Choi and Je2008).
In addition to direct effects of Bt plants on pest performance and behavior, some indirect effects may occur. Bt crops may facilitate the reduction of insecticide use to control pest outbreaks (Sisterson et al., Reference Sisterson, Biggs, Manhardt, Carrière, Dennehy and Tabashnik2007), as reported after the adoption of Bt cotton in Arizona (Carpenter & Gianessi, Reference Carpenter and Gianessi2001; Cattaneo et al., Reference Cattaneo, Yafuso, Schmidt, Huang and Rahman2006). It is likely that insecticide use on soybeans can be reduced where MON 87701×MON 89788 is grown. Currently, S. eridania is controlled by insecticides applied against other pests such as A. gemmatalis and C. includens. A reduction in insecticides to control these caterpillars may favor S. eridania outbreaks, which would increase its pest status in soybeans. However, a reduction in insecticide sprays may preserve biological control agents naturally present in the area, thus reducing the chances for S. eridania outbreaks. Despite the importance of this matter, these hypotheses could not be studied before the commercial release of Bt soybean MON 87701×MON 89788, as large-scale field trials would be necessary.
No impact was observed with regard to the studied parasitoid T. remus. Many studies have examined the effects of Bt proteins on predators and parasitoids through tritrophic pathways using prey or hosts that are sublethally affected in some manner after exposure to Bt proteins (low quality) (Naranjo, Reference Naranjo2009). Our findings differ from those because they report the absence of direct Bt effects on a host (S. eridania) that is not susceptible to Bt proteins (high-quality host) (table 2). When studies are performed with pests susceptible to B. thuringiensis, parasitoid development might be impaired indirectly by a decrease in host quality, but may not be due to Bt itself (Romeis et al., Reference Romeis, Meissle and Bigler2006). Hansen et al. (Reference Hansen, Gabor, Lovei and Szekacs2012) reported that female parasitoid emergence of Lariophagus distinguendus Forster (Hymenoptera: Pteromalidae), an ectoparasitoid of coleopteran larvae and pre-pupae, was lower on Sitophilus zeamais (Colepotera: Curculionidae) fed with Bt corn. Other studies of susceptible hosts have reported similar negative impacts of Bt plants on parasitoids (Liu et al., Reference Liu, Sun and Zhang2005a , Reference Liu, Zhang, Zhao, Cai, Xu and Li b ; Zhang et al., Reference Zhang, Fie, Cui and Li2006; Sanders et al., Reference Sanders, Pell, Poppy, Raybould, Garcia-Alonso and Schuler2007). When non-susceptible hosts are used, negative impacts on the parasitoid are not reported. This confirms that there is currently no indication that Cry1Ac directly affects parasitoids or other Hymenopterans (Dhillon & Sharma, Reference Dhillon and Sharma2013). It is important to emphasize that based on the mode of action of Bt protein (gut-active), it is unlikely that the Cry1Ac, found on Bt soybean MON 87701×MON 89788, would be transferred from S. eridania larvae to adults to eggs. Therefore, T. remus exposure to Cry1Ac would be zero or very low (close to zero) in this study and direct effects are excluded. Any possible effect would be indirectly associated to the Bt protein insertion.
We conclude that the effect of Bt soybean MON 87701×MON 89788 on S. eridania development and reproduction is small, and favorable to pest development. These differences are less likely to directly result from the toxin presence but indirectly from unintended changes in plant characteristics caused by the insertion of the transgene or the breeding steps following transformation. Our results should be viewed as an alert that S. eridania populations may increase in Bt soybeans, but on the other hand, no adverse effects of this technology were observed for the egg parasitoid T. remus which can help to prevent S. eridania outbreaks on these crops.
Acknowledgements
The authors wish to thank Embrapa Soybean, the sponsor agencies CAPES, CNPq and FAPESP for the financial support; and Monsanto Brazil Ltda for providing the seeds for accomplishing this study. This paper was approved for publication by the Editorial Board of Embrapa Soja as manuscript number 23/2013.
