Despite numerous advantages of using genetically modified insect-resistant plants, there are still concerns about the unknown effects associated with this technology (Kouser and Qaim, 2011; Nunes, 2010). Several studies show that the effect this technology may have on non-target organisms is still unclear (Faria et al., 2007; Liu et al., 2005; Naranjo, 2005; Silva, 2013; Whitehouse et al., 2007). Thus, the large-scale use of genetically modified crops for resistance to pests can pose a risk to biodiversity as a result of possible effects on non-target organisms (Dutra et al., 2012), which underscores the importance of conducting studies that assess such interactions.

Non-target organisms are defined as species that may be exposed to Bt proteins for a long time, but which are not direct targets of transgenic technology (Andow and Hilbeck, 2004). Among these organisms, one of the most important groups are the pollinating insects that may be both directly exposed to the toxins of these plants, through consumption of the plant, nectar, or pollen (Nunes, 2010), as indirectly through the food chain when arthropods feed on herbivores or honeydew from insects that have fed off transgenic plants (Groot and Dicke, 2002; Faria et al., 2006).

There is evidence that the expression of Bt toxins in transgenic plants can affect (directly or indirectly) the populations of non-target species (Birch, 1997; Monnerat and Bravo, 2000) by reducing the populations of pollinating insects and floral visitors, including beetles, butterflies, and a number of beneficial arthropods, which can lead to reduced biological pest control (Hong et al., 2008). In Bt maize that expresses the Cry1Ab toxin, as does the soybean used in the present study, a negative effect on the non-target species Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) and its larval parasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae) was determined (Vojtech et al., 2005). Similar negative effects were also observed on the natural enemy Pirata subpiraticus (Araneae: Lycosidae) in cultures of transgenic rice expressing the same protein Cry1Ab (Chen et al., 2009). Other studies also show that the survival of bees feeding on pollen of Bt cotton expressing this toxin was negatively affected (Liu et al., 2000). Therefore, the objective of the current study was to determine the characteristics of the population dynamics of species of floral-visiting insects in Bt and non-Bt soybean Glycine max (L.) Merrill cultivars.

Experimental area

The experiment was conducted at the experimental farm of the Federal University of Grande Dourados, Dourados-MS, Brazil (22°14′ 20.51″south, 54° 59′M 58.4″ west; altitude 394 m). The studied areas comprised two fields of 1.0 ha each. One field was sown with the cultivar DM 6563 Intact (MON 87701 × MON 89788) and the other with the cultivar BMX Potência RR (M 8360 RR). Both cultivars, which have a similar phenological cycle, were sowed on 5 November, 2014.

Survey of floral visitors

The collection of floral visitors took place throughout the flowering period (26 December, 2014 to 11 January, 2015) of the cultivars. The sampling method was adapted from that of Pires et al. (2006a). The collection of floral visitors was done to determine the abundance and diversity of these insects in the Bt and non-Bt soy, as well as visitation characteristics and species composition for each period in both cultivars.

Four evaluations were carried out every three or four days and in each assessment. Active insect collection was carried out every 15 min for 12 h in each cultivar. Insects were collected using an insect net or directly in a deadly vial. All insects found resting on or taking off from flowers were captured. After the 15 min of collection, the dead insects were placed in an envelope, stating date/time, collector, and treatment and then stored in a styrofoam box to be taken to the Entomology Laboratory of the Federal University of Grande Dourados (UFGD) for later sorting.

Identification of floral visitors

The insects were identified to the lowest taxonomic level possible using specific keys for the groups and confirmed with experts in the field.

Statistical analysis

To analyze the diversity of the two communities studied, the Shannon-Wiener function was calculated (Shannon and Weaver, 1949), as well as Simpson’ index (Pinto-Coelho, 2002). To obtain representative gradients of the floral visitors community structure, based on the species composition at each time and day of collection, the normality of the data of all individuals of each species in each day and time of collection, Kolmogorov-Smirnov, and Shapiro-Wilk tests were used. Then, comparative analysis was performed between the different schedules and treatments using the Kruskal-Wallis test.

Both cultivars flowered for approximately 15 days. During flowering, 549 species of floral-visiting insects (Class Insecta) were collected, comprising 8 orders, 30 families, and 92 species. Of this total, 279 individuals (50.81%) were present in the Bt cultivar, whereas 270 (49.18%) were collected from flowers of non-Bt cultivar (Table 1). No significant differences were noted in the abundance of insect species in relation to the cultivars, as similar numbers of individuals were observed in both cultivars. The Hymenoptera, to which the largest number of collected specimens belonged, showed the highest abundance among all of the orders collected, both in the Bt cultivar where 122 individuals were sampled (43.72% of the total found in this cultivar), as well as in the non-Bt cultivar that presented 121 individuals of this order (44.81%). Among the specimens from this order collected, representatives of the Apidae family were the most abundant, representing 40.86 and 38.14%, respectively, of all insects found in the Bt and non-Bt cultivars, totaling 9 species in both cultivars, and Apis mellifera was the most abundant.

Diptera had the highest diversity, with 15 species in the non-Bt cultivar and 17 species observed in the Bt cultivar, with Hymenoptera having 22 species in the Bt cultivar and 16 in the non-Bt cultivar. Coleoptera had 10 species in the Bt cultivar and 13 species in the non-Bt cultivar and Lepidoptera had 9 in the Bt cultivar and 11 species in the non-Bt cultivar. Hemiptera had six species in both cultivars, while Neuroptera, Mantodea, and Dermaptera had only one species each. Although, the distribution of species was very similar between the two evaluated cultivars, the results of the Shannon-Wiener and Simpson tests demonstrated that the Bt cultivar had greater diversity (H = 1.30280; D = 0.86471) than the non-Bt cultivar (H = 1.22871; D = 0.84557).

The greatest number of species was observed in the third evaluation on January 6 with a total of 34 species in the BMX Potência and 35 in the DM 6563 IPRO (Figure 1). The first (December 30) and the fourth (9 of January) evaluations had nearly equal number of species, while at the second evaluation (02 January) was noticed the lowest value of the species found during the period of sampling.

The greatest number of floral visitor species in the soybean cultivars occurred between 8:00 and 11:15 am. The least species visitation to the soybean flowers occurred between 12:00 and 15:15 pm (Figure 2). Among the 103 species collected in the two studied soybean cultivars, A. mellifera L. had the highest number of individuals (102 individuals in the cultivar Intacta and 91 in the cultivar BMX Potência), representing 36.55 and 33.70%, respectively, of the total insects found in both crop cultivars (Figure 3). The total number of individuals of A. mellifera observed during the period of the evaluated day (from 07:00 am to 18:15 pm) (Figure 4) did not indicate normality by the Kolmogorov-Smirnov and Shapiro-Wilk tests, that evaluated block (evaluation days) and treatment (evaluated times). Therefore, the nonparametric Kruskal-Wallis test (α = 5%) was performed. The variation in the abundance of A. mellifera in the different classes of times was determined and no significant differences were noted between the classes of times in both cultivars.

An important disturbance called Colony Collapse Disorder (CCD) has been the focus of many studies globally due to the disappearance of individuals that form bee colonies, as seen by the absence of dead bees in apiaries, the rapid decrease in the number of adult bees, and the lack of cleptoparasites (Cox-Foster and van Engelsdorp, 2009). One of the most important probable causes of this disorder is the effect of chemical insecticides on or near the colonies. Therefore, in efforts to protect bee species, mainly A. mellifera, from CCD, the data obtained in this study regarding the time of least visitation to the flowers, can determine the most sustainable insecticide applications, when necessary.

Based on the results of the current study, it can be concluded that the Cry1Ac toxin from Bt did not affect the population dynamics of the species of flower-visiting insects in soybean crops. Therefore, it is possible to add that this technology led to no significant differences in diversity, species composition, frequency, richness, or time of occurrence among the species of floral visitors to the culture.

The authors have not declared any conflict of interests.

The authors would like to thank FUNDECT/CAPES, chamada nº 44/2014-PAPOS-MS/Manoel A. Uchoa-Fernandes.