Testing Pollen of Single and Stacked Insect-Resistant Bt-Maize on In vitro Reared Honey Bee Larvae

Abstract

The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.

Effects of multiple Bt proteins and GNA lectin on in vitro-reared honey bee larvae

“…Recently conducted pollen feeding trials, in which in vitro-reared third-instar larvae were exposed to 2 mg pollen of the Bt maize variety "Mon89034 × Mon88017" during 5 days, showed 100% survival (Hendriksma et al, 2011b) and thus are fully in line with results from worst-case exposure scenarios obtained in the present study. Similarly, the overall mean weight of prepupae and also mean prepupal weight at the highest applied purified protein doses are in perfect range with the pollen feeding test (Hendriksma et al, 2011b).…”

“…Similarly, the overall mean weight of prepupae and also mean prepupal weight at the highest applied purified protein doses are in perfect range with the pollen feeding test (Hendriksma et al, 2011b). Our results on single Bt proteins further complement the less standardized colony level studies on single Cry1Ab or Cry1F maize pollen (Hanley et al, 2003) and the purified Cry3B protein (Arpaia, 1996), for which also no deleterious effect by Cry protein were found on honey bee Figure 2.…”

Protocol for the in vitro rearing of honey bee (Apis mellifera L.) workers

“…Hendriksma, Härtel, and Steffan-Dewenter (2011a) and Hendriksma, Härtel, Babendreier, von der Ohe, and Steffan-Dewenter (2012) demonstrated control bee survival of >90% up to the termination of the test at the prepupal stage of development. Krainer, Brodschneider, Vollmann, Crailsheim, and RiessbergerGallé (2016) were able to achieve high survival through adult emergence, but this level of success has historically been atypical.…”

Testing dose-dependent effects of stacked Bt maize pollen on in vitro-reared honey bee larvae

“…Later on in the season, (10, 11, and 25 September 2012), honey bee larvae were collected via grafting (Aupinel et al 2005(Aupinel et al , 2009). Usually, young larvae do not directly receive pollen in their food (Malone et al 2002), thus we started our experimental pollen treatments with third instar (L3) larvae (Hendriksma et al 2011b). We reared a total 1,002 larvae that were divided more or less equally over the treatment groups, Table I-S in the Online Ressource shows exactly how larvae were distributed.…”