Lack of Lethal and Sublethal Effects of Cry1ac Bt-Toxin on Larvae of the Stingless Bee Trigona Spinipes

Lack of lethal and sublethal effects of Cry1Ac Bt-toxin on larvae of the stingless bee Trigona spinipes Maria Augusta P. Lima, Carmen Silvia S. Pires, Raul Narciso C. Guedes & Lucio Antonio O. Campos Apidologie Official journal of the Institut National de la Recherche Agronomique (INRA) and Deutschen Imkerbundes e.V. (D.I.B.) ISSN 0044-8435 Volume 44 Number 1 Apidologie (2012) 44:21-28 DOI 10.1007/s13592-012-0151-z 1 23 Your article is protected by copyright and all rights are held exclusively by INRA, DIB and Springer-Verlag, France. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Apidologie (2013) 44:21–28 Original article * INRA, DIB and Springer-Verlag, France, 2012 DOI: 10.1007/s13592-012-0151-z Lack of lethal and sublethal effects of Cry1Ac Bt-toxin on larvae of the stingless bee Trigona spinipes 1 2 3 Maria Augusta P. LIMA , Carmen Silvia S. PIRES , Raul Narciso C. GUEDES , 4 Lucio Antonio O. CAMPOS 1Departamento de Biologia Animal, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil 2EMBRAPA Recursos Genéticos e Biotecnologia, Pq Estação Biológica, Asa Norte, Cx. Postal 02372, Brasília, DF 70849-970, Brazil 3Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil 4Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil Received 20 January 2012 – Revised 18 May 2012 – Accepted 4 June 2012 Abstract – Stingless bees, particularly Trigona spinipes, are important pollinators in tropical ecosystems and are potentially affected by environmental contaminants. In this study, we tested the possible negative effects on T. spinipes larvae of the ingestion of a diet contaminated with Cry1Ac Bt-toxin. This toxin is expressed in genetically modified cotton plants. A method of rearing stingless bee larvae is described in this paper. The larvae were provided with either pure larval diet, diluted larval diet, or larval diet diluted in a Cry1Ac solution compatible with the lethal pest-exposure level (50 μg/mL). Cry1Ac ingestion did not impair the development of worker larvae, but the diluted diet slightly increased larval mortality. These results indicate that harmful effects on stingless bee larvae due to the ingestion of pollen-expressed Cry1Ac toxin are unlikely under field conditions. Bacillus thuringiensis toxin / transgenic plants / environmental impact / native pollinator / risk assessment 1. INTRODUCTION face an increased risk of exposure to the toxin (Han et al. 2010). The cultivation of genetically modified (GM) Cotton flowers are frequently visited by crops is expanding rapidly worldwide, particu- several bee species to harvest pollen and nectar larly in Brazil. The increase in Brazil’s cultivat- (Arpaia et al. 2006). Foraging for these resour- ed area over the past few years is the highest in ces on Bt cotton may potentially affect these the world (James 2011). Transgenic cotton species, which are important because of their expressing Bacillus thuringiensis toxins (Bt pollination service for most terrestrial ecosys- cotton) was first planted in 1996 and is tems, including agroecosystems (Malone et al. currently the third largest crop cultivated world- 2002; Klein et al. 2007). Therefore, the envi- wide (James 2011). Due to the high levels of ronmental risk analysis of Bt cotton should expression of the Cry1Ac toxin in the pollen of include an assessment of the potentially harmful Bt cotton, the pollinators foraging on this crop effects of ingested pollen and nectar containing the expressed Bt protein Cry1Ac. This assessment should focus on a worst-case scenario, in Corresponding author: M.A.P. Lima, which the species facing a high risk of exposure [email protected] should be subjected to the highest doses that are Manuscript editor: Monique Gauthier possible in practice (Wilkinson 2004). Author's personal copy 22 M.A.P. Lima et al. The honey bee Apis mellifera Linnaeus is 2004). Because foraging stingless bees may the most frequently used pollinator in risk collect pollen from Bt plants and feed this assessment studies of Bt toxins due to its pollen to the larvae, these larvae can poten- economic value as a pollinator. There are few tially suffer lethal and sublethal effects from studies assessing the toxicological effect of Bt such exposure. The present study was con- toxins on bee larvae, even in the honey bee ducted to test this hypothesis. The objectives (Hanley et al. 2003; Babendreier et al. 2004; of the study were to develop a suitable Lerhman 2007;Limaetal.2011). Neverthe- methodology to assess the effects of Bt toxin less, the extrapolation of the results of such from GM plants on the worker larvae of studies to other bee species is questionable due stingless bees and to assess the risk of ingested to the nutritional, behavioral, and physiological Cry1Ac Bt-toxin on larval development. The differences among bee species. stingless bee Trigona spinipes Fabricius was Stingless bees (Hymenoptera: Apidae: used as a model because of its wide distribu- Meliponini), a group of eusocial bees, are tion and frequent occurrence on cotton flowers important pollinators in tropical and agricul- in Brazil. These characteristics make it a tural ecosystems (Slaa et al. 2006;Dick potential target for the expressed toxin (Arpaia 2001) and they are similar to most of the et al. 2006). In addition, caste determination in solitary bees in that they store large amounts this species is dependent on the amount of of pollen in their nests (Wcislo and Cane food ingested by the females (Buschini and 1996). The pollen is one of the primary Campos 1995). The colonies are usually large, components of the larval diet of meliponines showing a high level of recruitment of nest- (Velthuis et al. 2003)andistheprimary mates for foraging and efficient exclusion of protein source for stingless (and solitary) bee competing pollinator species (Almeida and larvae. The nutrition furnished by high-protein Laroca 1988;Niehetal.2004a,b, 2005; pollen produces larger adult workers, which Lichtenberg et al. 2011). These traits further are generally considered more efficient foragers increase the risk of exposure of the larvae of and pollinators (Klostermeyer et al. 1973;Pyke this species to the Bt toxins expressed in the 1978;Quezada-Euánetal.2011). pollen and nectar of GM cotton plants, and Unlike honey bee larvae, which are fed on this increased risk serves to justify the present glandular secretions produced by the nurse study. bees (Babendreier et al. 2004), stingless bee larvae ingest large amounts of pollen. There- 2. MATERIALS AND METHODS fore, the risk of exposure of stingless (and solitary) bee larvae to Cry toxins is higher than 2.1. Insects that of honey bee larvae and should be assessed. Unfortunately, despite the ecological Five colonies of the stingless bee species T. spinipes and economic importance of stingless bees in were collected in the field in Viçosa County (MG, tropical areas, there are no studies in stingless Brazil; 20°45′ S and 42°52′ W) from January through bees assessing the toxicity of GM plants June 2006 and maintained under field conditions at expressing Bt toxins. the experimental apiary of the Federal University Toxicological assessments of Bt proteins of Viçosa (Viçosa, MG, Brazil). These colonies expressed in GM plants in stingless bees are were used to collect larval diet and eggs for the needed, with a primary focus on the larval toxicological bioassays. stage. The development of suitable research The bioactivity of the Cry1Ac Bt-toxin was protocols is necessary to perform such verified following Lima et al. (2011)usingthe assessments. Tests with purified toxins are velvetbean caterpillar Anticarsia gemmatalis an initial step required for the risk assess- (Hübner) (Lepidoptera: Noctuidae), which is sus- ment of GM plants in bees (Malone et al. ceptible to this toxin. The caterpillars were reared Author's personal copy Does Cry1Ac Bt-toxin harm stingless bee larvae? 23 on a solid artificial diet based on beans and wheat with 36 μL of diet to allow the worker larva to germ under controlled conditions of 25±2 °C complete its development. The polyethylene micro- temperature, 70±5 % RH and 12 h photoperiod. plates containing the artificial brood chambers were maintained at 34±1 °C, 98±2 % RH and 24 h 2.2. Insecticidal bioactivity of Cry1Ac scotophase until the end of the feeding period. The artificial brood chambers were subsequently re- The toxin Cry1Ac used was obtained from the moved and transferred to conditions of 34±1 °C, Biochemistry Department of Case Western Reserve 70±10%RH,and24hdarktosimulatenatural University (Cleveland, OH, USA). The purified conditions (Sakagami 1981). All materials used to toxin was stored in lyophilized form at −15 °C collect the larvae, to store and collect the larval diet, and eventually dissolved in double-distilled (sterile) and to set up the bioassays were autoclaved or water to be mixed with the insect diet for the sterilized in germicidal UV light in a biosafety bioassays. The activity of the toxin was tested on chamber. second-instar velvetbean caterpillars. These caterpillars were continuously exposed to the Bt toxin- 2.4. Determination of pollen ingestion contaminated diet for 7 days at 34±1 °C and 98± by stingless bee larvae 2 % RH, environmental conditions typical for rearing the larvae of T. spinipes. This environment The values of the amount of pollen ingested simulated the conditions under which stingless bee per larva and the amount of toxin present in the larvae would be exposed to the toxins.

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