The Effects of Pollen Consumption of Transgenic Bt Maize on the Common Swallowtail, Papilio Machaon L
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ARTICLE IN PRESS Basic and Applied Ecology 7 (2006) 296—306 www.elsevier.de/baae Bt maize effects on Papilio machaon The effects of pollen consumption of transgenic Bt maize on the common swallowtail, Papilio machaon L. (Lepidoptera, Papilionidae) Andreas Langa,Ã, Eva Vojtecha,b aBavarian State Research Centre for Agriculture, Institute of Plant Protection, Lange Point 10, D-85354 Freising, Germany bInstitute of Environmental Sciences, University Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland Received 10 March 2005; accepted 31 October 2005 KEYWORDS Summary Zea mays; Effects of exposure to maize pollen of event Bt176 (cultivar ‘‘Navares’’) on the Bacillus thuringien- larvae of the European common swallowtail (Papilio machaon L.) were studied in the sis; laboratory. First instar larvae were exposed to different pollen densities applied to Cry1Ab toxin; leaf disks of Pastinaca sativa L. for 48 h. Pollen densities applied in this study were in Transgenic maize; the range recorded from the field. Larvae which were exposed to higher Bt maize Bt176 event; pollen densities consumed more pollen and had a lower survival rate. The LD with Non-target insect; 50 regard to larvae surviving to adulthood was 13.72 pollen grains consumed by first- Butterflies; instar larva. Uptake of Bt maize pollen led to a reduced plant consumption, to a Side effects; lower body weight, and to a longer development time of larvae. Effects on pupal Risk assessment weight and duration of the pupal period were present but less pronounced and smaller than effects on larvae. Larvae having consumed Bt-maize pollen as first instars had a lower body weight as adult females and smaller forewings as adult males. We conclude that possible effects of Bt maize on European butterflies and moths must be evaluated more rigorously before Bt maize should be cultivated over large areas. & 2005 Gesellschaft fu¨r O¨kologie. Published by Elsevier GmbH. All rights reserved. Zusammenfassung L1-Larven des Schwalbenschwanzes (Papilio machaon L.) wurden im Labor mit Pollen von Bt-Mais (Event 176, Sorte ‘‘Navares’’) gefu¨ttert. Die Larven waren 48 h lang verschiedenen Pollenkonzentrationen auf Futterpflanzenpla¨ttchen (Pastinak) ausge- setzt. Die im Laborversuch applizierten Pollendichten entsprachen bekannten Freilandwerten. Je ho¨her die Pollendichte war desto mehr Pollen fraßen die Raupen, ÃCorresponding author. University of Basel, Institute of Environmental Geosciences, Bernoullistr. 30, CH-4056 Basel, Switzerland. Tel.: +41 61 2670477; fax: +41 61 2670479. E-mail address: [email protected] (A. Lang). 1439-1791/$ - see front matter & 2005 Gesellschaft fu¨r O¨kologie. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.baae.2005.10.003 ARTICLE IN PRESS Bt maize effects on Papilio machaon 297 und eine ho¨here Konsumption von Bt-Pollen fu¨hrte zu geringeren U¨berlebensraten. Die LD50 bezogen auf geschlu¨pfte Schmetterlinge betrug 13.72 von L1-Larven gefressene Pollenko¨rner. Fraß von Bt-Maispollen reduzierte die Fraßleistung und das Ko¨rpergewicht der Larven und verla¨ngerte ihre Entwicklungszeit. Effekte auf Puppengewicht und Verpuppungsdauer waren weniger stark ausgepra¨gt. Die Konsumption von Bt-Maispollen durch Larven fu¨hrte zu einem geringeren Ko¨rperge- wicht der adulten Weibchen und zu kleineren Vorderflu¨geln bei adulten Ma¨nnchen. Es wird eine ausfu¨hrlichere Risikobewertung vor einem fla¨chendeckenden Anbau von Bt-Mais in Europa empfohlen. & 2005 Gesellschaft fu¨r O¨kologie. Published by Elsevier GmbH. All rights reserved. Introduction Langenbruch, 2001, 2003; Felke, Lorenz, & Lan- genbruch, 2002; Hansen Jesse, & Obrycki, 2000; Transgenic Bt maize varieties are used worldwide Hellmich et al., 2001; Losey et al., 1999; Stanley- with an estimated area cropped of 9.1 million Horn et al., 2001; Wraight, Zangerl, Carroll, & hectares (James, 2003). The Bt maize commercially Berenbaum, 2000; Zangerl et al., 2001). There is available in Europe has been engineered with genes clearly an urgent need for testing further non- of the soil bacterium Bacillus thuringiensis target butterflies as the effect of Bt can vary widely Berliner (Bt) var. kurstaki, and expresses the among different species (Felke et al., 2002). This is insecticidal delta-endotoxin Cry1Ab specific against especially important in Europe where one of the lepidopteran species like the European corn borer, two Bt maize events (i.e. Bt176 and MON810) Ostrinia nubilalis Hu¨bner (Koziel et al., 1993). registered by the European Community is the event After oral uptake by a susceptible species, the 176, which produces high toxin amounts in pollen microbial toxin binds to specific receptors in the (Lang, Ludy, & Vojtech, 2004; Sears et al., 2001), mid-gut epithelium of the larvae causing cell lysis and which is already cultivated in the field, e.g. on leading to the death of the insect (Whalon & 32,000 ha in Spain in 2003 (Lumbierres, Albajes, & Wingerd, 2003). Due to their selective impact on Pons, 2004). target organisms living in maize fields, Cry1Ab In assessing potential Bt effects on Lepidoptera it toxins of Bt maize are generally considered to be is essential to consider components other than the safe for most non-target organisms. However, the immediate larval mortality. This has been ac- Bt toxin is not species-specific and may harm other counted for by recording consequences of Bt pollen species closely related to the target species, e.g. consumption on several sublethal parameters such other moths and butterflies in the case of Bt var. as body weight, consumption rate or development kurstaki (Glare & O’Callaghan, 2000). Bt toxins are time of the larvae. In order to assess possible produced in most tissues of the Bt maize, and impacts at the population level, however, informa- pollen with toxin may be transported by wind into tion about components of relative fitness is adjacent areas, deposited on plants, and consumed significant, thus survival experiments should last by larvae of non-target species feeding on these at least one generation and include parameters of plants. Initiated by the publication of Losey, Rayor, the adult stages (Andow & Hilbeck, 2004). and Carter (1999), a series of studies were The European common swallowtail Papilio ma- conducted and published in the USA to assess the chaon Linne´ is a regular butterfly species occurring potential side effects of Bt maize on non-target in agricultural land in central Europe, and fre- lepidopteran larvae which focused on the Monarch quently reproduces on host plants occurring on field butterfly, Danaus plexippus Linne´ (results summar- margins (Ebert & Rennwald, 1991; Lang, 2004). ized in Sears et al., 2001). Although a growing Here, we report about laboratory experiments that number of papers have been published recently, quantify the effect of Bt maize pollen consumption the available assessments of Bt maize pollen on larvae of the common swallowtail. Larvae of P. effects on butterflies (Papilionoidea and Hesper- machaon were fed with differing amounts of Bt176 ioidea) is based on only half a dozen species maize pollen and the resulting effects analyzed worldwide, in Europe only one non-target and with respect to the survival of the larvae, deter- three secondary pest species have been studied so mining lethal doses and concentrations (LD and LC far, and pupal and adult stages have mostly not values), feeding and growth inhibition as well as been considered (Anderson, Hellmich, Sears, Su- development time of the larvae, pupal weight, and merford, & Lewis, 2004; Dively et al., 2004; Felke & adult weight and size. ARTICLE IN PRESS 298 A. Lang, E. Vojtech Material and methods surfaces counted under a stereo-microscope (50 Â ). Additionally, two treatments were carried Insects out with conventional non-Bt maize pollen in order to check whether maize pollen itself affects larvae Eggs of P. machaon were obtained either by our (see below). own breeding culture or from commercial butterfly A single larva was placed on a leaf disk into the breeders (all larvae stemmed from adults caught in wells of the plates. Filled plates were covered with Bavaria or in Switzerland). Eggs were held in the glass and placed inside a plastic box with standing laboratory under standardized conditions of 25 1C, water to maintain humidity close to 100% in order 50–60% relative humidity and at a day–night cycle to prevent leaf disk dehydration. The plates were of 16:8 h. For the bioassays only first instar larvae controlled after 24 h and if a disk was completely were used within 15–28 h after hatching. After eaten, it was replaced with a new untreated disk. hatching, larvae were first kept on leaves of wild After 24 and 48 h leaf consumption of larvae was carrot (Daucus carota L.), and then starved 4–5h recorded. The transparent plates were placed onto prior to the bioassay in order to standardize their a 1-mm scale paper, and the number of empty hunger level. squares counted, i.e. the squares that were no longer covered by leaf disk. This number was transformed into the proportion eaten of leaf Bioassays surfaces (sum of day 1 and day 2 yielding nine categories: 0%, À12.5%, À25%, À37.5%, À50%, All experiments were carried out in the labora- À62.5%, À75%, À87.5%, À100%). Then, the amount tory under the conditions described above, except of consumed leaf area (mm2) was calculated from for relative humidity which was close to 100% the the proportion of leaf surface eaten. After 48 h the first 48 h (see below). Bioassays involved exposure pollen left on leaf disks and in the wells was of swallowtail larvae to Bt maize pollen for 48 h, counted (larvae were checked for adhering pollen and were performed with 96-well plastic plates grains carefully), and the larvae of each treatment (125 Ã 82 mm, diameter of well ¼ 7 mm). Leaf disks were transferred to an insect cage (length * width * of wild parsnip (Pastinaca sativa L.) with differing height: 30 Ã 30 Ã 60 cm).