Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees

Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties

Dohrmann AB, Küting M, Jünemann S, Jaenicke S, Schlüter A, Tebbe CC. Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties. The ISME journal. 2012;7(1):37-49.Ribosomal 16S rRNA gene pyrosequencing was used to explore whether the genetically modified (GM) Bt-maize hybrid MON 89034 × MON 88017, expressing three insecticidal recombinant Cry proteins of Bacillus thuringiensis, would alter the rhizosphere bacterial community. Fine roots of field cultivated Bt-maize and three conventional maize varieties were analyzed together with coarse roots of the Bt-maize. A total of 547 000 sequences were obtained. Library coverage was 100% at the phylum and 99.8% at the genus rank. Although cluster analyses based on relative abundances indicated no differences at higher taxonomic ranks, genera abundances pointed to variety specific differences. Genera-based clustering depended solely on the 49 most dominant genera while the remaining 461 rare genera followed a different selection. A total of 91 genera responded significantly to the different root environments. As a benefit of pyrosequencing, 79 responsive genera were identified that might have been overlooked with conventional cloning sequencing approaches owing to their rareness. There was no indication of bacterial alterations in the rhizosphere of the Bt-maize beyond differences found between conventional varieties. B. thuringiensis-like phylotypes were present at low abundance (0.1% of Bacteria) suggesting possible occurrence of natural Cry proteins in the rhizospheres. Although some genera indicated potential phytopathogenic bacteria in the rhizosphere, their abundances were not significantly different between conventional varieties and Bt-maize. With an unprecedented sensitivity this study indicates that the rhizosphere bacterial community of a GM maize did not respond abnormally to the presence of three insecticidal proteins in the root tissue.The ISME Journal advance online publication, 12 July 2012; doi:10.1038/ismej.2012.77

Quantification of Cry1A.105 (A) and Cry3Bb1 (B) from mid- and hindgut samples of nurse bees exposed to Bt maize (treatment BT), other conventionally bred maize varieties (DKC, BEN) or other pollen sources (Phacelia, control); n indicates the numbers of replicate samples analyzed.

<p>Each individual sample is represented by a circle. Samples below the detection limit were set to zero.</p

Schematic figure on the incidence (A) and abundance (B) of bacterial phylotypes detected by T-RFLP based on 16S rRNA genes.

<p>The T-RF patterns for each treatment, i.e., exposure to Bt maize (BT), two conventional maize varieties (DKC, BEN) and mixed pollen sources including Phacelia (PHA), are based on 24 replicates from individual bees. Frequencies of incidences and abundances are indicated by squares and correlate with the grey scale. Abundance values in <b>B</b> indicate % of a particular T-RF in relation to total TRFs of the corresponding TRFP-profiles. Abundance values were averaged only from scored T-RFs. Significant differences in abundances of frequently occurring T-RFs are indicated with coloured boarder lines. Bacterial phylotypes indicated by the particular T-RFs were identified by DNA-sequencing (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059589#pone.0059589.s004" target="_blank">Table S2</a>).</p

Copy numbers of bacterial 16S rRNA genes in gut material from nurse bees as determined by qPCR.

<p>Different letters on top of columns indicate significant differences. Nurse bees originated from colonies exposed to Bt maize MON 89034 × MON 88017 (treatment BT), conventional maize varieties (DKC, BEN) or other pollen sources including Phacelia (PHA).</p

Correlation between the contents of Cry proteins for Cry1A.105 and Cry3Bb1, and maize pollen detected in the hindgut of nurse bees from colonies caged in field plots with Bt maize MON 89034 × MON 88017 during anthesis.

<p>Correlation data in the graph excluded values below the detection limit (DTC).</p

Nonmetric multidimensional scaling (NMDS) ordination plot of β-diversity patterns of bacterial community differences represented as Bray–Curtis distances of T-RFLP profiles.

<p>Stress values (0.21, 0.25) indicate that the distance between points in the ordination plot is a good representation of the degree of similarity between the bacterial communities in each sample. Each point represents the gut bacterial community obtained an individual nurse bee. Treatments: BT, exposure to pollen of Bt maize, DKC and BEN to conventional maize and PHA to other pollen sources including Phacelia.</p

Response of nurse bees after a 9 d exposure period either to Bt maize (treatment BT), or two conventional maize cultivars (DKC, BEN), or controls with <i>ad libitum</i> access to different pollen sources from colonies kept at a Phacelia field (PHA).

<p>The survival (A) was indicated by the retrieval rate of marked bees, their weight (B) was determined at the moment of their retrieval. Microscopic analysis of bee hindguts was performed to calculate a weighted average degree of maize pollen digestion (C). The error bars indicate 95% confidence intervals. *indicates significant difference of a specific treatment.</p

Hypothetical numbers of sporulated cells of <i>B. thuringiensis</i> strains expressing natural Cry proteins required for the detection of a Cry1A.105 equivalent by ELISA.

a<p>refers to an extraction volume of 300 µL; DTC, detection threshold was 0.5 mg mL^−1.</p