Use of an innovative T-tube maze assay and the proboscis extension response assay to assess sublethal effects of GM products and pesticides on learning capacity of the honey bee Apis mellifera L

Abstract

Transgenic Cry1Ac+CpTI cotton (CCRI41) is a promising cotton cultivar throughout China but side effects and especially sublethal effects of this transgenic cultivar on beneficial insects remain poorly studied. More specifically potential sublethal effects on behavioural traits of the honey bee Apis mellifera L. have not been formally assessed despite the importance of honey bees for pollination. The goal of our study was to assess potential effects of CCRI41 cotton pollen on visual and olfactory learning by honey bees. After a 7-day oral chronic exposure to honey mixed with either CCRI41 pollen, imidacloprid-treated conventional pollen (used as positive sublethal control) or conventional pollen (control), learning performance was evaluated by the classical proboscis extension reflex (PER) procedure as well as a T-tube maze test. The latter assay was designed as a new device to assess potential side effects of pesticides on visual associative learning of honey bees. These two procedures were complementary because the former focused on olfactory learning while the latter was involved in visual learning based on visual orientation ability. Oral exposure to CCRI41 pollen did not affect learning capacities of honey bees in both the T-tube maze and PER tests. However, exposure to imidacloprid resulted in reduced visual learning capacities in T-tube maze evaluation and decreased olfactory learning performances measured with PER. The implications of these results are discussed in terms of risks of transgenic CCRI41 cotton crops for honey bees.

Fig. 1

a The single T-tube used for the conditioning sessions (C1, C2, C3): cotton swab with 20% sucrose was used as a reward (unconditioned stimulus) at the end of the blue coloured arm (conditioned stimulus). These two stimuli were used together to establish CS/US association in honey bees (i.e. associate learning for blue colour). b The assembled T-tube maze used in the evaluation session (E): this device was used to test whether the bees could successfully negotiate to the last blue section through the maze after the conditioning sessions

Fig. 2

Percentage of individuals (n = 80 bees tested per group) (a sub-sample of surviving active bees selected for this test, n = 20, 4 replicates) succeeding in navigating the single T-tube and assembled T-tube maze to obtain the reward at the end of the blue arm after a 7-day period of oral exposure to food containing conventional pollen (control), Cry1Ac+CpTI pollen, or imidacloprid-contaminated (48 ppb) conventional pollen. Dashed line indicates innate response of bees to the blue colour. Session C2, C3 and E refer to the second and third conditioning sessions and the evaluation session respectively. A permuted Fisher exact test was used to compare treatments, * P < 0.05, ** P < 0.01, NS not significant (when no treatments differed from the control for a given trial, NS is reported only once)

Fig. 3

Percentage of honey bees (n = 80 bees tested per group) showing a PER (a sub-sample of surviving active bees selected for showing a PER before the conditioning, n = 20, 4 replicates) during conditioning trails (C1–C3) and extinction trails (E1–E5 after a 7-day period of oral exposure to food containing conventional pollen (control), Cry1Ac+CpTI pollen, or imidacloprid-contaminated (48 ppb) conventional pollen. A permuted Fisher exact test was used to compare treatments, * P < 0.05, ** P < 0.01 indicated a significant difference with the control, NS not significant (when no treatments differed from the control for a given trial, NS is reported only once)