Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum

Abstract

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

Figure 1. [¹²⁵I]-α-Bungarotoxin specific binding on pea aphid.

Saturation curves (A) and Scatchard plots (B) for [¹²⁵I]-α-Bungarotoxin (α-Bgt) specific binding. Membranes were extracted from whole parthenogenetic adults of pea aphid Acyrthosiphon pisum LSR1. Results are means of four experiments. Error bars represent the SEM.

Figure 2. [³H]-imidacloprid specific binding on pea aphid.

Saturation curves (A) and Scatchard plots (B) for [³H]-imidacloprid specific binding. Membranes were extracted from whole parthenogenetic adults of pea aphid Acyrthosiphon pisum LSR1. Results are means of four experiments. Error bars represent the SEM.

Figure 3. Neonicotinoids inhibition of [¹²⁵I]-α-Bungarotoxin specific binding.

Inhibition curves were determined on membranes of whole parthenogenetic adults of pea aphid Acyrthosiphon pisum for three neonicotinoids: A) imidacloprid (IMI), B) thiamethoxam (TMX) and C) clothianidin (CLT). Results are means of four experiments. Error bars represent the SEM.

Figure 4. Neonicotinoid inhibition of [³H]-imidacloprid specific binding.

Inhibition curves were determined on membranes of whole parthenogenetic adults of pea aphid Acyrthosiphon pisum for (A) α-Bungarotoxin (α-Bgt) and three neonicotinoids: (B) imidacloprid (IMI), (C) thiamethoxam (TMX) and (D) clothianidin (CLT). Results are means of four experiments. Error bars represent the SEM.

Figure 5. MRM chromatograms.

Chromatograms of thiamethoxam (A) and its metabolite clothianidin (B) in 13,400 pea aphid larvae exposed to thiamethoxam at LC₅₀ for 48 h. Intensity represents the peak area of the detected signal.

Figure 6. Expression level of nAChR mRNA subunits according to developmental stages of the pea aphid.

Quantitative experiments were performed on whole individuals in triplicate. Results are mean of three independent experiments. Relative expression ratio were calculated relative to first-instar nymphs and normalized with external reference gene luciferase. Statistical analysis (One-Way ANOVA) was carried out using GraphPad Prism 5 software. For each subunit, expression ratio statistically different according to larval stage are designated by different letters.

Figure 7. Expression levels of nAChR mRNA subunits after neonicotinoid exposure.

Experiments were assessed on whole survivingl larvae exposed to neonicotinoids at LC₅₀ for 48h. Aphids were intoxicated with imidacloprid (A) thiamethoxam (B) or clothianidin (C). Each qPCR experiment was performed in triplicate and results are represented as the mean of four to seven independent experiments after normalization with actin and ribosomal rpl7 gene. Error bars represent the SEM. Results are expressed in % of the expression level in control conditions (no insecticide, corresponding to 100%). Statistical analysis (t-test, α = 0.05) was carried out using Graphpad Prism 5 software.