Video tracking of bees using a vertical arena.

<p><b>A,</b> Examples of paths followed during 3 minutes by four individual young bees (day 1 after emergence). <b>B,</b> Superimposed paths followed by eighty individual bees. Overall, arena sides were more frequently visited. <b>C,</b> Locomotor ability measured at day 1, 2 and 6 after emergence (bees kept in an incubator). Mean distance (± S.E.M) covered by bees slightly increased from 3.2 to 3.8 meters between day 1 and 2 (p<0.01, n = 138 and 63 respectively) and did not significantly further increase as shown at 6 days after emergence (n = 38).</p

Evidence for locomotor deficits after exposure to a sublethal dose (SLD_48h) of a pyrethroid or a neonicotinoid but not a phenylpyrazole.

<p><b>A</b>, The average (± S.E.M) relative distance covered by young bees is significantly decreased 6±2h after exposure to either a SLD_48h of cypermethrin (2.5 ng/bee), tau-fluvalinate (33 ng/bee) or tetramethrin (70 ng/bee). <b>B</b>, A significant decrease in distance is observed after exposure to a SLD_48h of thiamethoxam (3.8 ng/bee) as well. <b>C</b>, The relative distance covered by bees after exposure to a SLD_48h of fipronil (0.5 ng/bee) is similar to the distance covered by control bees. In the case of fipronil, whereas early deleterious effects cannot be evidenced by the locomotion assay, an increased mortality is observed five days after exposure. For cypermethrin, n = 19 control and n = 20 exposed bees respectively. For tau-fluvalinate, n = 12 control and n = 19 exposed bees respectively. For tetramethrin, n = 20 control and n = 20 exposed bees respectively. For thiamethoxam, n = 19 control and n = 19 exposed bees respectively. For fipronil, n = 19 control and n = 20 exposed bees respectively.</p

Dose-response curve of permethrin-modified sodium channels in ALNs.

<p>Percentage of permethrin-modified channels, calculated according to Eqn 3, from the permethrin-induced tail current amplitude measured 3ms after a single pulse (of 3 ms in duration, see inset). The percentage of modified channels increases as a function of permethrin concentration (filled circle, n = 7, 10, 25 and 9 at 0.1, 1, 10 and 50 µM respectively). The same curve but recorded in ORNs (from Kadala et al. 2011) is shown for comparison (empty circles, n = 3, 9, 14, 5).</p

Use-dependent sommation of modified channels and tail current inactivation kinetics in ALNs.

<p>Mean time course of pyrethroid-modified channels along with protocol shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#pone-0112194-g002" target="_blank">Figure 2</a> (n = 7, 10 and 10 ALNs for cypermethrin, permethrin and tetramethrin, respectively). The percentage of modified channels is calculated from tail currents amplitude according to Eqn 3. Whereas cypermethrin and permethrin induce an increase in % of modified channels, tetramethrin shows the opposite effect. B2. Kinectics of tail currents estimated by the R600 value, i.e. the percentage of residual tail current 600 ms after the end of the tenth pulse of the 10-pulse protocol. Tetramethrin induces faster decaying tail currents than cypermethrin or permethrin.</p

Pyrethroids decrease deactivation rate constants.

<p>A. State model used to fit the experimental traces (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#s2" target="_blank">Methods</a> and text for details). Pyrethroids can only bind to open channel. Once bound they modify channel kinetics to and from open and inactivated states by given factors, i.e. KFwr, Kbck, KiF, KIB, Kin and Kout by p, q, r, s, t, and u respectively to give KFwrb, Kbckb, KiFb, KIBb, Kinb and Koutb. Voltage dependence is not changed. B-C-D. Fitting of experimental traces obtained under differents pyrethroids (cypermethrin, permethrin, tetramethrin) using the above model allows to determine the changes in the different kinetic parameters. Scale bar 200 pA (B and C) or 500 pA (D) and 150 ms. E. Radargraph of the changes (logarythmic scale) in the different kinetic parameters produced by pyrethroids. Fitting for KpyrF KpyrB, the binding and unbinding rate constants at a single concentration (10 µM), are used to calculate Kd.</p

Effect of pyrethroids on the use-dependent decrease in the sodium current peak.

<p>Mean evolution of the relative amplitude of the peak current along with protocol shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#pone-0112194-g002" target="_blank">Figure 2</a> (current normalized to the amplitude obtained at the first pulse of the train) from ALNs in a control solution (empty triangles) and after exposure to pyrethroids (filled circles). All pyrethroids tested amplify the decrease in current amplitude and tetramethrin (n = 10) has a stronger effect than cypermethrin (n = 6) or permethrin (n = 10).</p

Use-dependent effects of type II (cypermethrin) and type I (permethrin and tetramethrin) pyrethroids in ALNs.

<p>Sodium current recordings in response to a 10-pulse train (3 ms, from −80 mV to −10 mV, 13 Hz) in three different ALNs in the presence of 10 µM cypermethrin (A), 10 µM permethrin (B) or 10 µM tetramethrin (C).</p

Properties of the voltage-gated sodium current from honeybee ALNs.

<p>A, Sodium current traces obtained from an ALN for voltage steps to the indicated potential from a holding potential of −80 mV. B, Mean relative I/V curve (n = 24). C, Mean steady-state inactivation curve (n = 12). D, Recovery from fast inactivation in ALN (n = 12). E, Use-dependent decrease in the amplitude of the sodium current observed in an ALNs submitted to a 10-pulses train at 13 Hz. I, Average decrease in the amplitude of the sodium current in ALNs submitted to a 10-pulse train at 13 Hz (filled triangles, n = 36) or 35 Hz (filled circles n = 7).</p