First published online November 17, 2005
Journal of Experimental Biology 208, 4451-4466 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01917
The insecticide pymetrozine selectively affects chordotonal mechanoreceptors
Jessica Ausborn1,
Harald Wolf1,*,
Wolfgang Mader1 and
Hartmut Kayser2
1 University of Ulm, Neurobiology Department, D 89069 Ulm,
Germany
2 Syngenta Crop Protection AG, Research and Technology, WRO-1004.4.46, CH
4002 Basel, Switzerland
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Fig. 1. Chemical structures of pymetrozine and its inactive phenyl analogue.
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Fig. 2. Locust, Locusta migratoria, before (A) and after (B,C) pymetrozine
application (0.5 µg g–1 body mass). The animal in A
assumes a typical resting posture, holding the body elevated above the
substrate with all the legs, sometimes except the hind legs, touching the
ground. (B,C) Note extended femur–tibia joints, lifted legs (in the
coxal joints) and lifted tarsi after pymetrozine application. Leg and body
posture appear uncoordinated and the body is not held above the substrate but
rather rests on the floor. In C, the conspicuous levitation of both hind legs
is evident. Body length of insects is 60 mm.
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Fig. 3. Feedback loop controlling femur–tibia joint position in insects. (A)
Anatomical situation, semi-schematic diagram of a locust middle leg. The means
of experimental access to elements of the joint control loop are indicated: a
clamp attached to a transducer (TD) allows stimulation of the femoral
chordotonal organ (fCO) via its apodeme (tendon); a suction electrode
(SE) records activity of fCO sensillae; a hook electrode (HE) monitors
discharges of motoneurons supplying the extensor (or flexor) tibiae muscle;
and movement of the tibia is monitored optically. (B) Cybernetic diagram of
the feedback loop for joint control. Anatomical correlates of the elements of
the control circuit are indicated (cybernetic terms in brackets). Numbers in A
and B indicate corresponding structures (e.g. 1, sensor, fCO). (C) Resistance
reflex generated in the quiescent animal. A ramp-and-hold stimulus delivered
to the fCO (bottom trace; arrow indicates imposed fCO elongation, mimicking
tibia flexion) elicits a spike discharge in the nerve supplying the extensor
muscle (top trace; spikes of the three innervating motoneurons are marked:
FETi, fast; SETi, slow extensor motoneuron; CI, common inhibitor). Note the
pronounced response to movement and the smaller maintained discharge due to
altered fCO position. Thresholds and bins for evaluation are indicated; for
details, see text.
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Fig. 4. Effect of pymetrozine on the resistance reflex response in the extensor
tibiae motor nerve. (A,B) near-threshold concentration. (A) Sample recording
from the middle leg's extensor nerve (i) before and (ii) after application of
10–8 mol l–1 pymetrozine, during
ramp-and-hold stimulation of the fCO (bottom trace; stimulus amplitude 240
µm, or  40°). (B) Peristimulus time histograms (PSTHs) were
constructed from the recording sample in A (i) before and (ii,iii) after
pymetrozine application (bin width 20 ms; ordinates normalised to number of
stimulus presentations, as in all following figures; stimulus, bottom trace).
The histogram in Bii was constructed from experiments where a tonic spike
discharge was observed after pymetrozine application (N=8), the
histogram in Biii from experiments without such tonic discharge
(N=2). (C,D) Effect of pymetrozine at higher concentrations
(10–5 mol l–1); same presentation as in A
and B. (C) Sample recording from the middle leg extensor nerve (i) before and
(ii) after pymetrozine application (stimulus, bottom trace). (D) PST
histograms constructed from the recordings (i) before and (ii) after
pymetrozine application (bin width 20 ms; stimulus, bottom trace). (E)
Increased stimulus amplitude [360 µm (60°); instead of 240 µm]
or (F) increased stimulus velocity (455 deg. s–1; instead of
45 deg. s–1) did not restore the feedback response abolished
by pymetrozine. Same recordings as in C and D, response before pymetrozine
application in Ci and Di.
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Fig. 5. Effect of pymetrozine on the resistance reflex in the middle leg
femur–tibia joint of the stick insect Cuniculina impigra. (A)
Tibia position (middle trace) in response to sinusoidal stimulation of the
femoral chordotonal organ (bottom trace; stimulus frequency 0.5 Hz; amplitude
400 µm, corresponding to 80° tibia movement; arrow indicates tibia
extension and fCO elongation). Top trace shows a flexor tibiae electromyogram.
Application of 10–6 mol l–1 (probably
diluted before actually reaching the fCO) pymetrozine was just before the
beginning of the sample shown. (B,C) In this animal, movement response
declined slowly within 1–2 mins of pymetrozine application. This allowed
measurement at two stimulus frequencies (0.02 and 0.05 Hz; filled circles).
The modified Bode diagram plots response amplitude (B) and phase lag (C)
versus stimulus frequency (diamonds, before pymetrozine). Note the
decrease in response amplitude and unaltered phase lag after pymetrozine
application. Arrow in B indicates stimulus situation depicted in A (0.5 Hz,
different animal).
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Fig. 6. Effect of pymetrozine on individual femoral chordotonal organ (fCO)
receptor cells. (A) Sample recording from the soma of an fCO receptor cell
(distal scoloparium) in the middle leg (i) before and (ii) after
10–5 mol l–1 pymetrozine application.
Stimulus shown in bottom trace (amplitude 240 µm, 40°). Note
response of this receptor cell to the elongation phase of the ramp-and-hold
stimulus, mimicking flexion of the femur–tibia joint. Scale bar, 5 s.
(B) PST histograms constructed from the recording depicted in A, as described
for Fig. 4. Broken line in A
marks the threshold used for spike selection in the construction of the time
histograms in B.
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Fig. 7. The threshold for pymetrozine action on the femoral chordotonal organ is
10–8 mol l–1. Response of the middle leg
femoral chordotonal organ (fCO) was monitored during ramp-and-hold stimulation
of the receptor apodeme [ordinate; spike counts during elongation (filled
circles) and relaxation (filled squares) ramps, baseline activity subtracted].
Different concentrations of pymetrozine were applied, starting with
10–9 mol l–1 and increasing in steps of
5-fold or 2-fold (abscissa) every 25 min until stimulus-related responses
had completely vanished. 10–5 mol l–1 was
applied as the final control concentration. Numbers of measurements per data
point (n=N) range from 6 (above-threshold concentrations) to
12 (threshold area). Median values and 25% percentiles are shown. Graph for
relaxation stimuli is offset to the right by 20% for better visibility of
percentiles. Note that calculation of medians from data obtained in several
individuals obscures the fact that in any given animal pymetrozine had an
all-or-none effect, although at slightly different concentrations; for
details, see text.
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Fig. 8. Intracellular motoneuron recording does not reveal residual
stimulus-related responses after pymetrozine application. (A) Recording taken
during pymetrozine application (10–7 mol
l–1, application started 30 s before sample shown in
Ai). Bottom trace, ramp-and-hold stimulation of femoral chordotonal organ
(fCO) (240 µm, arrow indicates fCO elongation); middle trace, intracellular
recording from FETi soma; top trace, EMG from flexor tibiae muscle. (Aii)
Brushing the abdomen (heavy arrow) leads to active movements; concomitant
synaptic input to FETi proves that the intracellular recording had not been
lost during the previous pymetrozine application. Note that the pronounced
depolarisation of FETi was transient, probably caused by the initial tonic fCO
discharge sometimes observed after pymetrozine application, and disappeared
after a few minutes. FETi membrane potential was approximately –50 mV at
rest and increased by 5.5 mV in the course of the sample recording. This
depolarisation apparently reduced and eventually prevented spike discharges
towards the end of the sample shown. (B) Stimulus-related averaging of the
intracellular FETi record before (top trace) and after (middle trace)
pymetrozine application demonstrates the absence of residual stimulus-related
input after pymetrozine. (C) Histogram of flexor tibiae activity before (top
trace) and after (middle trace) pymetrozine application illustrates the
absence of stimulus-related discharge in FETi's antagonist. B and C show data
from the same recording as in A; bottom traces as in A; note response of FETI
to fCO elongation and of flexor tibiae muscle to fCO relaxation. Scale bars:
(A) 10 mV, 5 s; (B) 5 mV, 250 ms; (C) 36 spikes bin–1 (bin
width 10 ms), 250 ms.
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Fig. 9. Pymetrozine was not observed to have effects on the campaniform sensillae
of the dorsal tibia (A), the wing hinge stretch receptor (B), the campaniform
sensillae on the subcosta vein (C) or the hair sensillae on the tegula (D).
Data before (top traces) and after (middle traces) pymetrozine application
(10–6 mol l–1) are shown for comparison;
stimulus application is shown in the lower traces [pressure on sensillae in A,
upward movement of the wing in B, pressure on the subcosta vein in C (note
that stimulus duration outlasted length of sample trace for lower recording),
stroking of tegula hairs in D (only stimulus onset shown here due to variable
stimulus durations)]. (i) Sample recordings; (ii) time histograms. Ordinates
of histograms normalised to number of stimulus presentations, bin widths 25 ms
in A, 10 ms in B and D, 5 ms in C. Note that in A the campaniform sensillae
respond strongly to the onset of cuticular pressure but just slightly to
maintained pressure and to termination of cuticle indentation. Arrow in B
indicates response of wing chordotonal organ sensillae to the stimulus.
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Fig. 10. The chordotonal organs associated with the wing hinge stretch receptor (A)
and the tegula (B) are affected by pymetrozine like the femoral chordotonal
organ (fCO). Data before (top traces) and after (middle traces) pymetrozine
application (10–6 mol l–1) are shown for
comparison; stimulus (lower trace in A; downward movement of wing); arrows in
B (onset of pressure on tegula base). (i) Sample recordings; (ii) time
histograms. Ordinates of histograms normalised to number of stimulus
presentations, bin widths 10 ms.
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Fig. 11. Pymetrozine was not observed to have effects on central mesothoracic motor
control circuits, or on higher control centres, e.g. in the subesophageal
ganglion. The response of the extensor (A) and flexor (B) tibiae nerves to
ramp-and-hold stimulation of the femoral chordotonal organ (fCO; bottom
traces, 240 µm or 40° fCO movement) was examined before and after
application of 10–6 mol l–1 pymetrozine to
the ventral nerve cord, but not to the fCO (hemolymph space of the leg
isolated by VaselineTM plug (see text). Sample recordings (i and ii) and
time histograms (iii and iv) are shown for the intact (control) situation (i
and iii) and after pymetrozine application (ii and iv). Bin widths of
histograms 10 ms; ordinates normalized to number of stimulus presentations.
Different individuals were used in A and B.
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Fig. 12. Patterned motoneuron activity during walking-like movements persists after
pymetrozine treatment. Data before (A) and after (B) pymetrozine application
(0.5 µg g–1 body mass) are shown for comparison. Ai and Bi
show sample recordings; leg position, top trace (anterior is to the top, i.e.
swing phases are represented by rapid upward strokes); electromyograms (EMGs)
of tibia extensor (M106) and coxa levator/protractor (M94/95) muscles, middle
and bottom traces, respectively. Aii and Bii show corresponding averages of
leg movement (top) and time histograms of EMG discharges (middle and bottom;
bin widths 10 ms, ordinate scales normalised to number of steps evaluated).
Zero on the abscissa marks the start of swing movement; reference lines in top
traces mark lateral leg position. Note the differences in leg movement and
timing of levator activity after pymetrozine application; for details, see
text.
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© The Company of Biologists Ltd 2005
