The insecticide pymetrozine selectively affects chordotonal mechanoreceptors
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
* Author for correspondence (e-mail: harald.wolf{at}biologie.uni-ulm.de)
Accepted 3 October 2005
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Summary |
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Key words: insecticide, pymetrozine, chordotonal organ, locust, leg motor control, chemical ablation
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Introduction |
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Pymetrozine, a pyridine azomethine (Fig.
1), represents a new chemical class of insecticides with a
remarkable selectivity for plant-sucking insects, such as aphids, whiteflies
and plant hoppers, due to its systemic action
(Kristinsson, 1994; Wyss and
Bolsinger, 1997). This fairly narrow biological spectrum seems to be related
to a novel mode of action. Observation of feeding behaviour in aphids
demonstrates that pymetrozine application results in immediate feeding
inhibition, followed by delayed death through starvation
(Harrewijn and Kayser, 1997
).
In detail, sucking aphids immediately withdraw their stylet from the plant
vascular system, while probing and stylet insertion are blocked in non-feeding
aphids, when pymetrozine is applied by injection or ingestion.
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It turns out that pymetrozine affects chordotonal organs, including the
femoral chordotonal organ responsible for femurtibia joint control,
with high potency and selectivity (see also
Ausborn et al., 2001). It
blocks stimulus-related responses and consequently abolishes joint control in
the context of postural reflexes. These effects may fully account for the
raising and stretching of legs in the locust. The results may have further
bearing on the interpretation of the feeding inhibition observed in
pymetrozine-treated insects, which is essential for the insecticide action on
plant-sucking pests.
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Materials and methods |
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Animals
Experiments were carried out under daylight conditions at room temperature
(2022°C). All behavioural and electrophysiological studies were
performed with fully mature adult locusts (Locusta migratoria L.),
both males and females taken from a crowded breeding colony at the University
of Ulm. Experiments for Fig. 5 were performed with adult female stick insects, Cuniculina impigra
(Redtenbacher) (syn. Baculum impigrum Brunner) also taken from a
breeding colony at the University of Ulm.
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For intracellular recordings from motoneurons and interneurons, the above preparation was modified to allow access to the thoracic ganglia. The body was opened by a dorsal midline incision before setting of the dentists' glue. Gut, fatty tissue, salivary glands and (diaphragm) muscles overlying the nervous system were removed. The thoracic ganglia were supported on a wax-coated steel platform for intracellular recording with an npi SEC-10L amplifier (Tamm, Germany).
Stimulation of the femoral chordotonal organ (fCO)
To stimulate the fCO (Burns,
1974; Field and Pflüger,
1989
), a window was cut into the anterior dorsal surface of the
femur, sparing the attachment sites of tibia muscles as far as possible.
This exposed the apodeme (`tendon') of the fCO, which was clamped into a
pair of forceps attached to a transducer (TD in
Fig. 3A; modified loudspeaker
with feedback system; Hofmann and Koch,
1985; 2 in Fig. 3B)
and cut distally (open-loop situation). Sinusoidal or ramp-and-hold stimuli
were applied, centred around a joint angle of 110120°. Stimulus
amplitudes ranged from 240 to 480 µm, corresponding to approximately
4080° tibia movement.
Application of compounds
In intact animals, pymetrozine and its phenyl analogue
(Fig. 1) were each applied by
injection through the abdominal intersegmental cuticle using a Hamilton
microsyringe. Stock solution of insecticide was 10 mmol l1
in DMSO; it was diluted 1:5 in water before injection. The final dose of
insecticide in the insect body was 0.5 µg g1 body mass
(Kaufmann et al., 2004). In
dissected preparations, the saline
(Usherwood and Grundfest,
1965
) in the body cavity was replaced with saline containing the
desired concentration of the compound. Again, both pymetrozine and phenyl
analogue were used, the phenyl analogue only being at 105
mol l1. Usual pymetrozine concentrations were
106 to 107 mol l1,
although the highest and lowest applied concentrations were
105 mol l1 and 109 mol
l1 when determining threshold concentrations
(Fig. 7; see also
Kaufmann et al., 2004
). Minor
dilution may have occurred due to residual saline in the preparation.
Pymetrozine application could be restricted to the nervous system in the body
cavity by plugging the lumen of the coxa with VaselineTM and thus
isolating the hemolymph space of the leg, avoiding pymetrozine exposure of the
fCO (experiments shown in Fig.
11).
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Recording technique
The above preparation not only exposed the fCO apodeme but also the nerve
supply of the extensor tibiae muscle
(Theophilidis and Burns,
1983). Neurograms of slow and fast extensor tibiae motoneurons
(SETi, FETi) were recorded by attaching
mono-(Schmitz et al., 1988
) or
bipolar hook electrodes (HE in Fig.
3A; 5 in Fig. 3B)
to a nerve branch entering the extensor muscle. Alternatively, and in
particular in the walking preparation described below, bipolar
electromyographic recordings of muscle activity were obtained. A pair of 30
µm V2A steel wires, lacquer-coated except for the cut end, was inserted
through small holes just through the cuticle and fixed with a
beeswaxresin mixture. The holes had been pierced into the attachment
sites of the muscles of interest with minuten pins. Amplification and storage
of electrophysiological data and movement recordings were conventional
[extracellular amplifiers, custom-made by Peter Heinecke, Seewiesen; data
recording, DRA-800 analoguedigital converter (CED Cambridge Electronic
Devices, Cambridge, UK) and Pioneer PDR-04 CD recorder (Pioneer Electronic,
Willich, Germany), SPIKE2 software (CED Cambridge Electronic Devices)].
Extracellular recordings of fCO activity were obtained by a suction
electrode (SE in Fig. 3A; 1 in
Fig. 3B). The electrode tip was
positioned on the distal scoloparium (the sensor of femurtibia joint
position; Field and Pflüger,
1989), and electric contact to cell bodies of sensory neurons was
established by application of moderate suction. Different regions of the
scoloparium were sampled until satisfactory recordings, of individual sensory
neurons where possible, were obtained.
To record discharges of the whole fCO (e.g. Fig. 7) or of the campaniform sensillae on the dorsal tibia (below; see also Fig. 9A), the leg nerve was severed close to its exit from the mesothoracic ganglion and inserted into a suction electrode. Since all sensillae in the middle leg send their axons through this nerve, it was possible to monitor input from several sensors in this way, although together with the background of many other spontaneously active sensory neurons.
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Stimulation of campaniform sensillae on the subcosta wing vein and the dorsal tibia
Head and posterior abdominal segments of the locust were severed, and gut
and salivary glands removed from the thorax. This exposed mesothoracic nerve
1C (nomenclature of nerves according to
Campbell, 1961), which
supplies the wing and wing hinge. This nerve was later lifted onto hook
electrodes for recording spike discharges of the campaniform sensillae, as
outlined above for the fCO. Wings, wing base and thoracic dorsum were
immobilised on a cork board with dentist glue. The ventral surface of one wing
was left free, however, to allow access to the campaniform sensillae on the
subcosta vein at the wing base (Gettrup,
1966
). This field of sensillae was stimulated by indentation with
a minuten pin. The pin was attached to minuten relay contacts, serving as a
stimulus monitor. Sometimes, specific stimulation of the campaniform sensillae
appeared questionable, for instance due to high spontaneous spike activity in
nerve 1C or incomplete inclusion of the wing hinge in the dentist glue. In
these cases, the nerve supply to other sensory structures of the wing and wing
base was cauterised, namely tegula and all wing veins except the subcosta, and
the subcosta distal to the stimulation site.
The group of campaniform sensillae on the proximal dorsal tibia was
stimulated in the same way, and its activity recorded through a suction
electrode on the severed leg nerve (above). The stimulation needle was aimed
at the centre of the medial dorsal group of 69 sensillae
(Mücke, 1991;
Newland and Emptage, 1996
). In
this way, probably all sensillae in the group were activated by the stimulus.
Care was taken, however, not to touch any hair sensillae in the vicinity.
Stimulation of the tegula hair field
The preparation used to examine wing campaniform sensillae was also used to
test the sensory hairs on the tegula
(Kutsch et al., 1980).
However, instead of the subcosta vein, a front wing tegula was left free of
dentist glue. Elimination of other wing sensors was not necessary in this case
because the tegula is inserted in soft membranous cuticle, allowing specific
mechanical stimulation with a minuten probe. The probe was moved by hand or by
a modified loudspeaker (above) to achieve controlled bending of the sensory
hairs located on the posterior-medial half of the tegula.
As a control, the chordotonal organ associated with the tegula
(Kutsch et al., 1980) was
stimulated by denting the anterior cuticle of the tegula, which is devoid of
hairs, or the adjacent membranous ligament
(Fischer et al., 2002
).
Stimulation of the wing hinge stretch receptor (SR)
The preparation just described for the examination of subcosta campaniform
sensillae and tegula hair field was also employed to study the wing hinge
stretch receptor (e.g. Möhl,
1985). However, nerve 1D of the mesothoracic ganglion was recorded
with the hook electrodes, since it supplies the wing hinge stretch receptor,
wing chordotonal organ (Pearson et al.,
1989
) and dorsal longitudinal muscles. The nerve was severed
proximally to eliminate motoneuron spikes. The thorax was mounted on its
ventral surface to leave the wing hinge area free to move. The stretch
receptor was stimulated by inserting the front wing into a brace attached to a
mechanical stimulator. The wing was moved through
100° between a
downstroke position,
30° ventral of the horizontal plane, and an
upstroke position,
70° dorsal of the horizontal.
As a control, the chordotonal organ associated with the wing hinge stretch
receptor was stimulated. Since this sensor is most sensitive to wing vibration
(Pearson et al., 1989), this
was achieved by touching the wing surface with a small paint brush. However,
even the wing movement used to stimulate the stretch receptor activated
chordotonal sensillae (Figs
9Bi,
10A), which allowed
stimulus-related evaluation.
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Data acquisition and evaluation
The data stored on CD were run out on a chart recorder (Yokogava ORP 1200)
or digitised with electrophysiology software for further evaluation (SPIKE2,
CED, Cambridge, UK; DATAPAC, RUN Technologies, Lake Oswego, OR, USA), for
instance construction of peri-stimulustime histograms (bin width
indicated in Fig. 3C). Window
discrimination (thresholds indicated in
Fig. 3C) was used to evaluate
the spikes of a particular neuron. Throughout the text, N represents
the number of animals used, and n represents the number of
measurements carried out in the course of an experiment in a given animal.
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Results |
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Response characteristics of the complete feedback loop
In a first set of experiments, and as a reference and control for
subsequent experiments, the function of the complete femurtibia joint
control loop was examined (N=20; see below). The sensor in the
feedback loop, the fCO, was stimulated by clamping the receptor apodeme into
an electromechanical transducer (see Materials and methods;
Fig. 3A). The response of the
feedback loop was monitored through movement recordings (middle trace in
Fig. 5A) and electromyograms
(or neurograms) from the flexor or extensor tibiae muscles (or their nerve
supplies) (top traces in Figs
3C,
4A,C,
5A).
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In summary, the effects of pymetrozine were not concentration dependent but rather exhibited an all-or-none behaviour. That is, pymetrozine effects were either absent or fully present, without intermediate states. Towards lower concentrations, the proportion of unaffected animals increased but the effects, where present, were the same as at higher concentrations (see also doseresponse curve below in Fig. 7).
Stimulus-related responses were never restored, neither by rinsing with saline for up to 2 h nor by increasing stimulus amplitude from 40° to 60° or 80° (Fig. 4E) or increasing stimulus velocity from 45 deg. s1 to 455 deg. s1 (Fig. 4F). Tibia extensor motoneurons and muscles were often tonically active just after pymetrozine application. The same held true for the flexor muscle, where recorded. These results are illustrated in Fig. 5A (experiment performed in a stick insect; see below): the receptor apodeme was sinusoidally stimulated throughout but the corresponding leg movement ceased within less than 60 s after pymetrozine application, the leg assuming an extended position. In parallel to the decrease in stimulus-related leg movement, the electromyogram of the flexor tibiae muscle ceased to show stimulus-related spike bursts (top trace in Fig. 5A). Fig. 4 shows corresponding experiments with ramp-and-hold stimulation of the fCO. The upper traces (Fig. 4Ai,Bi,Ci,Di) give sample recordings and peri-stimulustime histograms of tibia extensor activity in the control situation, while the lower traces (Fig. 4Aii,Bii,Cii,Dii) illustrate the tonic spike discharge and the lack of stimulus-related responses after pymetrozine application.
Cybernetic analysis of the feedback loop after pymetrozine application (e.g. measurement of amplitude and phase relationships for the construction of Bode diagrams) usually proved futile since the effect of pymetrozine was rapid enough to abolish stimulus-related responses within a few stimulus cycles, or even a single cycle at low stimulus frequencies. In the stick insect Cuniculina impigra, however, the effect of pymetrozine sometimes developed more slowly (Fig. 5A), allowing measurement of a partial Bode diagram (filled circles in Fig. 5B,C). This is perhaps due to the longer and more slender legs of these insects, providing more gradual access of the applied insecticide to the fCO inside the leg. It is evident in these experiments that pymetrozine affected response amplitude but not response phase, indicating that the timing in the reflex pathway, and thus synaptic transmission, is not strongly influenced by pymetrozine, if at all.
It should be noted in this context that, in C. impigra, similar responses to pymetrozine were observed as in the locust, although they were examined in less detail (N=26). This is true in particular for leg posture, responses and response thresholds of the middle leg fCO, effects on the fCO feedback loop, and walking behaviour.
No effect on the joint control loop was observed after application of the biologically inactive phenyl analogue of pymetrozine (at 105 mol l1, including 0.1% DMSO; see Fig. 1). This held true for both locusts and stick insects.
Sensory coding by the fCO
The response of the sensor in the femurtibia joint control loop, the
fCO, was studied by recording from individual receptor cells with suction
electrodes (Fig. 6A) while
stimulating the receptor apodeme with ramp-and-hold movements. Rather
unexpectedly, the receptor cells of the fCO were affected by pymetrozine,
basically in the same way as the complete feedback loop
(Fig. 6A,B). Stimulus-related
responses of fCO receptor cells were abolished within a few seconds following
105 mol l1 pymetrozine application
(N=4; 5x108 mol l1
pymetrozine had the same effects, although fewer animals were affected; see
above). All physiological types of receptor cells were affected, those
responding to joint position, to the velocity of joint movement (exemplified
in Fig. 6) or to a combination
of these parameters (e.g. Burns,
1974; Field and Pflüger,
1989
; detailed analyses for the stick insect in
Hofmann et al., 1985
;
Büschges, 1994
). This
held true for stimulus velocities between 108 and 2730 µm
s1 (18455 deg. s1) and stimulus
amplitudes between 240 and 480 µm (4080°). Acceleration
sensitivity was not examined specifically, although the response to vibration
stimuli (Field and Pflüger,
1989
; Stein and Sauer,
1999
) was affected in the same way as that to ramp-and-hold
stimuli (see also Fig. 10A). The receptor cells were either silent or, sometimes, spontaneously active
after pymetrozine application. Spontaneous discharge is seen in the original
recording (Fig. 6Aii) and the
corresponding time histogram (Fig.
6Bii), together with the clear absence of stimulus-related
activity. Spontaneous discharges, if present, usually occurred during the
first few minutes after pymetrozine application.
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Intracellular motoneuron recordings
The recordings from the fCO receptor cells suggested that pymetrozine acts
on these sensory neurons to abolish stimulus-related responses of the joint
control loop. These experiments could not clarify, though, whether or not
all of the 80 or so fCO neurons in the distal scoloparium are
affected, and thus the sensory effects of pymetrozine might be sufficient to
explain the observed cessation of feedback responses. In the locust,
intracellular recordings from motoneurons, and particularly from the fast
(FETi) and slow (SETi) (N=9) extensor tibiae motoneurons were made to
detect any possible remaining subthreshold feedback responses.
Mechanoreceptors of the fCO, in addition to their projections onto spiking and
nonspiking interneurons, contact leg motoneurons monosynaptically, although
usually with low efficacy (Field and
Burrows, 1982; review in
Burrows, 1996
). Therefore,
residual activity of receptor cells after pymetrozine application should be
visible in intracellular motoneuron recordings, at least after
stimulus-related averaging.
Two notable observations were made in the intracellular motoneuron recordings in addition to the electromyograms. First, not even the smallest sub-threshold stimulus-related synaptic potentials were detected in leg motoneurons after pymetrozine application (Fig. 8B). This is evident in original recordings (middle trace in Fig. 8A) as well as in stimulus-related averages (middle trace in Fig. 8B; control before pymetrozine application in top trace), which should reveal even the smallest stimulus-related synaptic input. Accordingly, stimulus-related spikes were also absent, even in flexor tibiae units that were often tonically active (middle trace in Fig. 8C), at least initially after pymetrozine application. This indicates that, indeed, all receptor cells of the fCO's distal scoloparium, i.e. all mechanoreceptor cells involved in joint control, are affected by pymetrozine. This holds for all three parameters of fCO signalling, joint position, movement velocity, and acceleration or vibration stimuli. Second, when the animals performed active movements (for example, in response to stimulation of the abdomen; heavy arrow in Fig. 8Aii; see also Fig. 12), the motoneurons received synaptic input and discharged spikes, even with insecticide present. Similarly, spike activity in the motoneurons remained unaffected by pymetrozine when elicited by current injection into the intracellularly recorded cell. This demonstrates that pathways involved in the initiation and control of active movement components are probably not, or not directly, affected by pymetrozine.
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The presence of motoneuron discharges and muscle activity during active
movement demonstrates that neuromuscular transmission and muscle function
remained basically unimpaired by pymetrozine, in agreement with previous
results (Kaufmann et al.,
2004). This had also been illustrated by our electromyographic
(extensor and flexor tibiae muscles), motor nerve (nerves 3B2c, 5B1c) and
movement recordings (see above).
Response characteristics of other sense organs
The action of pymetrozine on locust mechanoreceptors other than the fCO was
examined in order to test whether or not pymetrozine exerts similar effects on
other sensory cells, and perhaps on mechanoreceptors in general. Campaniform
sensillae on the dorsal tibia (e.g.
Pringle, 1938) and on the
subcosta wing vein (Gettrup,
1966
) and hair sensillae on the tegula organ of the wing base
(Kutsch et al., 1980
) belong
to the same class of cuticular mechanoreceptors as the chordotonal sensillae
(type I sensillae; e.g. Ramirez and
Pearson, 1990
). The front wing stretch receptor
(Möhl, 1985
) was studied
as a representative of a different group of mechanoreceptors (type II
sensillae; Ramirez and Pearson,
1990
). It was a favourable coincidence that chordotonal organs are
associated with both tegula and stretch receptor, allowing to test the
admittedly unlikely possibility that pymetrozine effects are specific
just for the femoral chordotonal organ.
Contrary to our expectations, neither campaniform sensillae (Fig. 9A,C) nor hair sensillae (Fig. 9D) were influenced in their response characteristics by pymetrozine [note similarity of top and middle traces, corresponding to control situation and pymetrozine application, in both original records (i) and time histograms (ii)]. Indentation of the campaniform sensillae on the tibia or on the subcosta wing vein elicited the same response with and without pymetrozine at 106 mol l1 (N=12 and N=9, respectively). While details of the response characteristic of the tibial campaniform sensillae were not readily discernible in original recordings due to background activity in the leg nerve (Fig. 9Ai), the results were very clear in histograms (Fig. 9Aii), particularly for the campaniform sensillae on the wing base (Fig. 9C). The same observations were made with stimulation of the hair sensillae (Fig. 9D) on the tegula organ (N=7). The response of the wing hinge stretch receptor (Figs 9B, 10A, large spikes) remained similarly unaffected (N=10).
By contrast, the chordotonal organs associated with the wing hinge stretch receptor and the tegula were affected by pymetrozine in the same way as the fCO (Fig. 10; N=16). No stimulus-related responses are discernible in the middle traces in Fig. 10, in both original records (i) and time histograms (ii). This demonstrates, first, that the experimental situation was adequate, i.e. that pymetrozine indeed reached the wing hinge area and tegula. Second, it also demonstrates that pymetrozine affects several, and by inference perhaps all, chordotonal sensillae.
The above results are corroborated by preliminary observations on the
(unaffected) responses of hair fields on the head, signalled by the
tritocerebral commissure giant neuron
(Bacon and Tyrer, 1978), and of
cercal hairs, signalled by giant neurons in the abdominal connectives
(Boyan and Ball, 1990
). As
noted above, mechanosensors on the leg other than the fCO also did not show
noticeable deficits after pymetrozine application.
Pymetrozine effects on central neurons and on higher control of local reflex pathways
Possible effects of pymetrozine on central neurons in the mesothoracic
ganglion, and on higher control of the local mesothoracic motor control
pathways, were assessed by restricting pymetrozine application to body cavity
and ventral nerve cord. The leg and fCO were isolated from the insecticide
with a VaselineTM plug in the coxa of the respective leg. In this way,
pymetrozine action on the sensory cells of the fCO was excluded, and major
effects on the central nervous circuitry for joint control in the mesothoracic
ganglion and on higher motor control centres in subesophageal ganglion or
brain (e.g. speculations by Kien,
1983), as far as they affect local processing in the mesothorax,
should become evident.
Fig. 11 illustrates that the resistance reflex in the mesothoracic femurtibia joint remained completely unaffected by pymetrozine application to the central nervous system (N=6) on the level of the present analysis. The reflex responses of extensor (Fig. 11A) and flexor (Fig. 11B) tibiae nerves were virtually identical before and after pymetrozine application. In original records (i and ii) as well as in peri-stimulustime histograms (iii and iv) no difference is discernible between the control situation (i and iii) and pymetrozine application (ii and iv). This is particularly remarkable in the case of the flexor tibiae nerve recording, since there were always several motor units present in flexor nerve recordings. And the reflex responses in all motor units remained unchanged, even in presumed slow motor units with a tendency to be tonically active at rest (the sample histograms in Fig. 11Biii,iv comprise all spike amplitudes just above background activity). This attests to the absence of even small pymetrozine effects.
Control application of pymetrozine to the fCO following these experiments reproduced the above result, i.e. those without isolation of the leg hemolymph space (see Results above).
Walking behaviour
Movements of the middle legs were recorded during tethered walking in
Locusta migratoria in order to examine, in more detail, possible
behavioural effects of pymetrozine. So far, only very coarse behavioural
observations had been made (e.g. Fig.
2; Kaufmann et al.,
2004). Although analysis of feeding behaviour would appear more
desirable, the neuronal basis of leg motor control is known in much more
detail, allowing more meaningful interpretation. Leg movements in walking were
compared before and after pymetrozine application (0.5 µg
g1 body mass) (Fig.
12). Walking movements in locust front and hind legs were affected
in ways similar to those observed in the middle legs, although the hind legs
were more often and more clearly lifted and extended and usually did not
participate in walking. This behaviour had initially been observed in
Locusta migratoria and Periplaneta americana by Kayser and
co-workers (Kaufmann et al.,
2004
). The following general observations were made
(N=11). The femurtibia joints were extended, the legs were
lifted and the body weight was, therefore, usually not supported; rather, the
body was dragged across the ground. Sometimes, front and middle legs did not
even touch the substrate. Leg coordination was impaired; in particular, there
were often almost or completely synchronous swing and stance movements of both
segmental legs, contrasting with the alternating stepping pattern in most
normal walking situations (e.g. Burns,
1973
). Fig. 2B,C
shows photographs of a locust taken
10 min after injection with
pymetrozine at 0.5 µg g1 body mass. Locomotion was almost
impossible for these animals.
Fig. 12 provides recordings
of middle leg movement and muscle activity before
(Fig. 12A) and after
(Fig. 12B) injection of
pymetrozine. Both movement and muscle activity were altered after pymetrozine
application (compare, for example, Burns,
1973; Burns and Usherwood,
1979
; Theophilidis and Burns,
1990
; Wolf, 1990
,
1992
). This was evident in the
brief walking sequences elicited by stroking the animal's abdomen (spontaneous
walking was much reduced or absent after pymetrozine). Starting from a lateral
to slightly posterior position, the leg was rapidly moved posteriorly just
before the swing, reaching the normal posterior extreme position, or a
slightly more posterior attitude (by an average 0.4 mm), before the swing
movement commenced. The swing movement itself and the subsequent beginning of
the stance were sometimes of normal speed, though usually also more rapid
[taking an average of 76 ms instead of 143 ms in the intact (control)
animals]. The stance phase usually did not reach the normal posterior extreme,
and often a small anteriorly directed movement occurred during its later part
(arrow in Fig. 12Bii), after
which the rapid posteriorly directed movement just mentioned heralded the next
swing. These altered leg movements were reflected in leg muscle activity, for
example in the much shortened discharges of the levator/promotor muscle (M94/5
according to Snodgrass, 1929
;
middle and bottom traces in Fig.
12A,B). Overall, the impression of leg movements was that of a
brief `paddling' back-and-forth movement around a laterally extended resting
position.
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Discussion |
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The response of the fCO to joint movement, including position- and
velocity-sensitivity (Burns,
1974; Hofmann et al.,
1985
), is abolished by pymetrozine. A tonic spike discharge may be
present, although usually transiently just after pymetrozine application. It
is, however, not related to mechanical stimulation. Apparently all chordotonal
sensillae are affected since no trace of residual stimulus-related activity
could be detected in any of our experiments, and particularly not in
intracellular motoneuron recordings (Fig.
8), which should reflect even sub-threshold activation of the
feedback loop.
In view of the effects of DMSO on insect neurons, as described by
Theophilidis and Kravari
(1994), it is important to
note that the effects attributed to pymetrozine in the present study were not
caused, nor even influenced, by the DMSO present in the applied dilutions of
the insecticide in saline (see Materials and methods). There are three lines
of evidence to support this interpretation. First, the maximum concentration
of DMSO in the animals was 0.1% (by volume), and was probably slightly lower
due to some dilution by residual hemolymph. It was thus below the lower limit
for neural effects reported by Theophilidis and Kravari
(1994
). Second, we explicitly
tested 0.1% DMSO in saline in control experiments without pymetrozine and did
not find any effects (Ausborn,
2001
). Finally, the inactive phenyl analogue of pymetrozine was
applied in the same solvent as pymetrozine, that is with the same DMSO
concentrations. Application of the phenyl analogue had no effect, even at the
highest concentrations (105 mol l1 and
0.1%, respectively). This experiment served as a control for both pymetrozine
and DMSO.
Pymetrozine has an all-or-none effect on the fCO and on the reflex
responses elicited by it. That is, either the full effect is present or there
is no effect at all. Dose dependency manifests itself only in an increased
number of unaffected animals at lower insecticide concentrations, while there
is no qualitative change in pymetrozine effects, when they are present. The
threshold for pymetrozine effects on the fCO is 108 mol
l1. This agrees well with previous observations on isolated
locust ganglia and foregut (Kaufmann et
al., 2004
), which exhibited similar all-or-none characteristics
with the same concentration threshold. This similarity in threshold supports
our present interpretation that the chordotonal organs are the primary site of
pymetrozine action.
Postural control in the femurtibia joint is, of course, impossible without sensory feedback from the fCO. This may explain the altered position of the femurtibia joint after pymetrozine injection (Fig. 2) and the apparent impairment of postural control. In particular, the tonic spike discharge often observed initially after pymetrozine application will signal to the central nervous system a certain, fixed leg position, which is translated into corresponding muscle activity and resulting joint posture. The fact that the legs are usually hyperextended after pymetrozine application argues for a predominance of tonic discharges in fCO afferents that signal flexed joint positions. And when the fCO falls silent after some time, the feedback loop appears to show some bias into the same direction.
Inactivation of femurtibia joint control may also affect
neighbouring leg joints, namely the tarsal and subcoxal joints, in the context
of interjoint reflexes (e.g. Field and
Rind, 1981; Heß and
Büschges, 1997
; Bucher et
al., 2003
). It is not clear, at present, whether the behavioural
effects of pymetrozine application can be attributed entirely to the absence
of fCO input and its ramifications through reflex pathways or whether
additional effects have to be considered. The present data do not provide more
specific suggestions for any additional effects.
Effects on central interneurons are one possibility here. They would have
to be minor by comparison with the impact on the fCO but might still have
behavioural consequences through amplification in motor control networks. No
effects on the central processing of femurtibia joint control signals
were observed in the present study, nor were there effects on sensory
receptors other than chordotonal organs. However, the present experiments
cannot rule out more inconspicuous effects, for instance on membrane
properties and spike shape. In fact, Riewe
(2001) observed slightly
increased membrane resistance and broadened action potentials after
pymetrozine application in the fast extensor tibiae motoneuron of the locust
Schistocerca gregaria. Studies by Kayser and co-workers
(Kaufmann et al., 2004
)
suggested effects on serotonergic control (see below).
During active, walking-like movements, severe deficits are evident, much
like in postural control. This concerns not just coordination between legs
often almost synchronous movements in the legs of one body segment
but also movements and joint positions in individual legs
(Fig. 12; see also
Kaufmann et al., 2004). These
are in general agreement with the deficits observed in postural control, for
instance more-extended femurtibia joints, lifted subcoxal joints and
lifted tarsi. More detailed interpretations are, again, not possible. Most
remarkable is the fact that after pymetrozine application, and with the
resulting complete absence of sensory feedback from the fCO, walking-like
movements are possible at all. The fCO plays a crucial role in current models
of leg motor control (reviews, for example, in
Büschges, 2005
;
Cruse et al., 1995
), for
instance the transition from swing to stance phases (see also review in
Pearson, 1993
). The present
results suggest that our current picture of insect leg motor control may not
be complete yet. In particular, fast walking, as in the escape situations
elicited here, and the interaction of centrally programmed movement modules
and shaping through sensory feedback, especially from the fCO, may merit
further scrutiny.
In a preceding study (Kaufmann et al.,
2004), pymetrozine enhanced spontaneous spike discharges of the
metathoracic and subesophageal ganglia in situ. Moreover, pymetrozine
effects were mimicked by serotonin, and pymetrozine and serotonin potentiated
each other's effects. These findings may be interpreted as indications for
central nervous effects of the insecticide. Considering the present results,
however, it appears equally possible that the sensory effects of pymetrozine
had been translated into altered spike discharges in the motor nerves of the
metathoracic and subesophageal ganglia. For instance, in a reduced preparation
that is not deafferented, the raising and stretching of the legs observed in
the intact animal (Fig. 2) may
well be reflected by an increased discharge of levator and extensor
motoneurons. Similarly, the serotonergic effects might result from the
modulation of chordotonal sensillae, or of other sensory cells, by serotonin
(e.g. Ramirez and Orchard,
1990
; Kloppenburg et al.,
1999
) in a way mimicking and potentiating pymetrozine effects.
Pymetrozine may well have other target sites in addition to the chordotonal
sensillae demonstrated here. This is suggested by the effects of the
insecticide on the motility of isolated foregut preparations, which is again
blocked by serotonin antagonists (Kaufmann
et al., 2004). It is completely unclear at present whether or not
these effects may be produced by similar cellular mechanisms, perhaps sensory
structures associated with the ingluvial ganglia.
Irrespective of the occurrence of by comparison, minor
effects on pathways in the central nervous system transmitting sensory
information from the fCO, it should be possible to predict deficits elicited
by pymetrozine through its action on chordotonal organs not studied here
(review in Field and Matheson,
1998). These are, for instance, Johnston's organs in the bases of
the antennae. Maintenance of antenna position and perception of air movement
should be impaired by pymetrozine when extrapolating the present findings.
Such experiments will provide critical tests for the conclusions outlined
here.
Having identified the primary mechanism of pymetrozine action in the
locust, it will be interesting to scrutinise the present results in the main
pest species affected by the insecticide, namely aphids, white flies and rice
hoppers. Even more significant will be the study of the cellular basis of
pymetrozine action, which is possible now with chordotonal sensillae
identified as the target neurons. This is all the more important in view of
the apparent specificity of pymetrozine for chordotonal sensillae, as opposed
to other mechanoreceptors. One prediction is possible already when comparing
our present results with pharmacological data on locust mechanoreceptors from
the literature, particularly Ramirez and Pearson
(1990). Fast sodium ion
channels responsive to phentolamine are ubiquitous in sensory neurons and can
be safely excluded as the site of action of pymetrozine, like all compounds
acting on spike propagation. What appears more promising is the fact that
chordotonal organs are the only insect sense organs with a fully developed
ciliary apparatus, the molecular components of which have recently received
notable attention (review in Field,
2005
). This may allow identification of the cellular processes
underlying pymetrozine action.
Considering this specificity, pymetrozine may prove useful as a tool for
the specific elimination of chordotonal organ input in insect neurobiology in
general. On the one hand, it is often difficult or impossible to remove a
particular sensory input in neurobiological experiments. On the other hand,
such experiments are essential, for example, when examining the roles of sense
organs and central nervous system in motor control (e.g.
Wolf and Pearson, 1988). At
least in the case of chordotonal organs, these problems may be outdated due to
the specific action of pymetrozine, in particular if this should prove true
for a broader spectrum of insect species.
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