Effects of temperature on tuning of the auditory pathway in the cicada Tettigetta josei (Hemiptera, Tibicinidae)

Abstract

The effects of temperature on hearing in the cicada Tettigetta josei were studied. The activity of the auditory nerve and the responses of auditory interneurons to stimuli of different frequencies and intensities were recorded at different temperatures ranging from 16°C to 29°C.
Firstly, in order to investigate the temperature dependence of hearing processes, we analyzed its effects on auditory tuning, sensitivity, latency and Q10dB. Increasing temperature led to an upward shift of the characteristic hearing frequency, to an increase in sensitivity and to a decrease in the latency of the auditory response both in the auditory nerve recordings (periphery) and in some interneurons at the metathoracic–abdominal ganglionic complex (MAC). Characteristic frequency shifts were only observed at low frequency (3–8 kHz). No changes were seen in Q10dB. Different tuning mechanisms underlying frequency selectivity may explain the results observed.
Secondly, we investigated the role of the mechanical sensory structures that participate in the transduction process. Laser vibrometry measurements revealed that the vibrations of the tympanum and tympanal apodeme are temperature independent in the biologically relevant range(18–35°C). Since the above mentioned effects of temperature are present in the auditory nerve recordings, the observed shifts in frequency tuning must be performed by mechanisms intrinsic to the receptor cells.
Finally, the role of potassium channels in the response of the auditory system was investigated using a specific inhibitor of these channels,tetraethylammonium (TEA). TEA caused shifts on tuning and sensitivity of the summed response of the receptors similar to the effects of temperature. Thus,potassium channels are implicated in the tuning of the receptor cells.

Full text

1834
Introduction
Like many other species of insects (von Helversen and von
Helversen, 1994; Stumpner and von Helversen, 2001) cicadas
have developed highly specialized systems of intraspecific
acoustic communication in the course of evolution. Acoustic
communication is important for several behaviors, such as
recognition of conspecifics, localization of the sender, mate
choice and predator detection. Precise recognition of the
species-specific signal is essential, especially when other sound
communicating species are present, because the cicada must
discern the song of its own species (Huber, 1984). Cicadas
evolved a sophisticated auditory pathway capable of a fine
discrimination of sound parameters, such as the spectral
content and the temporal pattern of a signal (Daws et al., 1997;
Fonseca and Revez, 2002a; Sueur and Aubin, 2002). Cicadas
have up to 2000 receptor cells per ear, with a high frequency
resolution that is maintained at the level of the central nervous
system (CNS) (Fonseca et al., 2000; Fonseca and Revez,
2002a). Tettigetta josei, the experimental model insect species
in the present study, exhibits this fine frequency resolution as
revealed by the response of its auditory ascending interneurons
which are sharply tuned and cover a large range of frequencies,
including around the peak of the calling song – 16
·kHz
(Fonseca et al., 2000).
The receptor cells are organized in a bulb-like auditory
organ, which is located lateroventrally in the first abdominal
segment (Doolan and Young, 1981; Michel, 1975). A rod, the
tympanal apodeme, provides the mechanical connection to the
oscillating tympanum which acts as a pressure difference
receiver (Fonseca and Popov, 1997). In T. josei, about 700
afferent fibers run into the auditory nerve, which is connected
to first order interneurons at the metathoracic–abdominal
ganglionic complex (MAC). More than a dozen auditory
ascending interneurons have been found in cicadas, together
with some local cells (Huber et al., 1990; Fonseca, 1994;
Fonseca et al., 2000).
Cicadas are essentially ectothermic insects (Fonseca and
Revez, 2002b; Sanborn et al., 1992) and therefore fluctuations
in environmental temperature might influence auditory
processing. T. josei is active during the day in June and July,
The effects of temperature on hearing in the cicada
Tettigetta josei were studied. The activity of the auditory
nerve and the responses of auditory interneurons to
stimuli of different frequencies and intensities were
recorded at different temperatures ranging from 16°C to
29°C.
Firstly, in order to investigate the temperature
dependence of hearing processes, we analyzed its effects on
auditory tuning, sensitivity, latency and Q
10dB
. Increasing
temperature led to an upward shift of the characteristic
hearing frequency, to an increase in sensitivity and to a
decrease in the latency of the auditory response both in the
auditory nerve recordings (periphery) and in some
interneurons at the metathoracic–abdominal ganglionic
complex (MAC). Characteristic frequency shifts were only
observed at low frequency (3–8·kHz). No changes were
seen in Q
10dB
. Different tuning mechanisms underlying
frequency selectivity may explain the results observed.
Secondly, we investigated the role of the mechanical
sensory structures that participate in the transduction
process. Laser vibrometry measurements revealed that the
vibrations of the tympanum and tympanal apodeme are
temperature independent in the biologically relevant range
(18–35°C). Since the above mentioned effects of
temperature are present in the auditory nerve recordings,
the observed shifts in frequency tuning must be performed
by mechanisms intrinsic to the receptor cells.
Finally, the role of potassium channels in the response of
the auditory system was investigated using a specific
inhibitor of these channels, tetraethylammonium (TEA).
TEA caused shifts on tuning and sensitivity of the summed
response of the receptors similar to the effects of
temperature. Thus, potassium channels are implicated in
the tuning of the receptor cells.
Key words: cicada, Tettigetta josei, hearing, temperature.
Summary
The Journal of Experimental Biology 210, 1834-1845
Published by The Company of Biologists 2007
doi:10.1242/jeb.001495
Effects of temperature on tuning of the auditory pathway in the cicada Tettigetta
josei (Hemiptera, Tibicinidae)
P. J. Fonseca* and T. Correia
Departamento de Biologia Animal e Centro de Biologia Ambiental, Faculdade de Ciências da Universidade de
Lisboa, Bloco C2, Campo Grande, 1749-016 Lisboa, Portugal
*Author for correspondence (e-mail: pjfonseca@fc.ul.pt)
Accepted 7 March 2007
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1835Effects of temperature on cicada hearing
usually singing at ambient temperatures from 22°C to 35°C.
Fonseca and Revez (Fonseca and Revez, 2002b) showed that,
at least in three species of cicadas, some temporal parameters
of their songs are deeply affected by changes in temperature.
Because hearing mechanisms depend on electrochemical
transduction which is likely to be affected by temperature, we
might expect the auditory pathway to be temperature sensitive.
In fact, Oldfield (Oldfield, 1988) discovered that, in the locust,
tuning and sensitivity of single auditory receptors are
temperature dependent. The characteristic frequency and
sensitivity of the auditory receptors increased while latency
decreased with increasing temperature. Auditory tuning
properties have also been found to be affected by changes in
temperature in a variety of vertebrates: amphibians (Dijk et al.,
1990), reptiles (Eatock and Manley, 1981; Smolders and
Klinke, 1984), hearing specialist fish (Fay and Ream, 1992)
and birds (Schermuly and Klinke, 1985). By contrast, in
mammals, no temperature-dependent changes in the
characteristic sound frequency measured in afferent fibers were
found (Gummer and Klinke, 1983). Moreover, no effects of
temperature on sensitivity and latency were found in hearing
generalist fish (Amoser and Ladich, 2006).
Frequency selectivity can be performed by mechanisms
extrinsic or intrinsic to the receptor cells (Dallos, 1992;
Fettiplace and Fuchs, 1999; Kennedy et al., 2005). Changes in
temperature might affect frequency analysis mechanisms at one
or both levels. Changes in the mechanical properties of the
tympanic membranes and/or the auditory organ, caused by
temperature variations, could not explain the shifts observed in
the characteristic sound frequency of locust auditory receptors
(Oldfield, 1988). Thus, these shifts might have been due to
changes in the intrinsic properties of the receptors. Intrinsic
mechanisms may depend on electrical properties of the
individual receptors (Fettiplace, 1987; Fuchs et al., 1988;
Hudspeth and Lewis, 1988).
The aim of this study was to analyze the effects of
temperature on tuning, sensitivity, latency, Q
10dB
and response
strength of interneurons and auditory receptors in the species
T. josei and to scrutinize some of the mechanisms that might
be responsible for the fine frequency tuning observed in the
auditory pathway of cicadas.
Materials and methods
Animals
Cicadas of the species Tettigetta josei Boulard 1982 were
collected from the end of May until July in south eastern
Portugal. The insects were transported in a cool box and kept
either on a feeding shrub at ambient temperature in the shade
(Lisbon University) or in a cooled environment at about 10°C
(University of Southern Denmark in Odense).
Electrophysiology
Dissection and recording: Cicadas were waxed, ventral side
up, to a holder 6·mm in diameter. The temperature of the holder
was varied with a Peltier element (Fig.·1) and controlled
through two thermocouples. One thermocouple measured the
temperature of the holder itself and allowed variations in the
range of 10–40°C, temperatures that the animal may face in its
natural environment. The second thermocouple was inserted
into the pool of insect saline where the metathoracic-abdominal
ganglionic complex (MAC) was kept during the experiments
(see below). This temperature sensor controlled a thermostat,
the set point of which was selected from a predefined value in
the range 16–30°C. Prior to each recording session the
temperature of the MAC was continuously monitored and was
allowed to stabilize at each of the set points selected (±0.5°C).
To expose the nervous system (auditory nerve and MAC),
the legs and wings were removed and the meso- and
metathoracic sterna were detached after cutting the integument
laterally and sectioning the apodeme bridges, uncovered by
carefully lifting the sterna. Insect saline was added to form a
pool where the MAC was maintained. This preparation was
alive for several hours. In order to minimize possible
systematic effects, the frequency of the stimulus and the
temperature of the cicada did not follow a monotonous
increasing or decreasing series, and some temperatures were
repeated during the experimental series. Moreover, in the TEA
experiments (see below) the activity at the auditory nerve could
be recovered after more than 3·h by washing the preparation
with insect saline.
For intracellular recordings the MAC was stabilized with a
metal spoon. The activity of the auditory nerve was recorded
with a hook made from an electrolitically sharpened tungsten
electrode. A silver wire indifferent electrode was placed in the
insect saline pool. The intracellular electrodes consisted of
60–100·M⍀ glass micropipettes (Clark GC100F-10; Reading,
UK) filled with Lucifer Yellow (Sigma, St Louis, MO, USA;
Cat. No. L-0259; 5% in LiCl 0.5·mol·l
–1
). After a successful
recording, and when allowed by stability, the dye was injected
by iontophoresis (–0.5 to –1.5·nA) in order to identify the
morphology of the neuron. The microelectrodes were
positioned with a Leitz micromanipulator (Germany). In order
to soften the sheath of the ganglion, collagenase (Sigma C-
0130) was applied to some preparations for 15–30·s,
immediately followed by a thorough wash. The intracellular
signal was amplified 10⫻ (Neuro Data model IR-283, Neuro
Data Instruments Corp., New York, USA), digitized (50·kHz,
12·bit resolution, low pass filtered at 5·kHz for intracellular
data and 10·kHz for extracellular recordings) with a
multichannel board (Digidata 1200, Axon Instruments, Foster
City, USA, controlled by Axoscope 9.0) and stored for later
analysis on the hard disk of a PC, along with the extracellular
recording of the auditory nerve (1000⫻ amplified with a lab
made amplifier), the sound stimulus and a time marker used as
trigger during analysis.
Sound stimulation: the stimuli consisted of pure tone pulses,
30·ms long with 2·ms ramps and produced at intervals of
120·ms. These stimuli, repeated five or 10 times at each of 13
sound amplitudes ranging from 30·dB to 90·dB SPL delivered
in 5·dB steps (re. 20·␮Pa), were presented at 16 different
frequencies ranging from 0.5·kHz to 24·kHz (0.5, 1, 1.5, 2, 3,
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1836
4, 5, 6, 8, 10, 12, 14, 16, 18, 20 and 24·kHz). The sound stimuli
were generated by a PC computer and delivered (Sound Blaster
Extigy, Creative Labs, Singapore, 96·kHz D/A conversion, 24
Bit, low pass filtered at 24·kHz) after amplification (Technics
SU-V500 M2, Matsuchita Electric Industrial Co., Osaka,
Japan) to a loudspeaker (Dynaudio D28/2, Skanderborg,
Denmark) at 24·cm from the head of the preparation. By
placing the loudspeaker in front of the cicada, the effects of
directionality of the auditory periphery (Fonseca, 1993;
Fonseca and Popov, 1997; Fonseca and Hennig, 2004) on the
variation of the responses of interneurons that might receive
input from either side of the animal were minimized. The
amplitudes of the sound stimuli were measured and equalized
using a Bruel & Kjær type 4135 s inch microphone (Naerum,
Denmark) at the position later occupied by the cicada. The
microphone was previously calibrated with a pistonphone
Bruel & Kjær type 4220 (Naerum, Denmark). Because the
sound field in an electrophysiological set up is always disturbed
by the presence of equipment close the preparation, echoes
were monitored and the sound field optimized by shielding
equipment and lining the Faraday cage with sound absorbing
material (cotton and Illbruck ‘super waffle’).
Effects of TEA on the auditory responses
In order to measure the effect of TEA on the tuning,
sensitivity, latency and Q
10dB
evaluated from the summed
responses of the auditory receptors, the insect saline bathing
the MAC, which effectively circulated through the insect body
as hemolymph, was replaced with a solution of 200·mmol·l
–1
TEA in insect saline. Recordings were made within 20–60·min
after the application of the TEA solution. Then the preparation
was repeatedly washed for a maximum of 2·h and 20·min with
P. J. Fonseca and T. Correia
insect saline to observe the possible reestablishment of the
auditory activity. The time course of the drug effect might
be different from animal to animal probably caused by
different timing for the drug to reach the cells within the
protective sheath of the insect nervous system. The
temperature in the laboratory ranged between 24–28°C.
Laser vibrometry
Laser vibrometry measurements were made at the
University of Southern Denmark (Odense, Denmark).
The cicada with legs, wings and opercula removed [the
effects of removing the opercula are small, see Fonseca and
Hennig (Fonseca and Hennig, 2004)], was waxed to a holder
(9·cm long, 2·mm across). This holder was in turn fixed to
a stand that allowed great freedom of movements for
positioning the cicada relative to the laser beam. In order to
enhance the reflection of the laser beam from the tympanum,
a few very small glass spheres, each weighing about 0.5·pg,
were applied on the tympanal ridge at the position to be
measured. These spheres did not affect the mechanics of the
tympanum even if applied to the very thin membrane.
Because we were interested in measuring any
temperature effect on the vibrations of the tympanum
likely to be analyzed by the auditory organ, the laser beam
was focused on the closest point externally available for
measurement, which is where the tympanal ridge connects to
the tympanal apodeme. Notice that in cicadas this apodeme
forms a relatively stiff rod that then connects to the bulb shaped
auditory organ. There were no relevant vibrations of the animal
holder. Moreover, the tympanal vibrations were measured
within the linear dynamic range of the tympanum which was
previously evaluated.
The sound stimulus consisted of a sine sweep burst with a
frequency span of 0 to 25·kHz and 3.5·ms long. The short
stimulus and the use of a rectangular force window ensured that
the measurement ended before any relevant reflections were
recorded. The beginning of the sampling of the vibrations was
delayed relative to the generation of the sound stimulus to take
into account the sound propagation from the speaker to the
preparation. The stimulus was generated by a HP35665A
spectrum analyzer (Hewlett Packard, Washington, USA),
amplified (Xelex DD8, Stockholm, Sweden) and delivered by
a loudspeaker (Dynaudio D28 AF, Skanderborg, Denmark) at
21·cm and ipsilateral to the measured tympanum. This audio
chain guaranteed that the stimulus produced had enough power
above 1·kHz. The analyzer computed the transfer function from
the stimulus to the recorded tympanal vibrations (laser dopler
vibrometer Dantec, Copenhagen, Denmark).
The temperature of the cicada was varied as described above.
Data analysis
The electrophysiological recordings were analyzed off line
using dedicated home made programs and conventional
spreadsheet software. The hearing thresholds were evaluated
from intensity response curves using as criterion the averaged
subthreshold activity plus three times the standard deviation
Peltier
element
Thermocouples
(setpoint)
Auditory
interneuron
recording
Auditory nerve
recording
Flowing water
Sound
stimulus
Fig.·1. Set-up used to control the body temperature of the cicada during
intracellular recordings of auditory interneurons and recordings of the
auditory nerve activity. The temperature of the animal holder was modified
with a Peltier element and controlled via two thermocouples. The sensor
in the holder kept its temperature within values compatible with the living
tissues (10–40°C) while the second thermocouple measured and was used
to control the temperature of the cicada body. The flowing water is needed
to add to or remove heat from the Peltier element.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1837Effects of temperature on cicada hearing
(Fonseca et al., 2000). For intracellular recordings the activity
was evaluated as the average number of action potentials
occurring in a response window defined as the stimulus
duration with a latency lag, for a certain frequency and
amplitude. Similarly, the activity on the auditory nerve was
estimated as the peak-to-peak amplitude of the averaged
recordings at each frequency and intensity which represents the
summed response of the receptors. Each stimulus was repeated
five times for the intracellular measurements and five or ten
times for extracellular recordings. The latency of the
interneurons’ response was measured from the beginning of the
sound stimulus to the first action potential. Although several
interneurons showed spontaneous activity, the beginning of the
response was usually unambiguous because it was often
accompanied by an EPSP (see Fig.·2). The latencies of the
auditory nerve response were measured from the beginning of
the stimulus marker to the peak of the averaged recording,
which was well defined because of receptor synchronization.
Both latencies were measured 20·dB above threshold. The
sharpness of tuning was estimated from the tuning curves
dividing the characteristic frequency by the frequency band
10·dB above threshold (Q
10dB
).
Statistic analysis
Data represented by quantitative and continuous variables
was tested using parametric methods of analysis of variance in
case the assumptions of linear model were verified. When this
was not the case, or the data consisted of quantitative discrete
variables, non parametric procedures were used. See Zar (Zar,
1999) for details on the methods. All computations were made
using the packages Statistica 6.0 and R 2.1.1 or a conventional
spreadsheet (Microsoft Excel).
The presence of effects caused by the change in temperature
on the characteristic frequency of the interneurons and the
summed response of the auditory receptors was tested using a
Kruskal–Wallis (KW) non parametric method. This was
followed by a Dunn’s post-hoc pairwise test to investigate
significant differences of characteristic frequency between
temperatures. The same method was applied to test the effect
of TEA on the characteristic frequency evaluated from the
summed response of the auditory receptors.
To investigate the effects of temperature on the sensitivity
of the interneurons and on the sensitivity revealed by the
summed response of the auditory receptors a bi-factorial
ANOVA, a one-way ANOVA or a Kruskal–Wallis test were
used depending on the nature of the data. The bi-factorial
ANOVA considers both the effects of the temperature and of
the individual cells on the sensitivity without an interaction
term. The effects of TEA on the sensitivity evaluated from
extracellular recordings were tested with a one-way ANOVA
followed by a Tukey’s test to investigate significant differences
of sensitivity between pairs of treatments.
The effects of the temperature and of the TEA on the latency
of the extracellular recording were assessed using one-way
ANOVA. For the influence of temperature on the latency of the
interneurons a Kruskal–Wallis test was used.
Whenever the presence of significant effects of temperature
on the sensitivity and latency were found, the significance of
the slopes of the individual linear regression lines was tested
using analysis of covariance (ANCOVA). This analysis was
done using as categories auditory interneurons or auditory
nerves. In case the slopes were not significantly different
among cells or individuals, then a general slope was computed.
To test the significance of increasing response strength of the
cells with temperature a bi-factorial ANOVA was used. Finally
possible changes of the sharpness of tuning (Q
10
) caused by
temperature were tested with a bi-factorial ANOVA, used on
intracellular and extracellular recordings, and with a one-way
ANOVA followed by a Tukey’s test, used on data from TEA
experiments.
Computations were made with values in dB, and not in a
linear scale, because the several observations at each condition
did not differ from a normal distribution.
Results
Effects of temperature on the tuning of interneurons
We obtained 70 recordings of auditory interneurons from a
total of 45 cicadas: 14 recordings from 13 insects tuned to a
20 ms
25 mV
A
B
C
Fig.·2. Examples of electrophysiological responses of the auditory
interneurons. The types varied from a phasic response (A) with a
single action potential, to a phasic-tonic (B) and a more tonic response
(C). The examples are from three cells recorded 20
·dB above threshold
at 6
·kHz and at 24°C.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1838
low frequency band from 0.5 to 2·kHz; 3 recordings from 3
cicadas tuned to a high frequency band from 14 to 20·kHz; 3
recordings from 3 cicadas tuned to 3–6·kHz, corresponding to
two physiological types (Fig.·2B,C); and 50 recordings from
36 cicadas with two sensitivity maxima, one at low (3–8·kHz)
and another at high frequency (14–24·kHz). Recordings were
made with temperatures ranging from 16°C to 29°C.
All 14 recordings tuned to low frequency (0.5–2·kHz) and
the three tuned to 3–6·kHz, a total of 24%, maintained their
characteristic frequency with changing temperature. Similarly,
the three recordings (4%) tuned only to high frequencies
(14–20·kHz) did not exhibit any clear tuning shifts with
temperature. From the remaining 50 recordings with two
sensitivity maxima, 29 recordings (41%) from 26 cicadas,
corresponding to at least three physiological (Fig.·2) and five
morphological types (Fig.·3), revealed changes in the
characteristic frequency (KW, H
4,91
=43.91, P<0.01). This
characteristic frequency shifted up to 3·kHz with changing
temperature (Table·1) at the low frequency (3–8·kHz)
sensitivity maximum, but not at the highest frequency range
(Fig.·4A,B). The sharpness of tuning at the characteristic
frequency evaluated by the Q
10dB
was not affected by
temperature (bi-factorial ANOVA, F
13,55
=1.28, P>0.1). From
the remaining 21 (30%) cell recordings from 18 cicadas, 18
maintained their tuning and another three increased their
characteristic frequency up to 24–26°C but at the highest
temperature tested they showed a lower than expected
frequency tuning (data not shown).
Cells repeatedly recorded at the same temperature in the
course of an experiment maintained their tuning despite some
changes in sensitivity occasionally observed. These might be
due to some deterioration of the cell response caused by very
long recordings or to other mechanisms allowing variable gain
(see Discussion).
Effects of temperature on the sensitivity of interneurons
At the low frequency band (3–8·kHz), 20 of the recordings
referred to in Table·1, exhibited a decreasing threshold, that is,
they became more sensitive with increasing temperature (bi-
factorial ANOVA, F
9,29
=15.87, P<0.01) (Fig.·4C). Their
P. J. Fonseca and T. Correia
100 µm
tj19-1 tj67-1 tj67-2 tj71-2
Fig.·3. Examples of four morphological types of auditory interneurons (see Table·1) (tj19-1, tj67-1, tj67-2, tj71-2) with two sensitivity maxima
that revealed a shift in the characteristic frequency in the range 3–8
·kHz. A fifth cell type (tj52-1) with a different morphology was only partially
stained and therefore is not shown. The cells were stained with Lucifer Yellow.
Table·1. Characteristic frequency of 29 responses of
interneurons from 26 cicadas exhibiting changes in their low
frequency tuning in the range 3–8·kHz with changes in body
temperature
Frequency
Recording* 16–17°C 18–19°C 20–21°C 22–24°C 25–29°C
1-1 (C) 4 6 and 8
3-1 (C) 3 4 6
10-3 (A) 3 4
14-3 (C) 4 4 5
19-1 (C)
†
55 566
20-2 (A) 4 5/6
23-2 (C) 3 3 4
27-1 (B) 4 5 6
27-2 (B) 5 6
30-3 (B) 4 5 6
31-1 (C) 5 5 5 6 6
33-1 (B) 5 5 6
37-1 (B) 5 5 6 6
45-1 (C) 3 5
50-5 (B) 5 5 8
51-1 (B) 4 5 6 6
52-1 (B)
†
55 66
54-1 (C) 5 6
54-2 (B) 3 5/6
55-1 (B) 4 6
56-1 (C) 3 5 5
61-1 (C) 5 6
64-1 (B) 5 6
64-2 (B) 4/5 6 6 6 6
67-1 (C) 3 3 6 6 6
67-2 (B)
†
3355
71-2 (C)
†
44 466
73-2 (B) 5 6
75-3 (B) 3 4 4 5 6
Frequency values are in kHz.
*A, B and C refer to the physiological response types of the
interneurons, as indicated in Fig.
·2.
†
Cells that were stained at the end of the experiment and all
revealed different morphologies as shown in Fig.·3.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1839Effects of temperature on cicada hearing
increase in sensitivity averaged about
2.2·dB·°C
–1
(ANCOVA, F
17,15
=0.52, P>0.05,
computed common slope: 2.2). In the remaining
recordings sensitivity did not seem to have any
direct relationship with temperature.
At the high frequency band (14–24
·kHz), the
threshold values were not significantly
different at different temperatures (one-way
ANOVA, F
12,70
=0.66, P>0.05). A similar result
was obtained for the cells that were tuned just
to a low frequency band (0.5–2kHz), where
sensitivity did not change significantly with
temperature (KW, H
8,35
=5.17, P>0.05).
Fig.·4A,B are examples of tuning curves of
two auditory interneurons whose characteristic
frequency and sensitivity changed with
temperature at the low frequency minimum. At
high frequency a smaller shift in sensitivity can
be seen.
Effects of temperature on the latency of
interneurons
Each cell responds to the stimuli with a given
delay, which depends on stimulus intensity and
on temperature. Fig.·5 shows an example of the
effect of temperature on the response of one
auditory interneuron. The latency decreased
with temperature while the strength of the
response increased, as revealed by an increase
in the number of spikes. Indeed, all the
recordings represented in Table·1 showed a
clear similar decrease in latency with
increasing temperature (Fig.·4D; KW,
H
13,82
=64.30, P<0.05). Latencies decreased
about 0.83·ms·°C
–1
(ANCOVA, F
24,32
=0.37,
P>0.05, computed common slope: 0.83).
Effects of temperature on response strength of
interneurons
The response strength of the 29 cells in
Table·1, evaluated as the average number of
action potentials per stimulus 20·dB above
threshold, increased with temperature in most of
the interneurons (Fig.·4E). This effect was
observed both at the characteristic frequency (bi-
factorial ANOVA, F
13,55
=6.19, P<0.01) and at
the second higher frequency sensitivity
maximum (bi-factorial ANOVA, F
13,46
=12.73,
P<0.01). In three of those 29 cells, the general
increasing tendency at the characteristic
frequency was accompanied by an oscillation of
the number of action potentials. In the other three
interneurons the increase in temperature was not
followed by a change in response strength. Only
one notable exception of a reduction with
increase in temperature was seen.
16°C
20°C
24°C
A
16°C
19°C
24°C
28°C
B
30
40
50
60
70
80
90
Threshold (dB SPL)
Latency (ms)
C
10
15
20
25
30
35
5
D
15 20 25 30
Temperature (°C)
10
8
6
4
2
0
Number of APs
N=29
E
15 20 25 30
Temperature (°C)
30
40
50
60
70
80
90
Threshold (dB SPL)
5100152520
30
40
50
60
70
80
90
Threshold (dB SPL)
5100152520
Frequency (kHz)
N=20
15 20 25 30
N=29
Fig.·4. Effects of body temperature on tuning, sensitivity, latency and response
strength of auditory interneurons of the cicada T. josei. (A,B) Tuning curves of
two interneurons exhibiting shifts in their tuning and sensitivity with body
temperatures ranging from 16°C to 28°C. Maximum effects are observed at
temperatures from 16–18°C to 24°C in the frequency range 3 to 8
·kHz. At higher
frequencies the characteristic frequency remains constant, but some effect on
sensitivity is still present. (C) Sensitivity at the characteristic frequency of 20
recordings, of the 29 cells listed in Table
·1, which exhibited an increased
sensitivity with temperature. The lines connect the sensitivities of each
interneuron. (D) Dependence of latency on temperature, obtained from 29
recordings of interneurons. Latencies were measured 20
·dB above threshold at
the characteristic frequency and decreased with increasing temperature. (E)
Dependence of the number of action potentials on the temperature in 29
recordings. At each temperature the number of action potentials is an average
of five stimulus presentations at the characteristic frequency and 20
·dB above
threshold.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1840
Effects of temperature on the tuning measured from the
summed response of the auditory receptors
The effect of temperature on tuning observed at the level of
the auditory interneurons was already present at the periphery.
Nineteen out of the 20 cicadas in which auditory nerve
recordings were analyzed (Table·2) showed a significant
change in the characteristic frequency up to 3·kHz with
changing temperature (KW, H
4,75
=48.18; P<0.01). The
characteristic frequency increased from 3–4·kHz at low
temperature to 5–6·kHz at high temperature. In contrast, and
along with the results obtained with intracellular recordings,
the effects at high frequency were negligible (Fig.·6A,B). The
sharpness of tuning at the characteristic frequency evaluated by
the Q
10dB
was not affected by temperature (one-way ANOVA,
F
13,67
=0.74, P>0.1).
Effects of temperature on the sensitivity evaluated from the
summed response of the auditory receptors
Changes in threshold at the auditory nerve level were
observed at the characteristic frequency. The sensitivity of the
recordings in Table·2 increased with temperature (one-way
ANOVA, F
13,68
=3.68, P<0.01). However, only 10 out of these
19 recordings showed a clear enhanced sensitivity with
increasing temperature (Fig.·6C), exhibiting a shift of
2.4·dB·°C
–1
(ANCOVA, F
9,20
=0.39, P>0.05, computed
common slope: 2.4).
At high frequency (14–24·kHz), the sensitivity did not seem
to vary significantly with temperature (one-way ANOVA,
F
13,63
=0.60, P>0.05; cf. Fig.·6A,B).
P. J. Fonseca and T. Correia
As shown in the previous tables and figures, the effect of
temperature was already present at the auditory periphery and
was maintained at the level of the first order interneurons.
Effects of temperature on the latency measured from the
summed response of the auditory receptors
The auditory response of all cicadas listed in Table·2,
measured from the auditory nerve recordings, became faster as
the temperature increased from 16°C to 28°C (Fig.·6D). The
latency decreased significantly (one-way ANOVA,
F
13,66
=12.40, P<0.01) about 0.48·ms·°C
–1
(ANCOVA,
F
18,42
=0.25, P>0.05, computed common slope: 0.48).
Effects of temperature on mechanosensory structures
The tympanum and the tympanal apodeme are the structures
that are driven into oscillation by the sound, and they transmit
the oscillation to the receptor cells at the auditory organ, where
transduction occurs.
Measurements by Doppler laser vibrometry in three cicadas
demonstrated that the vibrations at the tympanum and the
tympanal apodeme were not significantly affected by
temperature, in the biologically relevant range (18–35°C).
The vibration velocity and the phase angles, measured at
different points on these structures, namely where the
tympanal apodeme attaches to the tympanal ridge (see an
example in Fig.·7), did not show any considerable change up
to 15·kHz. Although some changes were observed at higher
frequencies, they were not observed in all cicadas nor in the
same cicada in other measurements on the ridge. Consistently,
no effect was seen in the frequency range where the strong
30 mV
20 ms
16°C
24°C
20°C
Fig.·5. Intracellular recording of an auditory interneuron showing the
variation in latency and strength of the response with temperature. The
sound stimulus at 6
·kHz was delivered 20·dB above threshold at each
temperature.
Table·2. Characteristic frequency measured from the summed
response of the auditory receptors in 19 cicadas
Frequency
Recording* 16–17°C 18–19°C 20–21°C 22–24°C 25–29°C
14-3 3 4 4 6
19-1 3/4 4 5 5
31-1 3 3 6 6 6
39-4 3 5 5 5
42-2 3 6 6
48-2 3 3 6 6
49-2 3 3 3 6
50-4 3 3 6 4–6
51-1 3 3 6
52-1 3/4 6 6
54-2 3 4
56-1 3 5 5
58-1 3 4 5 4/5
63-5 4 4 6 6
64-2 3 4 5 6 6
65-3 3 6 6 6
67-1 3 5 6 6 6
71-2 3/4 4 4–6 4–6 6
75-3 3 3/4 4–6 6 6
Frequency values are in kHz.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1841Effects of temperature on cicada hearing
effects of temperature were detected in the nervous system
(3–8·kHz).
Effects of tetraethylammonium (TEA) on tuning, sensitivity
and latency evaluated from the summed response of the
auditory receptors
TEA is an inhibitor that blocks nonspecifically a variety of
potassium channels. An application of TEA (200·mmol·l
–1
)
diluted in insect saline and applied to the body cavity entered
the nervous system but did not block all the potassium
channels, since auditory processing was affected but not
abolished. Moreover, the effects of this drug could be removed
by repeatedly washing the preparation with insect saline.
The tuning evaluated from the auditory nerve recordings on
10 cicadas kept at room temperature (24–28°C; Table·3) were
modified by TEA application (KW, H
2,30
=18.19; P<0.01). The
characteristic frequency usually decreased in the presence of
the drug, with shifts of up to 3·kHz and, when TEA was washed
out, the auditory nerve responses recovered
(Dunn’s post-hoc test) and frequency tuning
was re-established (Fig.·8A,B). The Dunn’s
pairwise post-hoc test did not find differences
in the characteristic frequency between the
initial condition and the washed preparation.
The sharpness of tuning at the characteristic
frequency evaluated by the Q
10dB
was not
affected by TEA (one-way ANOVA,
F
2,27
=1.68, P>0.1).
Regarding sensitivity (Fig.·8), the threshold
increased with the presence of TEA and
decreased after the TEA has been washed out
(one-way ANOVA, F
2,27
=12.80, P<0.01).
Again a Tukey’s post-hoc test revealed no
differences in the sensitivity before TEA
application and after washing the preparation.
The effects on tuning and sensitivity paralleled
the effects of reducing temperature (compare
Fig.·8A with Fig.·6A). By contrast, latency did
not appear to change significantly with the
different treatments (one-way ANOVA,
F
2,27
=1.57, P>0.1).
The effect of TEA on tuning was only
observed at low frequencies (3–8·kHz).
Sensitivity exhibited stronger changes at low
frequencies, but some effect seemed also to be
present at high frequencies (Fig.·8A). The shift
on the tuning curves caused by the presence of
TEA was similar to that caused by low
temperatures. Hence, both the application of
TEA and changes in temperature affected the
frequency selectivity measured at the auditory
nerve in a similar manner and at the same
frequency range (3–8·kHz).
Discussion
Our results demonstrate that about 50% of the recordings of
auditory interneurons with two sensitivity maxima showed
temperature dependence (Fig.·4), in contrast to interneurons
tuned to very low (0.5–2·kHz) or to high frequency
(14–20·kHz). Moreover, in the cells with two sensitivity
maxima the effect on tuning was only observed at the low
frequency range (3–8·kHz) and not at the second higher
frequency sensitivity maximum (14–20·kHz). The
characteristic frequency in the 3–8·kHz range increased up to
3·kHz with increasing temperature, and this temperature-
dependent shift in tuning was already present in the auditory
nerve recordings (Fig.·6). This suggests that the temperature-
induced modifications are effected through frequency
selectivity mechanisms at the level of the receptor cells or on
the biophysics of the ear. The same shifts caused by
temperature on auditory tuning were observed in lower
vertebrates (Dijk et al., 1990; Eatock and Manley, 1981; Fay
and Ream, 1992; Schermuly and Klinke, 1985; Smolders and
C
18°C
28°C
A B
19°C
24°C
28°C
D
16°C
Latency (ms)
10
15
20
25
30
35
5
Temperature (°C)
30
40
50
60
70
80
90
Threshold (dB SPL)
5100152520
Frequency (kHz)
15 20 25 30
15 20 25 30
N=19N=10
30
40
50
60
70
80
90
Threshold (dB SPL)
5100152520
30
40
50
60
70
80
90
Threshold (dB SPL)
N=18
Fig.·6. Effects of body temperature on tuning, sensitivity and latency evaluated from
recordings of the auditory nerve of the cicada T. josei. (A) Averaged and (B) example
tuning curves measured at different temperatures ranging from 16°C to 28°C. There is
a strong effect in the characteristic frequency and sensitivity in the range 3–8
·kHz, but
not at higher frequencies. Error bars indicate the standard deviation. (C) Sensitivity at
the characteristic frequency measured in the 10 cicadas, from 19 recordings (see
Table
·2), which exhibited increased sensitivity with temperature. The lines connect the
sensitivities evaluated from each auditory nerve recording. (D) Dependence of latency
on temperature measured in 19 cicadas. Latencies were measured 20
·dB above threshold
and decreased with increasing temperature.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1842
Klinke, 1984) and in the locust that, to our knowledge, is the
only report in insects (Oldfield, 1988).
Frequency analysis may be performed by extrinsic
mechanisms, prior to the receptor cell transduction. This is
currently accepted in insects, where the mechanics of the
tympanal structures is considered to be responsible for
frequency analysis [e.g. crickets (Ball and Hill, 1978;
Kleindienst et al., 1983), locusts (Michelsen, 1971) (but see
Windmill et al., 2005)], and is recognized as the primary
mechanism in mammals, where the place of maximal vibration
at the basilar membrane varies systematically with sound
frequency, although here complemented by cellular
amplification (Dallos, 1992; Kennedy et al., 2005). In other
systems frequency analysis can be performed by intrinsic
receptor mechanisms [e.g. electrical tuning (Fettiplace, 1987;
Fuchs et al., 1988; Hudspeth and Lewis, 1988)].
Mechanical properties of the tympanum (tympanal ridge and
apodeme) are likely important components of the frequency
selectivity of the cicada auditory receptors. However,
measurements on those structures by laser vibrometry showed
no significant vibration dependence on temperature in the low
frequency range up to 15
·kHz (Fig.·7). Hence, it seems that the
mechanical properties of the peripheral structures do not
explain the effects of temperature on the frequency selectivity
of the receptors in T. josei, observed in the range 3–8
·kHz.
Studies performed in the turtle, where measurements of
basilar membrane motion using laser interferometry revealed
no position-dependent mechanical tuning (O’Neill and
Bearden, 1995), indicated electrical tuning of the receptors
P. J. Fonseca and T. Correia
(hair cells) as the prime mechanism for frequency
selectivity. By contrast, in the mammalian cochlea,
filtering is primarily performed by mechanical
properties of the basilar membrane, enhanced by
electromechanical amplification mechanisms of the
outer hair cells (Dallos, 1992) and possibly the
stereocillia bundles (Kennedy et al., 2005). In the
electrical tuning of receptor cells, first described for
the turtle auditory papilla, the filter is totally intrinsic
to the hair cell, where the receptor potential is
modulated by voltage-dependent ionic currents that
generate a series of damped oscillations of the
membrane potential. If the sound frequency of the
stimulus matches the resonant oscillations of the
membrane potential an amplified response will arise
(Fettiplace, 1987). This electrical tuning, observed in
hair cells of a hearing specialist fish (Sugihara and
Furukawa, 1989), amphibians (Hudspeth and Lewis,
1988), reptiles (Fettiplace, 1987) and birds (Fuchs et
al., 1988), might also be present in insect receptor
cells (Oldfield, 1984; Oldfield, 1988).
Because higher temperatures increase molecular
motion and speed of chemical reactions, we would
expect higher temperatures to increase the neurons’
excitable state. Indeed, kinetics of ion channels has
for a long time been known to be highly temperature
sensitive (Hodgkin et al., 1952). Moreover,
characteristics of BK channels were recognized as the rate-
limiting step for determining the frequency of electrical tuning.
BK channels, which belong to the potassium channel family,
are large conductance voltage and Ca
2+
-activated potassium
channels whose activity is regulated by membrane voltage
and/or intracellular Ca
2+
. Higher frequency tuning was
accompanied by an increase in the number and speed of the BK
channel kinetics (Fettiplace and Fuchs, 1999). Therefore, if
higher temperatures enhance the ion channel kinetics, increased
Point of LDV
measurement
Tympanum
Auditory organ
Tympanal
apodeme
–180
–90
0
90
180
–40
–20
0
16°C
30°C
–30
–10
10
Vibration velocity (dB)Phase angle (deg.)
5100152520
Frequency (kHz)
Fig.·7. The typical effect of body temperature, in one of three males measured,
on the vibrations of the tympanal apodeme measured by laser Doppler vibrometry
(LDV). There is no clear effect on the vibration velocity (presented in arbitrary
units) and the phase angle, especially in the frequency range 3–8
·kHz, where a
strong effect of body temperature on auditory tuning and sensitivity was measured
in the nervous system. The diagram on the right is of a the tympanum, tympanal
apodeme and auditory organ, indicating the point where the laser beam was
focused.
Table·3. Characteristic frequency evaluated from auditory
nerve recordings of ten cicadas kept at room temperature
(24–28°C) treated with 200·mmol·l
–1
tetraethylammonium
Frequency
Animal Before TEA TEA (200·mmol·l
–1
) Washed
tj3 6 3 6
tj5 5 3/4 5
tj10 6 4 6
tj11 5 4 6
tj12 6 4 4/6
tj13 4 3 6
tj14 6 3 4
tj15 6 4 6
tj18 4 4 6
tj20 6 4 6
Frequency values are in kHz.
TEA, tetraethylammonium.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1843Effects of temperature on cicada hearing
frequency at the electrical oscillation of the membrane potential
will arise and thus will result on an upward shift of the tuning
of the cell. Furthermore, the electrical resonance of the hair
cells was shown to be highly temperature sensitive in the
leopard frog saccular hair cells (Smotherman and Narins, 1998)
and in the chick cochlea (Fuchs and Evans, 1990). If a similar
mechanism is present in T. josei, it might explain the
temperature effects observed at low frequency (3–8·kHz),
despite the fact that electrical tuning has been restricted to even
lower frequencies. At high frequency (14–24·kHz) this
mechanism is unlikely since it would require too high
frequency oscillations of the membrane potential.
In order to assess the effects of potassium ion channels on
hearing of T. josei, we used 200·mmol·l
–1
tetraethylammonium
(TEA) to block potassium channels. This drug caused a shift
in tuning similar to that caused by temperature variations
(Fig.·8, compare with Fig.·6A,B; see also Fig.·4A,B). Thus,
TEA and temperature are likely to have affected tuning
mechanisms in the same way. Low temperature and TEA might
have affected the kinetics or number of active potassium
channels available. Hence, it is likely that those channels play
an important role in frequency selectivity in the auditory
receptors of T. josei.
Because higher temperatures make the neuron more
excitable we would expect that sensitivity would increase with
temperature. Accordingly, the interneurons’ auditory
thresholds decreased with increasing temperature at low
frequency (3–8·kHz) but kept constant at very low (0.5–2·kHz)
and high frequencies (14–24·kHz). Again, this effect was
already present at the level of the receptors. Unexpectedly, in
three cells a decrease in sensitivity occurred at the highest
temperature tested. This might be explained by a
hyperpolarizing mechanism, similar to the one present in
crayfish motoneurons, caused by an excess of sodium
extrusion at high temperatures [Aréchiga and Cerbón
(Aréchiga and Cerbón, 1981) in Smolders and Klinke
(Smolders and Klinke, 1984)]. In the locust auditory
receptors most of the cells recorded maintained their
sensitivity relatively constant, in contrast to 20% of
them that increased their sensitivity (Oldfield, 1988).
Our recordings on T. josei revealed that the very low
(0.5–2·kHz) and high (14–24·kHz) frequency ranges
seemed to be less affected by temperature. If receptor
cells are tuned in to those frequency ranges, they
should remain relatively temperature insensitive as
well. If so, it would indicate different tuning
mechanisms among the receptor cells. Unfortunately
it has not yet been possible to confirm this hypothesis
in cicadas.
TEA affected sensitivity in the same way as a
reduction in temperature, causing an increase in the
threshold of the auditory nerve responses. This
suggests that blocking of potassium channels
interferes with the transduction mechanisms involved
in the generation of the receptor potential.
In T. josei some recordings of a single preparation
showed differences in sensitivity at the same temperature (up
to 30·dB). Thus, some oscillations of the auditory threshold
were temperature independent. Variations in sensitivity might
arise from several causes. Hennig et al. (Hennig et al., 1994)
discovered that the folding of the tympanum that occurred
during singing caused an increase in auditory thresholds by
about 20·dB. Cicadas are thus able to adjust their hearing
threshold within this range. De-tension accompanied by
folding of the tympanum occurs when the cicadas prepare to
sing and is probably a mechanism to protect the tympanum
and the auditory receptors from damages that might be caused
by the high pressures created in the abdomen during singing.
This state might affect measurements but is normally short,
unless the cicada starts singing (P.J.F., unpublished
observations). This condition could be easily detected in our
recording of the auditory nerve that includes the axon of the
tymbal motoneuron. In addition, the sensitivity of a sensory
system can usually be raised or lowered by efferent neuronal
connections, which may intervene at various sites in the
sensory system (Reichert, 1992) and might also be present in
cicadas. Furthermore, insects’ tympanal membrane vibrations
may be affected by ventilation and abdominal movements in
such a way that sensitivity to external sounds can be reduced
(Meyer and Elsner, 1995). These effects, however, are
rhythmic and usually do not last long enough to interfere
markedly with a stimulation series used in
electrophysiological experiments.
In contrast to the weak effect of TEA on latency, temperature
caused a decrease in the response delay of interneurons and
auditory receptors. Notice that temperature strongly affected
the conduction velocity of the neurons, an important
component of the time lag measured. This could be seen in the
distinct decrease of the latency in the response observed at the
Normal
200 mmol l
–1
TEA
Washed
AB
Frequency (kHz)
30
40
50
60
70
80
90
Threshold (dB SPL)
5100152520 5 100152520
N=10
Fig.·8. Effects of 200·mmol·l
–1
tetraethylammonium (TEA) on tuning and
sensitivity evaluated from recordings of the auditory nerve of the cicada T. josei.
(A) Averaged and (B) example tuning curves measured before and after drug
application, and the effect of repeatedly washing with insect saline. TEA resulted
in a downward shift of the characteristic frequency and a reduced sensitivity.
Sensitivity and tuning were reestablished after repeatedly washing the
preparation with insect saline for up to 2
·h 30·min. Error bars in A indicate the
standard deviation. Recordings were made at ambient temperature of 24–28°C.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1844 P. J. Fonseca and T. Correia
auditory nerve and especially at the interneurons, which are
located further away in the auditory pathway, while the
application of TEA did not produce any relevant effect on the
time lag from sound stimulus to the nerve activity. A similar
result was observed in the neostriatal neurons of the rat brain
(Bargas et al., 1989). However, TEA affected sensitivity,
causing an increase in the threshold of the auditory nerve
responses. This might be due to a blocking of potassium
channels involved in the generation of the receptor potential,
while the effect on the speed of axonal conduction, mostly
dependent on sodium channel excitability, was probably
largely unaffected by this chemical but strongly modified by
temperature.
Finally, in T. josei the tuning shift caused by temperature
might generate a mismatch between relevant behavioral stimuli
and the characteristic frequency in the 3–8
·kHz range.
Nevertheless, this should not cause any real constraint on this
communication system because, (1) insects typically
communicate at signal-to-noise levels well above thresholds,
(2) in this cicada the calling song peak is at a much higher
frequency (16
·kHz) and (3) singing usually occurs at
temperatures above 22°C.
Much work has already been performed, in lower vertebrates
and insects, in order to identify the mechanisms responsible for
tuning of auditory receptors. From our experiments we can
conclude not only that the tuning of the auditory receptors is
temperature dependent but also that the potassium channels are
likely implicated in the tuning of receptor cells in this cicada,
at least in the frequency range 3–6
·kHz. However, further
studies on the transduction mechanisms and on the
characterization of the channels at the auditory receptors are
needed to clarify their role in this system.
We thank Axel Michelsen, Matthias Hennig and two
anonymous referees for their comments on an earlier version
of the manuscript. We are thankful to Axel Michelsen for his
help in obtaining the laser vibrometry measurements and to
Ana Isabel Santos for providing support with the TEA
experiments. To Manuel do Carmo Gomes and Jorge Cadima
for his advice with statistical analysis and Jorge Maia Alves
for his help with the set up for temperature control. Fundação
para a Ciência e Tecnologia and Fundação Caloust
Gulbenkian supported this work.
References
Amoser, S. and Ladich, F. (2006). Year-round changes of ambient noise in
freshwater habitats and temperature effects on hearing in fishes. J. Acoust.
Soc. Am. 120, 3056.
Ball, E. E. and Hill, K. G. (1978). Functional development of the auditory
system of the cricket, Teleogryllus commodus. J. Comp. Physiol. 137, 27-
38.
Bargas, J., Galarraga, E. and Aceves, J. (1989). An early outward conductive
modulates the firing latency and frequency of neostriatal neurons of the rat
brain. Exp. Brain Res. 75, 146-156.
Dallos, P. (1992). The active cochlea. J. Neurosci. 12, 4575-4585.
Daws, A. G., Hennig, R. M. and Young, D. (1997). Phonotaxis in the cicadas
Cystosoma saundersii and Cyclochila australasiae. Bioacoustics 7, 173-188.
Dijk, P., van Lewis, E. R. and Wit, H. P. (1990). Temperature effects on
auditory nerve fiber response in the American bullfrog. Hear. Res. 44, 231-
240.
Doolan, J. M. and Young, D. (1981). The organization of the auditory organ
of the bladder cicada, Cystosoma saundersii. Philos. Trans. R. Soc. Lond. B
Biol. Sci. 291, 525-540.
Eatock, R. A. and Manley, G. A. (1981). Auditory nerve fibre activity in the
Tokay Gecko. II. Temperature effect on tuning. J. Comp. Physiol. 142, 219-
226.
Fay, R. R. and Ream, T. J. (1992). The effects of temperature change and
transient hypoxia on auditory nerve fibers response in the goldfish
(Carassius auratus). Hear. Res. 58, 9-18.
Fettiplace, R. (1987). Electrical tuning of hair cells in the inner ear. Trends
Neurosci. 10, 421-425.
Fettiplace, R. and Fuchs, P. A. (1999). Mechanisms of hair cell tuning. Annu.
Rev. Physiol. 61, 809-834.
Fonseca, P. J. (1993). Directional hearing of a cicada: biophysical aspects. J.
Comp. Physiol. 172, 767-774.
Fonseca, P. J. (1994). Acoustic communication in cicadas (Homoptera,
Cicadoidea): sound production and sound reception. Ph.D. thesis, University
of Lisbon, Portugal.
Fonseca, P. J. and Hennig, R. M. (2004). Directional characteristics of the
auditory system of cicadas: is the sound producing tymbal an integral part
of directional hearing? Physiol. Entomol. 29, 400-408.
Fonseca, P. J. and Popov, A. V. (1997). Directionality of the tympanal
vibrations in a cicada: a biophysical analysis. J. Comp. Physiol. A 180, 417-
427.
Fonseca, P. J. and Revez, M. A. (2002a). Song discrimination by male cicadas
Cicada barbara lusitanica (Homoptera, Cicadidae). J. Exp. Biol. 205, 1285-
1292.
Fonseca, P. J. and Revez, M. A. (2002b). Temperature dependence of cicada
songs (Homoptera, Cicadoidea). J. Comp. Physiol. A 187
, 971-976.
Fonseca, P. J., Munch, D. and Hennig, R. M. (2000). How cicadas interpret
acoustic signals. Nature 405, 297-298.
Fuchs, P. A. and Evans, M. G. (1990). Potassium currents in hair cells isolated
from the cochlea of the chick. J. Physiol. Lond. 429, 529-551.
Fuchs, P. A., Nagai, T. and Evans, M. G. (1988). Electrical tuning in hair
cells isolated from the chick cochlea. J. Neurosci. 8, 2460-2467.
Gummer, A. W. and Klinke, R. (1983). Influence of temperature on tuning
of primary-like units in the guinea pig cochlear nucleus. Hear. Res. 12, 367-
380.
Hennig, R. M., Weber, T., Huber, F., Kleindienst, H. U., Moore, T. E. and
Popov, A. V. (1994). Auditory threshold change in singing cicadas. J. Exp.
Biol. 187, 45-55.
Hodgkin, A. L., Huxley, A. F. and Katz, B. (1952). Measurements of current
voltage relationships in the membrane of the giant axon of Loligo. J. Physiol.
Lond. 116, 424-448.
Huber, F. (1984). The world of insects: periodical cicadas and their behavior.
Alexander von Humboldt Mittl. 43, 24-31.
Huber, F., Kleindienst, H. U., Moore, T. E., Schildberger, K. and Weber,
T. (1990). Acoustic communication in periodical cicadas: neuronal
responses to songs of sympatric species. In Sensory Systems and
Communication in Arthropods (ed. F. G. Gribakin, K. Wiese and A. V.
Popov), pp. 217-228. Basel: Birkhäuser Verlag.
Hudspeth, A. J. and Lewis, R. S. (1988). A model for electrical resonance
and frequency tuning in saccular hair cells of the bull-frog, Rana
catesbeiana. J. Physiol. 400, 275-297.
Kennedy, H. J., Crawford, A. C. and Fettiplace, R. (2005). Force generation
by mammalian hair bundles supports a role in cochlear amplification. Nature
433, 880-883.
Kleindienst, H., Wohlerds, D. W. and Larsen, O. L. (1983). Tympanal
membrane motion is necessary for hearing in crickets. J. Comp. Physiol.
151, 397-400.
Meyer, J. and Elsner, N. (1995). How respiration affects auditory sensitivity
in the grasshopper Chorthippus biguttulus (L.). J. Comp. Physiol.
A 176,
563-573.
Michel, K. (1975). Das tympanalorgan von Cicada orni L. (Cicadina,
Homoptera). Eine licht- und elektronenmikroskopische untersuchung.
Zoomorphologie 82, 63-78.
Michelsen, A. (1971). The physiology of the locust ear. II. Frequency
discrimination based upon resonances in the tympanum. Z. Vergl. Physiol.
71, 63-101.
Oldfield, B. P. (1984). The role of the tympanal membranes in the tuning of
auditory receptors in the tettigoniidae (Orthoptera: Ensifera). J. Exp. Biol.
116, 493-497.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

1845Effects of temperature on cicada hearing
Oldfield, B. P. (1988). The effect of temperature on tuning and physiology of
insect auditory receptors. Hear. Res. 35, 151-158.
O’Neill, M. P. and Bearden, A. (1995). Laser feedback measurements of
turtle basilar membrane motion using direct reflection. Hear. Res. 84, 125-
138.
Reichert, H. (1992). Introduction to Neurobiology. New York: Oxford
University Press.
Sanborn, A. F., Heath, J. E. and Heath, M. S. (1992). Thermoregulation and
evaporative cooling in the cicada Okanagodes gracilis (Homoptera:
Cicadidae). Comp. Biochem. Physiol. 102A, 751-757.
Schermuly, L. and Klinke, R. (1985). Change of characteristic frequency of
pigeon primary auditory afferents with temperature. J. Comp. Physiol. A
156, 209-211.
Smolders, J. W. T. and Klinke, R. (1984). Effects of temperature on the
properties of primary auditory fibres of the spectacled caiman, Caiman
crocodilus (L.). J. Comp. Physiol. 155, 19-30.
Smotherman, M. S. and Narins, P. M. (1998). Effect of temperature on
electrical resonance in Leopard Frog saccular hair cells. J. Neurophysiol. 79,
312-321.
Stumpner, A. and von Helversen, D. (2001). Evolution and function of
auditory systems in insects. Naturwissenschaften 88, 159-170.
Sueur, J. and Aubin, T. (2002). Acoustic communication in the palearctic red
cicada Tibicina haematodes: chorus organization, calling song structure, and
signal recognition. Can. J. Zool. 80, 126-136.
Sugihara, I. and Furukawa, T. (1989). Morphological and functional aspects
of two different types of hair cells in the goldfish sacculus. J. Neurophysiol.
62, 1330-1343.
von Helversen, O. and von Helversen, D. (1994). Forces driving coevolution
of song and song recognition in grasshoppers. In Neural Basis of Behavioral
Adaptations (ed. K. Schildberger and N. Elsner), pp. 253-284. Stuttgart:
Fischer.
Windmill, J. F. C., Göpfert, M. C. and Robert, D. (2005). Tympanal
traveling waves in migratory locusts. J. Exp. Biol. 208, 157-168.
Zar, J. H. (1999). Biostatistical Analysis. New Jersey: Prentice Hall.
THE JOURNAL OF EXPERIMENTAL BIOLOGY