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Time-frequency composition of mosquito flight tones obtained using Hilbert

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Article  in  The Journal of the Acoustical Society of America · October 2014

DOI: 10.1121/1.4895689 · Source: PubMed

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Time-frequency composition of mosquito flight tones obtained
using Hilbert spectral analysis
Andrew Aldersley
Bristol Centre for Complexity Sciences, University of Bristol, Queen’s Building, University Walk,
Bristol BS8 1TR, United Kingdom
Alan Champneysa) and Martin Homer
Department of Engineering Mathematics, University of Bristol, Merchant Venturers Building,
Woodland Road, Bristol BS8 1UB, United Kingdom
Daniel Robert
School of Biological Sciences, University of Bristol, Life Sciences Building, 24 Tyndall Avenue,
Bristol BS8 1TQ, United Kingdom

(Received 20 February 2014; revised 23 July 2014; accepted 25 August 2014)

Techniques for estimating temporal variation in the frequency content of acoustic tones based on
short-time fast Fourier transforms are fundamentally limited by an inherent time-frequency trade-
off. This paper presents an alternative methodology, based on Hilbert spectral analysis, which is
not affected by this weakness, and applies it to the accurate estimation of mosquito wing beat fre-
quencies. Mosquitoes are known to communicate with one another via the sounds generated by
their flapping wings. Active frequency modulation between pairs of mosquitoes is thought to take
place as a precursor to courtship. Studying the acoustically-based interactions of mosquitoes there-
fore relies on an accurate representation of flight frequency as a time-evolving property, yet con-
ventional Fourier spectrograms are unable to capture the rapid modulations in frequency that
mosquito flight tones exhibit. The algorithms introduced in this paper are able to automatically
detect and extract fully temporally resolved frequency information from audio recordings.
Application of the technique to experimental recordings of single tethered mosquitoes in flight
reveals corroboration with previous reported findings. The advantages of the method for animal
communication studies are discussed, with particular attention given to its potential utility for
studying pairwise mosquito interactions. VC 2014 Acoustical Society of America.

[http://dx.doi.org/10.1121/1.4895689]
PACS number(s): 43.80.Ka, 43.60.Hj, 43.80.Ev [ANP] Pages: 1982–1989

I. INTRODUCTION at least several species of mosquito (Gibson and Russell,

2006; Cator et al., 2009, 2010; Cator and Harrington, 2011;
As a prominent vector of numerous viruses and para-
Warren et al., 2009; Pennetier et al., 2010). Audio record-
sites, mosquitoes pose a major health risk to millions of indi-
ings of tethered pairs of mosquitoes have revealed the exis-
viduals across the globe. Disease is transmitted when the
tence of a dynamic interaction between the two. Males and
female mosquito bites her target to obtain a blood meal, a
females tend to reduce the difference between their flight fre-
protein supply vital to egg development. While the relation-
quencies prior to contact on the wing, a phenomenon that
ship between the mosquito reproductive cycle and their pro-
can occur at harmonic components beyond the fundamental
pensity to spread disease is well established, there remain
flight tone.
large gaps in our knowledge of the mating ecology of these
Despite significant growth in the mosquito bioacoustics
insects. Understanding mosquito breeding behaviors is a
literature, there remain aspects of their frequency-based
vital component of any targeted population control strategy
interactions that have not been consistently described or
proposed to mitigate the effects of mosquito-borne illnesses
quantified with sufficient detail. This can partly be attributed
on humans (Howell and Knols, 2009; Alphey et al., 2010).
to a lack of numerical and statistical analysis, an issue that
It has long been known that male mosquitoes in the
motivates the research described in this paper.
search for a mate are attracted toward the sounds produced
Information transfer between mosquitoes appears to
by the beating wings of nearby females (Johnston, 1855;
take place both in the frequency and time domains (Jackson
Roth, 1948; Wishart and Riordan, 1959; Belton, 1994;
and Robert, 2006). Consequently, a tool that offers an accu-
G€opfert et al., 1999; Charlwood et al., 2002). Recent
rate time-frequency representation of wing beat content is
ground-breaking behavioral experiments have highlighted
required to accurately study mosquito interactions. Previous
the key role played by audition in not only sexual identifica-
studies have generally extracted frequency information from
tion, but also the courtship rituals and selection practices of
audio recordings of mosquito flight through the use of a
Fourier transform. Breaking up the original input into
a)
Author to whom correspondence should be addressed. Electronic mail: smaller overlapping intervals in the time domain generates a
a.r.champneys@bristol.ac.uk short-time Fourier transform (STFT), and hence the

1982 J. Acoust. Soc. Am. 136 (4), October 2014 0001-4966/2014/136(4)/1982/8/$30.00 C 2014 Acoustical Society of America
V
frequency spectrum for local sections of the data. When
implemented in a spectrogram (the squared modulus of the
STFT), this technique allows for the reconstruction of the
time evolution of the signal’s frequency content.
However, one significant limitation of the STFT is that
it requires a trade-off between frequency resolution (the
scale at which nearby frequencies can be distinguished) and
time resolution (the interval over which frequency changes
can be seen). Using a smaller window to partition the signal
allows for better resolution in the time domain but results in
a poorer frequency resolution. Similarly, a wider frequency
bandwidth necessarily reduces temporal accuracy.
Spectrogram representations of the time-frequency plane are
often “blurred” as a result, and rendered unsuitable for the
detection and analysis of rapid frequency modulations in
bioacoustic signals (DiCecco et al., 2013), including mos-
quito flight tones.
Besides the STFT, there are several alternative techni-
ques able to characterize the evolution of frequency in time.
Hilbert spectral analysis (HSA, Huang et al., 1998), which
yields the instantaneous frequency of a signal, is a particu-
larly useful example of such a method. The application of
HSA is especially effective in the context of mosquito flight
tones, due to its utility in non-stationary systems that display
a highly variable frequency (Boashash, 1992). In this paper
we present a quantitative method for the extraction of fre-
quency content from mosquito wing beat recordings, based
on HSA. Key features of our approach include automated
identification of fundamental flight frequencies and har-
monic overtones, application of time-varying filters to
reduce noise interference, and an algorithm to detect flight
cessation. This methodology achieves a significant improve-
ment over previous studies in both the time and frequency
resolution of mosquito flight tones.
II. DATA ACQUISITION
A. Procedure
Experimental specifications were motivated by similar
previous studies (Gibson and Russell, 2006; Cator et al.,
2009; Warren et al., 2009; Pennetier et al., 2010). A colony
of the species Aedes aegypti was initiated using laboratory
eggs obtained from the London School of Hygiene and
Tropical Medicine. Individual adult mosquitoes were anes-
thetized and fixed to the rounded end of an insect pin on the
dorsal surface of the thorax (a procedure detailed in Cator
et al., 2009), such that the subject was able to beat its wings
and move its head unimpeded.
Mosquitoes were placed in an upright manner to mimic
as closely as possible the natural flight position. Flight tones
were recorded using an electret condenser microphone (FG-
23329-C05 microphone, Knowles Electronics, Itasca, IL)
placed directly underneath the individual at a distance of less
than 1.5 cm (as in Cator et al., 2009; Warren et al., 2009),
beyond the reach of physical contact. Flight was initiated by
gently blowing on the insect, or by stroking its legs. Data
were logged at a sample rate of 40 kHz with the LabVIEW
SignalExpress software package (National Instruments,
Austin, TX), using a National Instruments USB data
J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
acquisition device, for duration of 60 s following the onset of
consistent flight.
Laboratory temperatures were held between 24  C and

28 C for the duration of recordings. Lighting and humidity
conditions were also monitored and controlled. Mosquitoes
were used at the beginning of their night period, running on
a D16:N8 circadian cycle. Each recording was taken in a
sound-proof booth to minimize noise interference from
external sources.
B. Data output
One example of the raw waveform of flight acoustics
recorded from a single mosquito, prior to any processing, is
displayed in Fig. 1. Variation in the flight tone amplitude is
low, indicative of consistent and stable flight.
III. FREQUENCY ANALYSIS METHODS
Resolving the frequency content of mosquito flight
tones requires the isolation of harmonic components fol-
lowed by frequency extraction. The algorithm presented here
is based fundamentally on the notion of instantaneous fre-
quency, a concept which provides excellent resolution in the
time-frequency plane.
A. Instantaneous frequency
The instantaneous frequency, xi ðtÞ, of a signal, xðtÞ,
derives from its Hilbert transform, HðxðtÞÞ. Following
Huang et al. (1998, 2009), the general principle of HSA is as
follows. We first take the Hilbert transform
ð
1 1 xðsÞ
ð ð Þ Þ ð
H x t ¼y t ¼ PÞ ds; (1)
p 1 t  s
where P indicates the Cauchy principal value of the integral.
The Hilbert transform is known to be well defined provided
that the data is Lebesgue integrable, or more specifically that
the time series is a function of class Lp , with 1 < p < 1.
Note that this mild requirement allows for the data to include
discontinuities, spikes, and random noise. This is a notably
FIG. 1. Amplitude profile of a single male mosquito’s flight. Inset depicts
cycle-by-cycle resolution of the waveform over 25 ms. The magnitude of
the amplitude is roughly constant, with little modulation, indicative of stable
flight.
Aldersley et al.: Frequency analysis of mosquito flight tones 1983
useful trait when handling experimentally derived acoustic
signals displaying non-stationary behaviors, as with mos-
quito flight tones. Under this definition, HðxðtÞÞ yields the
imaginary part of the analytic pair, yðtÞ, of the real data, xðtÞ,
so that we have a unique analytic signal given by
zðtÞ ¼ xðtÞ þ jyðtÞ ¼ ai ðtÞejhi ðtÞ ; (2)
where ai ðtÞ and hi ðtÞ describe, respectively, the instantane-
pffiffiffiffiffiffiffi
ous amplitude and phase associated with xðtÞ, and j ¼ 1.
The instantaneous frequency is then defined as the time de-
rivative of hi ðtÞ;
dhi
x i ðt Þ ¼ : (3)
dt
Note that because hi is a function of time, so too is xi .
B. Isolating harmonic frequencies
The notion of an instantaneous frequency is only mean-
ingful when applied to mono-component functions
(Boashash, 1992), that is, data with a narrow bandwidth
(Huang et al., 1998). For multi-component signals, such as
the acoustic tones generated by mosquito flight, we must
therefore filter each of the harmonic frequencies prior to
HSA. We have developed an automated procedure based on
peak-frequency estimation that allows us to identify and iso-
late the various harmonic frequency components contained
within the sound profile of mosquito flight. The approach con-
sists of several stages, described below.
Given a raw acoustic recording, an initial estimation of
its frequency content is performed using Discrete Fourier
analysis. The signal is high-pass filtered (using a 4 order
Butterworth filter) to remove low-frequency components
(300 Hz), which can cause noise interference, prior to
Fourier transformation. From the resultant Fourier spectrum
we identify the peak corresponding to the mosquito’s funda-
mental wing beat frequency, which we find to be the largest
FIG. 2. Fourier spectrum of a lone male mosquito’s flight tone, depicting
frequencies up to 6.5 kHz. The fundamental wing beat frequency is the larg-
est peak, located here at approximately 600 Hz. Harmonic overtones are
clearly resolved above the background noise threshold up to the 9th compo-
nent, with each appearing progressively weaker in amplitude. These data
indicate that each harmonic peak is an integer multiple of the fundamental
frequency.
1984 J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
amplitude component in every case (Fig. 2). The prominence
of this harmonic in the flight tones that we have recorded
lends it well to application in the remainder of the algorithm.
Isolation of the fundamental and harmonic overtones
from the main body of the signal is facilitated by the applica-
tion of a bandpass filter, which is defined by a center-
frequency and bandwidth. We take an automated approach
to the specification and design of these filter parameters (see
Fig. 3). The first step is to approximate the distribution of
the Fourier spectrum about the chosen frequency component.
The spectrum is normalized by dividing through by its stand-
ard deviation (this improves function fitting). We then fit ei-
ther a skew Gaussian or a skew Laplace distribution to the
data; the choice of function is made by considering the kur-
tosis of the spectrum about the given frequency peak. We
find that data with a kurtosis  10 are best approximated by
the skew Gaussian, whereas the skew Laplacian consistently
fits data with a kurtosis >10.
The fitting procedure and subsequent filter parameter
estimation is equivalent whichever distribution is used. The
scale and location parameters of the fitting function are opti-
mized to the data using a least squares approach, recursively
tuning values to minimize error. The peak of the distribution
identifies the center point of the transfer function for the fil-
ter. The bandwidth (i.e., the upper and lower cutoff frequen-
cies) is obtained using the frequencies at full-width, fraction
p% maximum of the fitted distribution (we typically choose
p ¼ 2% for a Gaussian fit, and p ¼ 5% for a Laplacian).
This process is outlined graphically in Fig. 3. The resultant
linear filter is subsequently applied to the audio recording.
The temporal dynamics of mosquito flight frequencies
are inherently non-stationary. With this in mind, an addi-
tional layer of sophistication must be introduced to our filter
process to ensure that it robustly captures wing beat fre-
quency information. In particular, a simple static filter
applied to an entire recording can lead to a sub-optimal fre-
quency response and, for instance, an increase in the noisi-
ness of the resultant instantaneous frequency functions (see
Fig. 4). One way to address this problem is to instead
FIG. 3. An example of parameter estimation for the time-varying filter. In this
instance, a Laplace distribution is fitted to the Fourier spectrum around the
fundamental flight frequency. The filter is defined by a center frequency xmax
and bandwidth xD . xmax is denoted by the peak of the Laplacian, while xD is
given by the frequencies at full-width, 5% maximum of the fitted curve.
Aldersley et al.: Frequency analysis of mosquito flight tones

FIG. 4. Applying a static filter to a non-stationary signal can lead to instabil-
ities in the Hilbert transform, resulting in a noisy response of xi ðtÞ. Using a
time-varying filter we are able to significantly reduce this noise.
Instantaneous frequency is plotted as a time-evolving function under two
different filter regimes. A static linear Butterworth bandpass filter, with pa-
rameters estimated in situ, has been applied to the audio signal, and the in-
stantaneous frequency, extracted via HSA, is plotted in gray. This is
compared with the output from the time-varying filter approach outlined in
Sec. III (black trace).
implement a time-varying filter, enabling us to enforce a
locally optimal bandwidth. In this instance, timescale selec-
tion is of primary importance.
In our procedure, we iterate over a given recording at
increments of time duration sw , and extract local filter pa-
rameters for each segment using the process outlined above.
We generally choose sw ¼ 1 s, a condition that satisfies two
requirements for our data: that the frequency content of the
subset is well preserved, and that frequency over the subset
is unimodally distributed with one dominant component.
Large frequency modulations in mosquito flight tones tend
to occur slowly, especially when the insects are flying alone
(Arthur et al., 2014).
A time-varying filter is specified using a set of one-
dimensional vectors to prescribe the center frequency and
bandwidth, respectively, at each corresponding time instant
in the recorded signal. Thus for each increment sw ¼ 1 s,
there are 4  104 filter points, the sample rate of our data
being 40 kHz. Linear interpolation of the filter parameters is
performed at each boundary sw to ensure a smooth parameter
transition between time steps, and to reduce the occurrence of
instabilities in the frequency response of the filter. The time-
varying filter can be repeatedly applied n times to achieve the
desired order (we choose n ¼ 3 as a suitable compromise
between computational time and noisiness of output).
Our method thus far has primarily focused on isolation
of the fundamental flight frequency. The process is easily
extended, however, to yield higher harmonic components.
Analysis of wing beat frequencies indicates that harmonic
overtones are located at integer multiples of the fundamental
wing beat tone, even during frequency modulation (Arthur
et al., 2014). This allows us to approximate and filter the sig-
nal in the general region of any given harmonic, based on
analysis of the fundamental tone. Once the mono-component
function is resolved, we apply the time-varying filter routine.
For A. aegypti mosquitoes, a minimal useful dataset may be
J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
comprised of the harmonic frequencies up to and including
the male second and female third overtones; convergent
behaviors are observed to take place at or below these fre-
quencies (Cator et al., 2009).
C. Extracting instantaneous frequency
After filtering the original data, we obtain the analytic
signal via the Hilbert transform. Computation of xi ðtÞ fol-
lows from the subsequently derived instantaneous phase
function. Finally, we smooth the output using a Wiener filter
with a window of size 200 data points; this removes low-
amplitude high-frequency instabilities generated by the
time-varying filter while preserving the temporal qualities of
the instantaneous frequency signal. The instantaneous fre-
quency data obtained for each mosquito provide a rich pool
of information that is unbounded by the trade-off between
time and frequency resolution necessarily imposed by
Fourier-based approaches. Our determination of xi ðtÞ is con-
tinuous in frequency space and limited temporally only by
the sampling rate at which the signals were recorded. Such
high-resolution data sets have previously been inaccessible
and largely unused in mosquito bioacoustics (exceptions are
Jackson and Robert, 2006; Jackson et al., 2009).
D. Detecting flight cessation
A frequent observation during experimentation was that
the tethered mosquito would cease flight for a duration last-
ing anything up to several seconds. While such periods
formed a relatively minor part of our total recorded time,
there was nevertheless a strong motivation to develop an
automated method for the removal of such dormant sections
of the data. The main reason for this was to preserve as
much useable data from each recording as possible; Hilbert
transformation of background noise contained in a recording
(i.e., when there is no mosquito flight) can lead to distortions
in future analysis.
Flight cessation is typified by a rapid—and sometimes
erratic—decrease in wing beat frequency and amplitude,
while the first moments of flight, often referred to as the
“startle” period (Gibson and Russell, 2006; Cator et al.,
2010), constitute high frequency and amplitude. Therefore,
to detect when a mosquito has stopped flying, we use the am-
plitude of the waveform of the recorded flight.
Similar to the process for estimating filter parameters,
the audio signal from a given experimental trial is bandpass
filtered to remove frequencies outside the expected range of
the tethered male/female fundamental flight tone (typically
those below 300 Hz and above 1100 Hz for A. aegypti) [Fig.
5(a)]. We then twice smooth the modulus of the resultant
signal using a Gaussian-weighted moving-average (effec-
tively applying a low-pass finite impulse response filter)
[Fig. 5(b)]. Taking the gradient of this smoothed function
allows us to identify points of rapid amplitude decline or
increase using a peak-finding algorithm [Fig. 5(c)]. Iterating
over maxima pairwise, we extract those punctuated by peri-
ods with a median smoothed magnitude less than Ac . A value
Ac ¼ 0:01 V consistently indicated flight cessation through-
out our data. This mechanism enabled us to remove silent
Aldersley et al.: Frequency analysis of mosquito flight tones 1985

FIG. 5. Automated detection and removal of intra-recording flight cessation.
(a) Raw (gray) and low-pass filtered (black) waveform. (b) Modulus (gray)
and twice-smoothed modulus (black) of the filtered signal. (c) Identification
of silent components, based on peak detection in the first-time derivative of
the smoothed function in (b). (d) Removal of the identified silent elements
from the xi ðtÞ trace.
components of the time-frequency representations for each
mosquito’s flight [Fig. 5(d)]. It avoids excessive pruning of
the audio signal and preserves the maximum amount of use-
able data.
IV. RESULTS
A. Frequency extraction of mosquito flight tones
We recorded individual flight tones of 27 male and 19
female A. aegypti. Frequency extraction using the combined
time-variant filter and HSA approaches presented here was
carried out for each insect, up to the second and third har-
monic frequencies for males and females, respectively.
Visual comparisons with spectrogram-based analyses—a
1986 J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
FIG. 6. Comparing a spectrogram-based frequency representation (gray) of
an individual male mosquito’s flight tone to that of the instantaneous fre-
quency (black) demonstrates superior resolution in the time and frequency
domains for the latter approach. Rapid frequency modulations are captured
in excellent detail, providing a much clearer visualization of local mosquito
wing beat frequency behaviors. Note that the time-axis is extended either
side of the instantaneous frequency data merely to enable adequate presenta-
tion and comparison to the spectrogram image.
sample of which are shown in Fig. 6—were made to confirm
accuracy of the resultant time-frequency representation.
During recordings some mosquitoes were observed to
adopt an erratic flight position, possibly in an attempt to free
themselves from the tether. This behavior produced a “saw-
tooth” pattern in the wing beat frequency trace, as noted by
Warren et al. (2009). Data sets in which this trait was
observed were pruned such that only periods of consistent,
“stable” flight, with a minimum duration of 20 s, were
included in the analysis.
B. Properties of mosquito flight
Our approach to frequency extraction yields a one-
dimensional time series—xi ðtÞ—for each harmonic compo-
nent with a high temporal resolution, limited only by the
sample rate of the original audio recordings. These data lend
themselves well to a thorough analysis of the properties of
male and female flight tone characteristics, an example of
which we now present.
As has been documented for A. aegypti (Clements, 1999;
G€opfert et al., 1999; Cator et al., 2009; Arthur et al., 2014),
we observed tethered males to fly at a higher fundamental fre-
quency than females. Average flight frequency ranged from
492.1 to 880.3 Hz across the different males recorded, with an
overall mean and standard deviation of 691.1 6 90.4 Hz. For
females, the average individual flight frequency was in the
interval 415.9 to 532.6 Hz, with a group-wide mean and stand-
ard deviation of 480.6 6 32.5 Hz. As in Arthur et al. (2014),
the variance in wing beat frequency was much lower for indi-
vidual mosquitoes than across the totality of data collected.
For males, the mean intra-recording standard deviation was
11.2 Hz, while for females it was 8.9 Hz. The smaller spread in
lone female flight tones is a result of their naturally lower wing
beat frequency.
The non-stationary and time-variant properties of mos-
quito flight tones can cause the usefulness of applied
Aldersley et al.: Frequency analysis of mosquito flight tones
“global” statistics to be somewhat diminished. To illustrate
any potential differences between male and female flight
characteristics it may be more informative to partition the
data into smaller, approximately-stationary subsets. Using
the fundamental flight component, we determine the array
( )
j xi  l  j ðsÞ
yi ¼ (4)
 j ðsÞ
l
i¼1;…;n
for the mosquito j, where xi represents the instantaneous fre-
quency at a given time instant, and l  j ðsÞ is the piecewise
moving mean taken at time intervals of s, evaluated over n
consecutive data points. We select s ¼ 1 second, giving
n ¼ 4  104 .
For each mosquito, yji represents the fractional deviation
from the local mean flight tone, standardized to account for
differences between individuals and the non-stationary fea-
tures of wing beat frequency. It enables us to aggregate flight
information from each recording across the male and female
groups. Gender specific “meta” time series are generated
separately, by combining yji for j ¼ 1; …; 15 mosquitoes,
selected at random from within our experimental database.
Figure 7 shows the probability density functions (PDFs) of
the aggregate yji for males and females in solo flight. The dis-
tributions are well approximated by Gaussian functions.
While broadly similar, there is a notable difference between
the sexes: male mosquitoes generally deviate more from
FIG. 7. PDFs showing the deviation from the mean fundamental flight tone
for a random selection (N ¼ 15) of single tethered (a) male and (b) female
mosquitoes, illustrated using gray bars. Black lines indicate subsequently fit-
ted Gaussian distributions.
J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
FIG. 8. Cumulative distribution functions (CDFs) for the aggregated male
and female wing beat frequency deviations presented in Fig. 7. Male mos-
quitoes exhibit a broader range of flight tone deviations, as evidenced by the
shallower slope of the CDF, when compared to females.
their local mean flight frequency than their female counter-
parts (Fig. 8).
V. DISCUSSION
We have developed a novel method for extracting flight
frequencies from acoustic recordings of tethered mosquitoes.
Our technique involves several steps, including frequency
estimation using Fourier transformation, the application of a
time-varying bandpass filter to isolate harmonic overtones,
and computation of instantaneous frequency content utilizing
the concepts of HSA. We also report on the design of an
algorithm able to detect and remove sections of data in
which mosquitoes cease to fly, which can be problematic
from an instantaneous frequency perspective.
The signal processing tools described are fully auto-
mated and have several advantages over Fourier-based meth-
ods that have typically been used in the study of mosquito
interactions in the frequency domain (Gibson and Russell,
2006; Cator et al., 2009; Warren et al., 2009; Pennetier
et al., 2010). The primary benefit is the absence of a trade-
off between temporal and frequency resolution, which ulti-
mately leads to a higher quantity and quality of wing beat
frequency data. Our technique offers significant improve-
ments on both these of fronts (Fig. 6), and, crucially, is able
to capture the rapid frequency modulations during mosquito
flight that are unclear in previous visualizations.
Spectrogram representations of frequency yield a data
matrix of frequency “power” as a time-evolving function.
Obtaining an accurate estimation of the largest amplitude
frequency components within the matrix at any given time
(the fundamental frequency and harmonic overtones of a
mosquito’s flight) requires the use of feature extraction tech-
niques, which may be limited by resolution in the time and/
or frequency domain. The procedure presented here, how-
ever, returns a readily available set of one-dimensional time
series (one for each harmonic component), negating the need
for further manipulation of the time-frequency plane. These
data facilitate subsequent analyses that go beyond simple
time series plots (for example, see Figs. 7 and 8) and allow
Aldersley et al.: Frequency analysis of mosquito flight tones 1987
us to explore further the nature of frequency as a property of
flight.
We recorded the flight tones of tethered lone male and
female A. aegypti mosquitoes for a duration up to 60 s. Male
and female wing beat frequencies obtained via HSA are stat-
istically consistent with those reported elsewhere for this
species, both in terms of global mean flight frequency and its
spread across individuals (G€opfert et al., 1999; Cator et al.,
2009; Arthur et al., 2014), indicating that our method does
indeed accurately capture the true frequency content of the
mosquito flight trace. Furthermore, by partitioning the data
into smaller short-time subsets, we have been able to account
for the inherent non-stationarity of mosquito flight. Our find-
ings reveal that wing beat frequencies are normally distrib-
uted about a local mean value. Male mosquitoes, however,
tend to deviate more from this mean than their female
conspecifics.
The significance of this qualitative difference between
male and female flight tone characteristics is unclear, yet
analyses of pairwise mosquito interactions point to wing beat
frequency as a critical factor in species and gender identifica-
tion (Gibson and Russell, 2006; Cator et al., 2009; Warren
et al., 2009; Pennetier et al., 2010), as well as in sexual selec-
tion (Cator et al., 2010; Cator and Harrington, 2011). Despite
this, there is a lack of quantitative investigation in this field,
and consequently the ubiquity and accuracy of the described
phenomena remain uncertain. For instance, during harmonic
convergence (Cator et al., 2009) between males and females,
how close do their flight tones have to be to result in a suc-
cessful duet? And what information do the more detailed and
dynamic properties of wing beat signals, such as high-
frequency tonal modulations and local variance, convey to
other individuals? In order to answer these questions, and
thus more comprehensively investigate interactions between
mosquitoes, a detailed realization of frequency as a time-
dependent property is required.
Arthur et al. (2014) recently presented two tools to sepa-
rate simultaneous flight tones during convergence behavior.
The first of these is based on the phase-derivative of spectral
maxima in the spectrogram, while the second uses polyspec-
tra to enhance time-frequency resolution. Despite offering
greater accuracy than the coarsely represented spectrogram,
these approaches are still inherently limited by similar fac-
tors. By taking an instantaneous frequency approach to the
extraction of mosquito flight tone data, we have demon-
strated a far superior clarity in time and frequency compared
to any method used to date in the mosquito bioacoustics lit-
erature. As illustrated in Fig. 9, convergence of flight tones
now has the potential to be studied in much greater quantita-
tive depth, owing to the quality of data available.
Experimentally, resolution of nearby harmonic frequencies
from different mosquitoes can be achieved by recording
each individual on a separate digital channel. This is in con-
trast to previous research, which has tended to log the duet
on a single microphone positioned equidistant from each
mosquito (Gibson and Russell, 2006; Cator et al., 2009;
Warren et al., 2009; Pennetier et al., 2010). In these studies
it is somewhat unclear how overlapping signals from unique
sources (observed during harmonic convergence) were
1988 J. Acoust. Soc. Am., Vol. 136, No. 4, October 2014
FIG. 9. (Color online) When positioned close together (around 2 cm apart),
male and female Aedes aegypti display convergence of their flight tones.
This behavior commonly takes place at the male 2 and female 3 harmonic
frequency components. When viewed using a spectrogram, resolution is
coarse and it is difficult to accurately prescribe signal ownership. Using the
instantaneous frequency representation of flight tone, resolution in the time-
frequency plane is significantly enhanced. Recording mosquitoes on sepa-
rate channels also allows us to assign the male (M) and female (F) flight
tones with precision. As in Fig. 6, the time-axis displays both the spectro-
gram alone (0–30 s), and the spectrogram overlaid with the instantaneous
frequencies of each mosquito (30–60 s). This allows thorough inspection
and clear comparison between the two methods.
assigned to particular individuals using spectrogram analy-
sis. Our method is able to unambiguously ascribe a given
xi ðtÞ function to its producer (Fig. 9).
The study of acoustic communication between mosqui-
toes, and its broader role in reproductive processes, is central
to our understanding of their behavioral ecology. It is hoped
that the analysis presented here will stimulate further investi-
gations into the nature of frequency-based interactions
between these insects.
The characterization of nonlinear and non-stationary
bioacoustic signals using HSA has arguably been underutil-
ized in the literature. Exceptions include the study of whale
clicks (Adam, 2006) and bat echolocation calls (DiCecco
et al., 2013). One possible reason for this under-utilization is
that one has to first isolate a narrow band around the fre-
quency of interest in the signal before applying the Hilbert
transform. The central frequency and width of the band is
likely to vary from species to species and between individu-
als of the same species. Thus, unlike the STFT there has,
until now, been no black-box method that can be easily
applied to different biological systems. In this work, we
have shown how to construct a highly optimized tool for the
extraction of frequency content contained within mosquito
flight tones. In principle this same methodology can be
adapted to work for many other bioacoustic communication
studies involving non-stationary harmonic functions.
ACKNOWLEDGMENTS
This work was funded by the Engineering and Physical
Sciences Research Council (EPSRC), Grant No. EP/
E501214/1. D.R. is supported by the Royal Society London.
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