Research Article

Journal of Orthoptera Research 2017, 26(1): 21-27

Phenotypic plasticity in color without molt in adult grasshoppers of the genus
Sphingonotus (Acrididae: Oedipodinae)

JUAN RAMON PERALTA-RINCON!, GRACIELA ESCUDERO!, Pim EDELAAR!

1 Universidad Pablo de Olavide, Seville, Spain.

Corresponding author: Pim Edelaar (edelaar@upo.es)

Academic editor: Corinna Bazelet | Received 19 December 2016 | Accepted 23 April 2017 | Published 28 June 2017

http://zoobank.org/66D9F013-B62E-48 17-9A58-AA4FCE3A2905

Citation: Peralta-Rincon JR, Escudero G, Edelaar P (2017) Phenotypic plasticity in color without molt in adult grasshoppers of the genus Sphingonotus
(Acrididae: Oedipodinae). Journal of Orthoptera Research 26(1): 21-27. https://doi.org/10.3897/jor.26.14550

Abstract

Homochromy (i.e. that individuals have a similar color as their envi-
ronment) is frequent in grasshoppers, and probably functions to reduce
detection by potential predators. Nymphs of several soil-perching grass-
hopper species are known to show color changes during development that
increase homochromy, with color being determined with each molt. While
this is well documented for young individuals, the only color change in re-
sponse to the environment that has been recorded for adult grasshoppers
of these species is an overall darkening of the individual when exposed
to dark surfaces. Whether grasshoppers can also adaptively change color
hue is relevant for our understanding of the evolution of locally adapted
crypsis. We therefore exposed two groups of adult grasshoppers to a bluish-
gray substrate or a reddish-brown substrate, and recorded their color over
time. Quantitative digital image analysis showed that adult soil-perching
grasshoppers remained capable of adapting to changes in the color of
their surroundings through a plastic response. Compared to nymphs, the
changes are not as strong and much slower. We suggest that color change
in adults occurs through the ongoing deposition of melanins, with eu-
melanin making individuals more bluish-gray and pheomelanin making
individuals more reddish-brown. The fact that color change is possible but
slow supports that other mechanisms, such as habitat choice or selective
predation, may also play a role in adapting local populations to substrate
color. In addition, the ability of these grasshoppers to produce different
melanins in response to the environment supports a previous suggestion
that they might be useful in the future development of animal models
to study melanin-related diseases like melanoma and Parkinson’s disease.

Key words

homochromy, crypsis, color change, image analysis, habitat choice,
pheomelanin, eumelanin

Introduction

Crypsis is a well known anti-predation mechanism observed in
a wide variety of species. It is common among plant- and ground-
dwelling insects such as mantises, phasmids, grasshoppers and
bush crickets. Adaptive phenotypic plasticity (the ability of a single
genotype to change its phenotype in res ponse to environment
cues) is often key to optimizing crypsis in animals whose habitat
is heterogeneous through space or time (Umbers et al. 2014,

Valverde and Schielzeth 2015, Kang et al. 2016). For instance, for
grasshoppers four types of color change have been recorded: green-
brown morph switching, color pattern changes, hue shifting and
blackening (Rowell 1972). While color pattern has often been found
to be determined mostly by genes and maternal effects (Karlsson
et al. 2009, Forsman 2011, Karpestam et al. 2012b), hue variation
is thought to be driven to a great extent by an adaptive plasticity
response to environmental cues, as the new overall color tends to
match that of the subject's surroundings (Ergene 1955, Yerushalmi
and Pener 2001, Valverde and Schielzeth 2015). There is also
evidence of blackening in response to temperature and exposure to
solar radiation as well as dark substrates (Forsman 2011, Karpestam
et al. 2012a, Valverde and Schielzeth 2015). Green-brown morph
determination and switching remains the less understood of the
four types of color change but there is positive evidence for both
genetic and environmental influences (Rowell 1972, Valverde and
Schielzeth 2015). The environmental cues that are used for adaptive
plasticity to enhance crypsis are often unknown, but effects of
temperature, humidity and/or visual input have been recorded
(Rowell 1972, Umbers et al. 2014, Valverde and Schielzeth 2015).

The family Acrididae (which contains amongst others the
band-winged grasshoppers and locusts) is the most studied group
of grasshoppers. This is partly because it is the largest and most
widespread family, can be responsible for tremendous agricultural
losses, and is used in many countries for human consumption.
They often show homochromy, and those species that perch on
the ground tend to strikingly resemble the color of their local sub-
strate. In his revision of this family’s variable coloration, Rowell
(1972) proposed three non-exclusive causes for this local adap-
tation in color: differential predation, color change, and habitat
selection. He deemed the first one immediately acceptable, and
additive effects of the other two seemed logical to attain an almost
perfect color match. However, studies regarding the color change
in Acrididae grasshoppers have normally been conducted with
nymphs or, at most, recently metamorphosed adults (Valverde
and Schielzeth 2015) while fully developed imagoes, though in-
deed cryptic, have not received as much attention.

Apart from neglect of plasticity in adults, another problem with
most earlier studies is that assessments of color change have been

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

22

done rather subjectively, either assigning individuals to discrete ar-
bitrary color levels or comparing the subject's color to standard
charts which do not allow true quantitative analyses. To overcome
these limitations, when possible, it is preferred to use objective dig-
ital image analysis as a less biased approach (Stevens et al. 2007).

We have used this approach to study the color matching of az-
ure sand grasshoppers, Sphingonotus azurescens (Rambur) (Orthop-
tera: Acrididae: Oedipodinae) colonizing distinctly colored novel
urban habitats in Seville, Spain. While this species naturally occurs
on open sand or clay soils with little to no vegetation, we have
recently found them to locally also use abandoned man-made sur-
faces (sidewalks, bicycle paths, asphalt roads) for perching, feed-
ing, courtship, and reproduction. We found that individuals using
these pavements were consistently more cryptic on their local pave-
ment than on other, adjacent pavements. This shows that these
grasshoppers are able to adapt their color to fine scale environ-
mental variation, even when it involves novel materials (asphalt,
bricks, tiles) that historically they have not interacted with (Edelaar
et al. in preparation). Given the degree of daily movement of these
grasshoppers (on average 12 meter/day, Edelaar et al. in prepara-
tion), this observed fine scale population differentiation in color
among pavements should quickly cease to exist, unless something
helps to maintain color divergence. Surprisingly, previous studies
show that this appears to be mainly due to habitat choice, since
selective predation and color change appeared to be too weak and
too slow, respectively, to explain the observed patterns (Edelaar et
al. in preparation). With respect to color change, this conclusion
rests on the observation that the response to a black background
is limited and slow, and virtually nonexistent when a white back-
ground is used (Edelaar et al. in preparation, see also Ergene 1953).
However, the different pavements do not only differ in darkness
but also in hue, and it has not yet been tested whether changes in
hue in response to background color are possible in adult azure
sand grasshoppers, nor in any other grasshopper species.

There is an additional reason why color plasticity in these
adult grasshoppers deserves a closer look. The reddish color dis-
played by azure sand grasshoppers has recently been reported to
be related to the presence of pheomelanin, a pigment formerly
thought to be restricted to vertebrates only. Interestingly enough,
this pigment's pathway features mixed-type melanins arising from
both dopamine and DOPA, a process that in vertebrates has only
been reported for neuromelanin (Galvan et al. 2015). This is im-
portant because of the link between neuromelanin and Parkin-
son's disease. Grasshopper (and maybe other insect) species with
this kind of biochemical pathway may therefore play a role in the
future investigation of this pigment’s link to this important dis-
ease (Galvan et al. 2015). Being able to change the production of
pheomelanin in adult grasshoppers by a change in background
might therefore be very useful.

For this variety of reasons, our main objective in this study was
therefore to test whether this species of acridid grasshopper is still
capable of changing in hue in response to background color, even
after reaching full maturity.

Methods

Study subject.— The azure sand grasshopper is a ground dwelling
species which predominantly inhabits little vegetated, xeric scrub-
lands and grasslands and perches on the bare soil rather than on
vegetation. They show a base coloration that varies from reddish-
brown to bluish gray, and these colors can vary from being very

J.R. PERALTA-RINCON, G. ESCUDERO AND P. EDELAAR

< Y IY

~s

| a = >.
SS

~
he

oa

Figure 1. Example of an image taken for color measurement. The
individual was held in place by the transparent plastic lid of the
Petri dish to obtain a correct position. Brightness and hue were
measured in the red diamond-shaped part of the thorax. The color
of a small area of the background paper was also measured (red
circle) as a reference gray standard to correct for lighting variation
among images.

pale to very dark. Color variation is continuous and there is no
known green-brown polymorphism in this species. Their base
color generally resembles that of the substrate surrounding the in-
dividual. They also show a variable pattern of dark markings, and
a pale band halfway down the anterior pair of wings and the hind
legs which helps to disrupt their outline and therefore provides
additional crypsis (Fig. 1).

Nineteen individuals were collected in late August and early
September 2013 in the vicinity of Dos Hermanas (Seville, Spain)
and kept as a group for two months in one of two transparent
plastic boxes (30 x 40 cm). These boxes were each filled with ei-
ther “blue” (fine bluish gray gravel) or “red” (red-brown earth)
substrate, in order to test if adult grasshoppers would change their
color accordingly. Individuals were either assigned to the treatment
“blue” (10 individuals - 8 males and 2 females) or “red” (9 individ-
uals - 5 males and 4 females). Individuals were marked by writing a
number on their anterior wing tips with a water- and light-resistant
marker pen. Food was a mixture of dried wheat bran (45%), dried
mosquito larvae (45%), and powdered milk (10%) (the species is
omnivorous). Bottled mineral water was available in the form of a
gel (ReptiGel). Ambient light was provided by regular fluorescent
ceiling lamps from 8:00 to 20:00 hours, and heat was provided
from below using heating mats for terrariums in order to keep am-
bient temperature between 35 and 40 degrees Celsius.

Image production and color measurement.— At the beginning of the
experiment and for the duration of the treatments, we regularly
took photographs of each individual to subsequently measure its
color (Fig. 1). The experiment was terminated after 49 days, when

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

J.R. PERALTA-RINCON, G. ES;CUDERO AND P. EDELAAR

most individuals had died of old age. In order to create compa-
rable images, all photographs were taken using the same setup: a
Pentax K-r DSLR camera with a dual lamp Mecablitz 15MS-1 dif-
fuser macro flash, mounted on an 18-55mm Pentax kit lens. The
lens was always locked at 55mm and the camera set to shoot in
RAW format with fixed manual settings (f/14 aperture, 1/50 shut-
ter speed, ISO 200). Each image featured a 90% reflectance gray
paper background to control for white balance and flash intensity
(Fig. 1). The grasshoppers were held in the same position (offer-
ing a full dorsal view, with the head pointing to the top of the
image) and at the same distance from the lens by pressing them
into a base of cotton wool with the transparent plastic lid of a
Petri dish. A diamond-shaped zone from the posterior part of the
grasshoppers’ pronotum was used to measure color over time for
each individual, as this area is flat and representative for overall
individual color (Fig. 1).

Any color difference between grasshoppers from different
treatments could also be due to soiling of the individuals with
fine dust from the substrates. In fact, this happened in a first trial
with finely mashed up red soil, so we restarted this treatment three
weeks later with newly collected individuals from the same area
using intact, natural pieces of red soil (which has caused a shift
in the timing of sampling for each treatment and may account
for differences in color between treatments at the beginning of
the experiment, since older grasshoppers tend to be darker, see
Fig. 2). We therefore thoroughly cleaned all subjects which were
still alive when the experiment was finished and compared the
color change between treatments (“Cleaned” dataset, N=5). To
clean the grasshoppers, individuals were first frozen to rapidly
kill them, and then rubbed with a piece of cotton dipped in wa-
ter with dishwashing detergent and cleared under running wa-
ter. Images were taken when the cleaned individuals where dry
again. Few grasshoppers reached this cleaning stage due to the
mortalilty inherent to working with older individuals, and only
live grasshoppers could be tested since color changes rapidly after
natural death.

Color was objectively measured using the color analysis fea-
tures of the Mica Toolbox plugin (Troscianko and Stevens 2015,
we used version 1.12 Mac) for imageJ (Schneider et al. 2012).
Briefly, camera RAW image files were transformed into multispec-
tral images using a Human cone-catch model. We used this model
since measures with a photospectrometer showed that neither
the grasshoppers nor the natural and urban substrates reflect UV
radiation. The XYZ values obtained from these images were con-
verted to the CIE-L*a*b* color space for easier interpretation of
luminosity and hue variation. CIE-L*a*b* defines colors using 3
independent axes: L for lightness, a for green-to-red hue and b for
blue-to-yellow hue. Higher L, a and b values describe a lighter,
redder, and yellower color, respectively. Preliminary experiments
indicated that color values were highly repeatable among images
taken for the same individual on the same day, so in this study
only one image was taken per individual per day.

Statistical analysis.— The statistical analysis software R (R Core
Team 2015) and the packages Ime4 (Bates et al. 2015), ggplot2
(Wickham 2016) and gridExtra (Auguie 2016) were utilized to
visualize data, test our hypotheses and plot results.

To test for a differential change in color between individu-
als exposed to the distinctly colored soils, a linear mixed model
was fitted to the “Change over time” measurements on each CIE-
L*a*b* axis. This model contained Substrate (red or blue), Sex,

23

Time (Days)

Time (Days)

Figure 2. Average changes in color over time (days since the start
of each treatment) for adult azure sand grasshoppers kept on
bluish-gray stones (blue symbols and lines) or reddish-brown soil
(red symbols and lines). Color is expressed in the three independ-
ent dimensions of the CIE-L*a*b* space (see text). Lines show
the predicted values for each CIE-L*a*b dimension according to a
fitted model containing Time, Substrate, Sex, Time:Substrate and
sqlime; we excluded the subject random effect for better visuali-
zation. Continuous lines and circles are for females while dashed
lines and triangles are for males. The “Red Earth” treatment had to
be restarted using grasshoppers from the same field location but
captured later in time. As a result, the timing of data collection is
out of phase between treatments, and L is initially slightly lower
for “Red Earth” individuals (since the species gets darker with age).

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

24

Time (since the beginning of the experiment, in days), Squared
time (to fit non-linear change) and the Time:Substrate interaction
as fixed effects. This interaction tests our main hypothesis that any
color change over time depends on the substrate. As a random
effect, we added Subject (individual identity) to correct for the re-
peated nature of the data.

To check that any differential color changes were not due to
soiling, we also fitted similar linear mixed models to the “Cleaned”
dataset. This model containing Substrate (the treatment), Status
(living at the beginning versus dead + cleaned at the end), sex and
Substrate:Status interaction as fixed effects and Subject again as a
random effect.

The statistical support for any effects on color was determined
by comparing the AIC value of our full model to that of a similar
model lacking the studied effect. Given a set of statistical models
that try to explain the same data, the AIC (Akaike Information
Criterion) gives an estimation of the relative quality of each one of
them, based on likelihood and with a penalty for including more
parameters. The lower the AIC value of a model, the stronger the
support for that particular model. For comparison with more tra-
ditional ways of testing for statistical significance, the AIC differ-
ence is presented with a p-value obtained by a loglikelihood-ratio
test that compared the same two models as described above.

Results

Regarding the color change of live grasshoppers over time,
substrate: color showed a non-significant effect on grasshopper
lightness (DAIC=0.04, p=0.16, c?=1.96, df=1), and no differential
change in lightness over time (Fig. 2). In contrast, hue values were
influenced by the substrate on which individuals were kept (Fig.
2): the Time:Subtrate interaction received considerable statisti-
cal support for both the green-red axis a (DAIC=-11.9, p<0.001,
¢?=13.9, df=1) and the blue-yellow axis b (DAIC=-8.4, p=0.001,
c’=10.4, df=1). Individuals kept on bluish stones turned to a duller,
more desaturated color while the ones kept on the red earth be-
came relatively more red and yellow colored.

Regarding the color change of grasshoppers after freezing and
cleaning, we also found strong statistical supports that substrate
color influenced adult grasshopper color, although the patterns
were somewhat different to those for live grasshoppers (Fig. 3).
The interaction Substrate:Status was strongly supported for L
(DAIC=-8.02, p=0.002, c*=10.02, df=1), for a (DAIC=-16.08,
p<0.001, =18.08, df=1) and for b (DAIC=-17.33, p<0.001,
(=19.34, df=1). Compared to the start of the experiment, and af-
ter being frozen, grasshoppers kept on red earth turned darker, a
bit redder and a bit less yellow, a pattern that is very similar to the
live grasshoppers. In contrast, grasshoppers kept on blue stones
became much darker, redder, and more blue, a pattern that is quite
different from the live grasshoppers. This conforms to our visual
assessments (see Fig. 4): they showed a much darker, purplish
color after being frozen and cleaned.

Discussion

Our results show that color change in response to the color
of the substrate is not restricted to nymphs but also occurs in ful-
ly developed S. azurescens imagoes (Figs 2-4). These changes are
similar in appearance to those we have seen in nymphs of this
species (Edelaar et al. 2017, pers. obs.), as well as those reported
for nymphs in other Acrididae species (Rowell 1972). Individuals
changed their color in such a way that they increased resemblance

J.R. PERALTA-RINCON, G. ESCUDERO AND P. EDELAAR

(at least to the human eye) to that of the substrate on which they
were kept. It is therefore likely that the observed color change is
due to direct visual input, and functions to increase crypsis and
reduce predation rate under field conditions, as suggested before
(Rowell 1972, Cox and Cox 1974, Edelaar et al. 2017). As far as we
know, such adaptive changes in color hue have not been recorded
before for adult grasshoppers, only for nymphs.

Apart from changes in hue, we also observed changes in light-
ness, with individuals in both treatments becoming darker over
time. This is in agreement with previous experiments we have per-
formed with this species (Edelaar et al. 2017, pers. obs.). These
have shown that nymphs are capable of adaptive plasticity in dark-
ness and can become darker or paler, depending on the rearing
substrate (and even more so when exposed to simulated predation
risk: Edelaar et al. 2017), while adults generally turn darker over
time, but more so when kept on a dark substrate. Whether the
observed darkening in this experiment is due to aging or due to
substrate matching is not known, but it is clear that adults can
change color over time.

Exactly how this is done cannot be addressed with our data,
but a likely explanation given the combined results and obser-
vations is that pigments can still be deposited in the cuticle of
adults, but cannot be removed afterwards. This would explain why
a medium gray adult placed on a white background will virtu-
ally stop darkening over time, but it will not become paler (i.e.
a poor cryptic coloration persists), whereas nymphs can become
paler when they molt. The color changes we observed in response
to the different substrates are then likely due to the deposition
of different pigments: more brownish ones when kept on brown
soil, and more gray ones when kept on gray stones. In both treat-
ments, such deposition of additional pigments to change an
individual’s hue is in line with the observed general darkening.
That several pigments are involved is also hinted at by the distinct
color differences observed after freezing and cleaning: apparently
the effects of freezing differed between the individuals from the
different treatments, because the procedure was identical for both
groups and the color change for individuals kept on gray stones
from darkish gray towards more violet would not be expected if
the color difference was simply due to external pollution. Moreo-
ver, the individuals from this same experiment were subsequently
used for pigment analysis (Galvan et al. 2015). That analysis not
only reported one of the first demonstrations of pheomelanin (the
brown version of melanin) in non-vertebrates, but it also found
that the grasshoppers kept on brown soil were rich in pheomela-
nin, while the grasshoppers kept on gray stones were rich in eu-
melanin (the black version of melanin) (see Galvan et al. 2015,
online Supporting Information). Hence, at least part of the chang-
es in color we saw in response to manipulated substrate color can
be explained by the presence (and probably de novo deposition) of
the different chemical forms of melanin.

A noteworthy observation is that the color changes seem to
become slower as time passes (Fig. 2). This might be because
grasshoppers were reaching the coloration they aimed for, but
alternatively the ability to adaptively change color decreases
with age or with the amount of pigment already deposited. Our
experience with this species is that, although similar in direction
and overall appearance, the adaptive color change is remarkably
slower in adults than it is in nymphs. This has behavioral and
ecological implications. Given that adults are around for a longer
time than nymphs, and that (larger, flying) adults are more
mobile than nymphs, one would expect that adults come into
contact with a greater range of differently colored substrates.

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

J.R. PERALTA-RINCON, G. ES;CUDERO AND P. EDELAAR 25

60-
Substrate
L 40 - -—— Blue Stone
=~ Red Earth
20-
Start End
Cleaned
50-
40 - Substrate

-—— Blue Stone

—— Red Earth

a 320-
20 -
10-

\

Start End
Cleaned

Substrate

Blue Stone

—— Red Earth

oO
No sa KD
Oo OC 0 Oo Oo
' ' ' i i]

Start End
Cleaned

Figure 3. Individual changes in color in the final surviving azure sand grasshoppers when kept on bluish-gray stones (blue lines, N = 2)
or reddish-brown soil (red lines, N = 3). The comparison is between the same individuals at the beginning of the experiment and once
frozen and cleaned at the end. Color is expressed in the three independent dimensions of the CIE-L*a*b* space (see text).

Yet local populations of adult grasshoppers are typically quite living on differently colored, adjacent pavements (e.g. dark
well matched in color to their local environments, even if these asphalt roads compared to pale sidewalks) are locally adapted
environments are very close to each other in space. Edelaar et al. in color, despite the fact that the observed degree of movement
(in preparation) describe a situation in which grasshoppers (on average 12 meter/day) would predict a homogenization of

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

26 J.R. PERALTA-RINCON, G. ESCUDERO AND P. EDELAAR

Figure 4. Change in color over time for two example individuals. From left to right: at the start of the experiment, 7 weeks later, and
after freezing and cleaning at the end of the experiment. Individual a. was kept on blue-gray substrate and individual b. was kept on
reddish-brown substrate. For visualization, the bars under the images show the average dorsal color as captured by the CIE-L*a*b*
values measured for each individual at each point in time.

color across pavements in one day. The fact that the plastic color individuals choosing their perching pavement as a function of
change as reported here is adaptive but relatively slow supports their own color in order to increase crypsis.

the conclusion that this local adaptation is in fact mostly The plasticity in the production of pheomelanin in grasshoppers
driven by the grasshoppers’ selection of the environment, with (bothnymphsand adults) might be relevant for future applied studies,

JOURNAL OF ORTHOPTERA RESEARCH 2017, 26(1)

J.R. PERALTA-RINCON, G. ESCUDERO AND P. EDELAAR

because in humans pheomelanin is associated with increased risk of
melanoma in the epidermis (Mitra et al. 2012) and to Parkinson's
disease in the brain (Spencer et al. 1998). Finding pheomelanin and
understanding its chemical pathways in invertebrates may allow the
development of new animal models for these diseases (Galvan et al.
2015). Having a simple manipulation technique available to vary
the production of pheomelanin (placing individuals on brown soil)
would add to the versatility of such a model.

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