Homochromy (i.e. that individuals have a similar color as their environment) is frequent in grasshoppers, and probably functions to reduce detection by potential predators. Nymphs of several soil-perching grasshopper 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 response 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 eumelanin 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.

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

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 substrate. In his revision of this family’s variable coloration, Rowell (1972) proposed three non-exclusive causes for this local adaptation 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 indeed 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 done rather subjectively, either assigning individuals to discrete arbitrary 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 digital image analysis as a less biased approach (Stevens et al. 2007).

We have used this approach to study the color matching of azure sand grasshoppers, Sphingonotus azurescens (Rambur) (Orthoptera: 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 surfaces (sidewalks, bicycle paths, asphalt roads) for perching, feeding, courtship, and reproduction. We found that individuals using these pavements were consistently more cryptic on their local pavement than on other, adjacent pavements. This shows that these grasshoppers are able to adapt their color to fine scale environmental 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 preparation), 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 background 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 displayed 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 (Galván et al. 2015). This is important because of the link between neuromelanin and Parkinson’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 disease (Galván 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.

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, c²=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 statistical support for both the green-red axis a (DAIC=-11.9, p<0.001, c²=13.9, df=1) and the blue-yellow axis b (DAIC=-8.4, p=0.001, c²=10.4, df=1). Individuals kept on bluish stones turned to a duller, more desaturated color while the ones kept on the red earth became 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, c²=10.02, df=1), for a (DAIC=-16.08, p<0.001, c²=18.08, df=1) and for b (DAIC=-17.33, p<0.001, c²=19.34, df=1). Compared to the start of the experiment, and after 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.

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).

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.

Our results show that color change in response to the color of the substrate is not restricted to nymphs but also occurs in fully 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 (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 lightness, with individuals in both treatments becoming darker over time. This is in agreement with previous experiments we have performed with this species (Edelaar et al. 2017, pers. obs.). These have shown that nymphs are capable of adaptive plasticity in darkness 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 observations 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 virtually 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 treatments, 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. Moreover, the individuals from this same experiment were subsequently used for pigment analysis (Galván 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 pheomelanin, while the grasshoppers kept on gray stones were rich in eumelanin (the black version of melanin) (see Galván et al. 2015, online Supporting Information). Hence, at least part of the changes 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. Yet local populations of adult grasshoppers are typically quite well matched in color to their local environments, even if these environments are very close to each other in space. Edelaar et al. (in preparation) describe a situation in which grasshoppers living on differently colored, adjacent pavements (e.g. dark asphalt roads compared to pale sidewalks) are locally adapted in color, despite the fact that the observed degree of movement (on average 12 meter/day) would predict a homogenization of color across pavements in one day. The fact that the plastic color change as reported here is adaptive but relatively slow supports the conclusion that this local adaptation is in fact mostly driven by the grasshoppers´ selection of the environment, with individuals choosing their perching pavement as a function of their own color in order to increase crypsis.

The plasticity in the production of pheomelanin in grasshoppers (both nymphs and adults) might be relevant for future applied studies, 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 (Galván 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.