We here show the effect of background colour and temperature on colour changes and darkening in G. sibiricus. There were no colour morph switches among 78 individuals tested, neither when exposed to matched nor to unmatched background. Colour morph switches have been widely reported in other orthopterans (Table 1), hence it is interesting that we cannot confirm this for our species. If this species is capable of switching colour at all, the probability of colour morph switch in individuals that mismatched their cage background was certainly very low for both rounds of trials (≤7 %). Additionally, we find that the amount of radiant heat affected colouration darkening within nymphal stages, with darkening at low amounts of radiant heat and lightening at high amounts of radiant heat. Imagoes tended to darken within the first week after final ecdysis independent of radiant heat treatment, suggesting that imagoes may face different life-history trade-offs than nymphae.
Colour morph switches have been previously reported, with several observations of the phenomenon in a diverse array of species (Table 1). Several of the species for which colour morph switches have been reported are members of the Acrididae (the family that also includes Gomphocerus), but none of them is a member of the subfamily Gomphocerinae . It is possible that the capability for colour morph switches has been lost somewhere in the branch of Gomphocerinae, but this remains speculative in the absence of information about other species. The lack of a mechanism for switching colour during nymphal stages in G. sibiricus may also be due to the really fine-grained structure of the habitat inhabited by the species. It would be very costly for an individual to move at all within the matrix of colours of the habitat if this would require an active colour switching to match their background, even if this could be done in a relatively short time window.
Background colour was visually perceivable to the developing individuals and based on previous studies, we assume visual perception to be the main input that triggers colour change to match the background (see  and references therein). However, it might be argued that our coloured paper was not of the right kind for triggering colour changes. Previous studies have used a large diversity of materials for the background manipulation, such as stones, sawdust, sand, coal, clay, paper and paint (Table 1). These different materials have typically elicited colour morph switches, which suggests that the effect does not depend on the exact kind of materials used as background. We consider it unlikely (albeit possible) that our paper type was so substantially different from previously used materials that it would not be suitable for triggering colour switches.
In previous experiments, individuals have been tested for different periods of time in order to assess colour morph switches, usually starting the experiments at early nymphal stages [27, 32, 47–49]. Colour morph switches have typically been reported to occur across nymphal stages, often quantified a few days after ecdysis, though detailed information on the exact timing of switches is usually lacking. The only exception to colour morph changes occurring within nymphal stages are changes to black colouration, which have been reported to occur during the imaginal stage . We started with our experimental treatment very early in the life of the grasshoppers (also in comparison with previous studies), giving scope for switches within and/or between developmental stages. Therefore it is rather unlikely that the duration of the treatment prevented colour switching.
Switches might also have been expected due to the radiant heat treatment, since brown individuals tend to be darker on average than green individuals and we would expect them to be better able to heat up if radiant heat is limiting. High temperature, as well as high humidity, high food moisture content and low individual density, are known to drive green body colouration in grasshoppers [14, 15]. We expect that in our experiment high radiant heat conditions would have served to cue individuals of a green habitat. High alpine habitats are typically characterized by high humidity regimes due to high condensation of air humidity at night, which is available as dew drops early in the mornings, and also due to high precipitation regimes . Under these conditions, high temperature and high humidity will promote vegetation growth and produce greener habitats than conditions of low temperature, where vegetation would have weaker growth. Such conditions could have promoted colour morph switches from brown to green, possibly as a means for habitat matching. In contrast, low radiant heat conditions would have served to cue individuals of a browner habitat with less flourishing vegetation. These conditions, in the case of green individuals, could have promoted colour morph switches from green to brown, either for habitat matching and/or to improve thermoregulatory capacity.
Green individuals on a brown background and under the low radiant heat treatment thus constitute the subgroup for which colour morphs switches from green to brown appear particularly advantageous. It is possible that the combination of relative high humidity, high food moisture content and individual housing (i.e., low population density) counteracted the effect of the background, hampering the occurrence of the colour morph switch [14, 15]. This is different for brown individuals on a green background and under the high radiant heat treatment, since for those individuals the combination of low density, high humidity, background mismatching and no need for improved heat absorption are all expected to favour colour morph switches towards green. Yet none of the 16 individuals under this suit of conditions switched colour.
Our limited sample size does not allow us to exclude the possibility that G. sibiricus is capable of colour morph switches under some conditions. Still it strongly points against frequent, general developmental switches in response to background colour and temperature. We had expected that if colour morph switches occurred in G. sibiricus, they would occur at the nymphal stages, given that matching the habitat background is expected to improve survival in natural conditions. It is possible that nymphs of G. sibiricus achieve homochromy even in the absence of developmental switches by actively seeking out matching (micro)habitats . Habitats of G. sibiricus are spatially highly heterogeneous and this might distinguish them from many of the other species that show developmental colour morph switches. Microhabitat variability might favour behavioural over developmental homochromy, while more global (temporal) variability in less structured habitats might favour developmental switches.
While the temperature treatment in our experiment did not elicit colour morph switches, it elicited a more subtle response in colouration, causing darkening under low radiant and lightening under high radiant conditions. Radiant heat can limit behaviour of individuals by hampering thermoregulation and this in turn can constrain activity levels, growth and development, and ultimately fitness [51–53]. An increase in the amount of melanin under the cuticle surface would improve thermoregulatory capability due to a difference in heat absorbance between black and brown or green colours, and this would help counteract the effect of a short window of radiant heat exposure. Hence the darkening that we found in the low heat treatment is in line with what would be expected for improved thermoregulation [39–41].
The lightening in colouration in response to the high radiant heat treatment can result from a trade-off between melanin as a colour pigment and other functions of melanin. Melanin plays a role in several functions in insects, such as immune defence, integumental colouration, wound healing and cuticle sclerotisation, among others (reviewed in ). It has also been documented that melanin production in insects can be costly, mostly because of the many possible functions of melanin, but also because of dietary limitations of melanin precursors or lack of enzymes necessary to process precursors [51, 55–57]. Therefore lightening of colouration can be seen as an option to avoid investing melanin in body colouration when it is not necessary for absorbing more radiant heat. This reasoning might give an adaptive explanation for the lightening in our high radiant heat treatments.
A change in darkness within nymphal stages implies a mechanism which allows individuals to adjust the amount of visible melanin in their epidermis during the relatively short time spanned between moults. Such a mechanism would include cells at the epidermis capable of spreading pigment granules under the cuticle surface, but also capable of withdrawing the pigment granules under proper stimulation [19, 58]. Relatively little is known about the physiology of colour changes in grasshoppers, but different physiological and morphological mechanisms have been described in other arthropods.
A very intuitive mechanism which could explain the changes in darkness under different radiant heat regimes is pigment dispersal and concentration within chromatophores [19, 59]. This type of cell is known to be present in several taxa, such as fish, reptiles, amphibians, crustaceans and bacteria . The shape of chromatophores is typically highly branched, allowing for pigment to disperse to the branches or contract to the centre to achieve colour change [19, 59, 61]. Another physiological mechanism involved in colour change is granule migration. Here granules of pigment are transported along microtubules which are perpendicular to the cuticular surface, and which branch distally, allowing the pigments to spread and therefore causing colour change. A striking example of this plastic mechanism is observed in the temperature-controlled daily changes in the colour of the chameleon grasshopper Kosciuscola tristis. In this case granules of pigment migrate from the epidermis when the grasshopper is exposed to temperatures above 25 °C, giving males a bright turquoise colouration . A similar mechanism is used in the stick insect Carausius morosus . The colour change that we found in G. sibiricus is much slower, but since granule migration might simultaneously explain changes in darkness in both directions, it might contribute to our observations.
The unpredictable climate in the habitat of G. sibiricus, characterized by long spans of time with few favourable climatic conditions, might explain the occurrence of a darkening and lightening mechanism. In this environment, given the duration of each nymphal stage (about one week, albeit likely to be substantially longer in the field) nymphae may need to adjust colouration even within nymphal stages to be able to cope with climatic variability. In species dwelling in habitats where radiant heat is a limited factor, energy balance and thus activity levels during early developmental stages could be hampered by limited sun exposure conditions. Being able to adjust colouration darkness could greatly improve the use of resources by an individual, in this case melanin which is a multipurpose and apparently costly pigment .