Cuticular melanisation and immune response in a butterfly: local adaptation and lack of correlation



    1. Department of Animal Ecology I, Bayreuth University, Bayreuth, Germany
    2. Department of Animal Ecology, Zoological Institute and Museum, Greifswald University, Greifswald, Germany
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    1. Department of Animal Ecology I, Bayreuth University, Bayreuth, Germany
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    Corresponding author
    1. Department of Animal Ecology, Zoological Institute and Museum, Greifswald University, Greifswald, Germany
      Klaus Fischer, Department of Animal Ecology, Zoological Institute and Museum, University of Greifswald, Johann-Sebastian-Bach-Str. 11/12, D-17489 Greifswald, Germany. E-mail:
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Klaus Fischer, Department of Animal Ecology, Zoological Institute and Museum, University of Greifswald, Johann-Sebastian-Bach-Str. 11/12, D-17489 Greifswald, Germany. E-mail:


1. Despite growing knowledge on the relationship between ecological variables and individual immune function, data on the spatial variability of immune defence in invertebrate natural populations are scarce.

2. Here, we use replicated populations of the butterfly Lycaena tityrus from different altitudes to investigate genetic variation in the melanin-based encapsulation response. As high- and low-altitude populations differ in cuticular pupal melanisation, we further tested for any associations between pupal melanisation and parasite resistance.

3. Although pupal melanisation was higher at higher compared with lower altitudes (and at a higher compared with a lower rearing temperature), any obvious relations to the encapsulation response were absent. Further phenotypic correlations within groups were significant in one out of four cases only, suggesting that in L. tityrus encapsulation operates largely independent of cuticular melanisation.

4. A significant interaction between altitude and temperature indicated that high-altitude animals show a stronger melanisation response than low-altitude ones at the lower temperature and vice versa, indicating local adaptation to different climates.


Over the past years, the evolutionary ecology of immune function has received substantial attention (Rolff & Siva-Jothy, 2003), and consequently our knowledge on the important contribution of immune defence to adult fitness has expanded considerably (Rolff, 2002; Rolff & Siva-Jothy, 2002; Schmid-Hempel, 2003, 2005; Stoehr, 2007; Kraaijeveld & Godfray, 2008; Vijendravarma et al., 2009). As an increased allocation to immune defence is expected to be associated with a reduction in other fitness components (Sheldon & Verhulst, 1996; Vijendravarma et al., 2009), an organism's optimal allocation strategy depends on the balance between the risks and costs of infection and the consequences of reallocation away from e.g. survival or reproduction (Bonneaud et al., 2003; Jacot et al., 2004; Schwartz & Koella, 2004; Little & Killick, 2007). Therefore, the regulation and control of immune responses are of great significance, and understanding how traits are integrated at the organismal level remains a fundamental problem at the interface of developmental and evolutionary biology.

An increasing number of studies has demonstrated that invertebrate resistance to infection can be highly variable in response to both biotic (Schmid-Hempel, 2003, 2005; Berggren, 2009) and abiotic factors (Ferguson & Read, 2002; Blanford et al., 2003). Nevertheless, data on the spatial variability of immune defence in invertebrate natural populations are still scarce (Cornet et al., 2009). In nature, geographically distinct populations often experience very different ecological conditions and selection pressures, and heritable genetic variation for resistance traits between populations is therefore expected (Brown, 2003). In this context, clinal variation is of special interest, as populations from different altitudes or latitudes experience predictable variation in ecological conditions, causing differentiation in fitness-related traits including immunity (Brodie et al., 2002; Baucom & Mauricio, 2007; De Block et al., 2008; Karl et al., 2008).

Against this background we investigated variation in the immune response of the butterfly Lycaena tityrus across natural populations from different altitudes. The high- and low-altitude populations chosen show clear variation in several fitness-related traits (e.g. development time and temperature stress resistance; Karl et al., 2008, 2009a), accompanied by genetic differentiation (Karl et al., 2009b). Such variation includes pronounced differences in pupal melanisation, with high-altitude pupae being substantially more melanised than low-altitude ones (Karl et al., 2009c). This system thus provides an excellent opportunity to investigate potential trade-offs between immune function and cuticular melanisation, as both rely on the same melanin-producing enzyme cascade (Carton & Nappi, 1997; Siva-Jothy, 2000). Melanin synthesis may in general involve costs as a result of the allocation of amino acid precursors (phenylalanine and tyrosine) and the production and regulation of a series of enzymes (True, 2003; Talloen et al., 2004), such that trade-offs may emerge based on resource limitations. Note though that in a number of phase-polyphenic species, cuticular melanisation is positively related to parasite resistance (Barnes & Siva-Jothy, 2000; Wilson et al., 2001). Similarly, in the dragonfly Calopteryx splendens, males with larger dark wingspots had a faster encapsulation rate indicating a better immunocompetence, with wingspots thus functioning as an indicator of male quality (Rantala et al., 2000). As pupal melanisation is further affected by developmental temperature in L. tityrus, we additionally use two different rearing temperatures to investigate plastic responses and possible altitude-by-temperature interactions, indicating local adaptation in immune defence.

To score the immune response, we used a standard technique for insects, viz. the encapsulation rate of an ‘artificial parasite’ (such as a nylon monofilament inserted into the animal's haemocoel; see for example Rantala et al., 2000; Civantos et al., 2005; Stoehr, 2007). A recent study showed that this method is biologically relevant, as it is associated with the resistance against real pathogens (Rantala & Roff, 2007). In insects, internal parasites are usually isolated within a melanised layer of haemocytes (Gillespie et al., 1997), and individuals with a greater ability to melanise their internal parasites are less susceptible to pathogenesis (Nappi et al., 1991). Thus, we here investigate variation in immune function across natural populations of L. tityrus, specifically testing for any trade-offs or positive relationships associated with genetic and environmental variation in pupal cuticular melanisation.

Materials and methods

Study organism and egg sampling

Lycaena tityrus (Poda, 1761) is a widespread temperate-zone butterfly, ranging from Western Europe to central Asia (Ebert & Rennwald, 1991). The species is bivoltine with two discrete generations per year in most parts of its range, although populations with one or three generations per year occur (Ebert & Rennwald, 1991; Tolman & Lewington, 1998). The principal larval host-plant is Rumex acetosa L., but some congeneric plant species such as R. acetosella L. and R. scutatus L. are utilised as well (Ebert & Rennwald, 1991; Tolman & Lewington, 1998).

Young females from replicated low- [Greifswald, Germany: 29 m above sea level, a.s.l. (54°02′N, 13°24′E; n = 15); Ueckermünde, Germany: 9 m a.s.l. (53°43′N, 14°01′E; n = 26)] and high-altitude populations [South Tyrol, Italy: 2010 m a.s.l. (46°43′N, 10°52′E; n = 19); Tyrol, Austria: 2050 m a.s.l. (46°52′N, 11°01′E; n = 23)] were caught in July/August 2008. All females were transferred to a climate chamber [27 °C, high humidity (c. 70%), LD 18:6 h light/dark cycle] at Bayreuth University for egg laying. For oviposition, females were placed individually in translucent plastic pots (1l) covered with gauze, and were provided with R. acetosa (oviposition substrate), fresh flowers (Crepis spec., Achillea millefolium, Bistorta officinalis, and Leucanthemum vulgare) and a highly concentrated sucrose solution (for adult feeding). Eggs were collected daily, then pooled across females and kept, separated by population, in small glass vials until hatching.

Experimental design

After hatching, larvae were randomly divided among two rearing temperatures (20 and 27 °C, LD 18:6 h throughout). They were placed in groups of five in translucent plastic boxes (125 ml), containing moistened filter paper and fresh cuttings of R. acetosa in ample supply. A total of 47 boxes was used (22 at 20 °C, 25 at 27 °C), thus effectively controlling for effects of common environment. Boxes were checked daily and supplied with new food when necessary. Resulting pupae were used to induce a non-specific encapsulation response. In arthropods, infiltrated macroparasites are enveloped in specialised cells (haemocytes) and the resulting capsule is subsequently melanised, which kills the parasite through asphyxiation and/or by the production of various toxic quinones (Sugumaran et al., 2000). To induce the encapsulation response, a nylon monofilament (ø 0.2 mm) of 2 mm length was used (hereafter referred as ‘implant’). After having roughened the surface of the implant with a fine rasp, the implant was sterilised in 70% ethanol (to reduce possible effects of bacteria on the encapsulation response; Stoehr, 2007) before being inserted into the haemocoel of the pupae. Therefore, a small hole was pricked into the cuticle using a sterilised dissection pin on the left side of the penultimate abdominal segment, through which the implant was inserted into the haemocoel. Afterwards, pupae were transferred back to their respective rearing temperature for 6 h in the dark, thereafter being frozen at −20 °C for later analysis. Throughout, 6- (at 20 °C) and 3- (at 27 °C) day-old pupae, respectively, were used to correct for differences in physiological age across temperatures (see Karl et al., 2008).

Digital analysis

Digital images of all pupae were taken dorsally, using a digital camera (Leica DC300, Leica Microsystems, Wetzlar, Germany) connected to a binocular microscope. Likewise, dissected implants were photographed. The resulting images were analysed using Image Tool public software (Version 3.0). Pupal area was measured as cross-sectional projection (mm2). To measure pupal melanisation, images were converted into black and white images using a threshold approach (i.e. all pixels above a certain threshold appeared black, all others white). Pupal melanisation was thereafter calculated as the proportion of black area relative to the total pupal area. The degree of encapsulation was determined as the fraction of melanised implant, using a threshold approach as described above.

Statistical analyses

Data on pupal area, pupal melanisation and the degree of implant encapsulation were analysed using nested analyses of (co-)variance (an(c)ovas) with replicate population nested within altitude. Replicates were treated as random effects, while altitude and temperature were considered fixed effects. Pupal area and melanisation were added as covariates when analysing the degree of encapsulation. Note that sexes cannot be distinguished in the pupal stage in this species, such that sex was not included as an additional factor. Throughout, minimum adequate models were constructed by removing non-significant interaction terms. Correlations were computed using Pearson's product moment correlations. All statistical tests were performed using JMP 4.0.0 (SAS Institute Inc., Cary, North Carolina) or Statistica 6.1 (Statsoft, Tulsa, Oklahoma). Least square means ± 1 SE are given in text.


Pupal area showed no significant variation between altitudes, but differed significantly across replicate populations and temperatures (Table 1, Fig. 1a). Animals reared at the lower temperature were larger than those reared at the higher temperature (20 °C: 38.6 ± 0.4 mm2 > 27 °C: 36.9 ± 0.4 mm2). In contrast, pupal melanisation differed significantly between altitudes and temperatures, but not between replicate populations. High-altitude pupae were considerably more melanised (18.6 ± 0.5%) than low-altitude pupae (6.9 ± 0.4%). Further, pupae were darker at the higher (14.2 ± 0.3%) compared with the lower temperature (11.3 ± 0.3%; Table 1, Fig. 1b). A significant altitude-by-temperature interaction indicates that low-altitude pupae responded much weaker (increase by 5.8% at the higher compared to the lower temperature) to different temperatures than high-altitude pupae (increase by 25.1%).

Table 1.  Nested an(c)ovas for the effects of altitude, replicate population (nested within altitude), and temperature on pupal area, pupal melanisation, and the degree of implant encapsulation in Lycaena tityrus.
  1. Pupal area and melanisation were added as covariates when analysing the degree of encapsulation. Minimum adequate models were constructed by sequentially removing non-significant interaction terms. Significant P-values are given in bold.

Pupal area    
 Replicate (altitude)211.812,2147.470.001
 Replicate (altitude)49.302,2131.930.148
 Altitude × temperature322.161,21325.21<0.001
 Replicate (altitude)5788.812,2111.760.175
 Altitude × temperature10187.901,2116.190.014
 Pupal area1777.051,2111.080.300
Figure 1.

Least square means (±1 SE) for pupal area (a), pupal melanisation (b), and the degree of implant encapsulation (c) for Lycaena tityrus from low- and high-altitude populations across two rearing temperatures (20 °C: white bars; 27 °C: black bars). Data were pooled across two replicate populations each.

Encapsulation rate was significantly higher at the higher (88.3 ± 4.2%) than at the lower rearing temperature (55.8 ± 4.1%), but was not affected by altitude or replicate population (Table 1, Fig. 1c). A significant interaction between altitude and rearing temperature indicates that the encapsulation rate was higher in high- as compared to low-altitude animals at the lower temperature, but vice versa at the higher temperature. Pupal area and pupal melanisation added as covariates did not significantly affect the degree of encapsulation. Finally, if tested separately for all altitude by temperature groups, a positive correlation between pupal melanisation and the rate of encapsulation was found in low-altitude pupae reared at 27 °C (r = 0.254, t = 2.1, P = 0.040, n = 65), whereas in all other groups correlations were not significant.


Studies involving clinal variation frequently suggested a positive correlation between body size and colder environments (James & Partridge, 1995; Van't Land et al., 1999). Recent studies, however, demonstrated that the associations between temperature and body size may be complex, ranging from positive to negative ones (Chown & Klok, 2003; Blanckenhorn & Demont, 2004). In line with a previous study (Karl et al., 2008), L. tityrus showed virtually no evidence for altitudinal variation in body size, which most likely reflects complex interactions between temperature, generation time, voltinism, and season length (Karl et al., 2008). However, as evidenced by (developmental) temperature-mediated variation in pupal size, L. tityrus does conform to the temperature-size rule (Fischer & Fiedler, 2000), caused by an increase in food intake as well as in the efficiency in converting ingested food into body matter at lower temperatures (Karl & Fischer, 2008).

Regarding pupal melanisation, high-altitude populations showed, as expected, a higher degree of melanisation compared with low-altitude ones (Karl et al., 2009c). Increasing melanisation with increasing altitude or latitude has also been found in other insects such as flies (Rajpurohit et al., 2008), butterflies (Espeland et al., 2007) or beetles (Rhamhalinghan, 1999), and is often interpreted in the context of the thermal melanism hypothesis (Clusella-Trullas et al., 2007). In contrast to the above patterns in wing or body melanisation though, a darker colouration in the pupal stage is more difficult to explain and might be related to cryptic coloration, disease resistance, or protection from ultraviolet radiation (Wilson et al., 2001; Lorioux et al., 2008) rather than temperature. Note in this context that pupal melanisation increased at the higher rearing temperature in L. tityrus (see also Karl et al., 2009c), thus challenging the thermal melanism hypothesis.

Despite the above clear variation in pupal melanisation across altitudes, there was no overall difference in encapsulation rate between high- and low-altitude populations. Thus, cuticular melanisation was neither traded off against the encapsulation response, nor was there a positive association as has been found in some other species, where the degree of cuticular melanisation was a strong indicator of resistance with darker individuals being more resistant than lighter ones (Verhoog et al., 1996; Barnes & Siva-Jothy, 2000; Rantala et al., 2000; Wilson et al., 2001). Note here that we did not use a stress treatment in our experiment, which may have facilitated the detection of a trade-off (e.g. Talloen et al., 2004), but that the natural variation in cuticular melanisation across populations was particularly large. Further, a positive phenotypic correlation between pupal melanisation and the encapsulation rate was evident in the low-altitude animals reared at 27 °C only, but not in any other group. As the time given for encapsulation was the same across temperatures, the lack of a correlation at 20 °C may be caused by the slower physiological reactions at the lower temperature, which probably also explains the general temperature effect with the higher temperature enabling a faster encapsulation rate. In high-altitude butterflies, however, there was no evidence for a positive correlation regardless of temperature. Note that the overall temperature effect could on principle also be affected by the specific experimental design used: while larvae reared at 27 °C remained at the oviposition and egg development temperature, the larvae reared at 20 °C experienced a temperature change potentially imposing thermal stress. However, it seems to be very unlikely that such a temperature change very early in ontogeny may have such a profound effect on an adult trait.

The significant interaction between altitude and temperature indicates that high-altitude animals show a stronger melanisation response than low-altitude ones at the lower temperature, but the opposite at the higher temperature. This clearly indicates local adaptation to different climates, with low-altitude animals outperforming high-altitudes ones at the higher temperature and vice versa. Further, low-altitude animals appear to be generally more ‘plastic’ with regard to the encapsulation response as compared with the high-altitude populations (cf. Fig. 1c), which is, however, not true for cuticular melanisation (Fig. 1b).

Another interesting issue is the lack of correlation between encapsulation response and body size, measured as pupal area here. In the house cricket, for instance, females base their mate choice on calling song characteristics, which ultimately leads to a preference for large males with high encapsulation ability (i.e. haemocyte load; Ryder & Siva-Jothy, 2001). In Gryllus bimaculatus and the earwig Forficula auricularia, in contrast, encapsulation was negatively correlated, but lytic enzyme activity positively correlated with body size (Rantala & Roff, 2005; Rantala et al., 2007). These results suggest that body size does not generally reliably reflect immune function, in particular because of the differential responses of different components of the immune function. Nevertheless, immune function does seem to be a largely condition-dependent trait, but body size may not necessarily be an appropriate proxy of condition (e.g. Schulte-Hostedde et al., 2001).

Note that we were not able to sex pupae in our study. Thus, we currently do not know whether there are any sex differences in either pupal melanisation or encapsulation response. While such a difference for pupal melanisation would be rather unexpected, females are often predicted to show a higher investment into the immune response than males (Kurtz & Sauer, 2001), as males are expected to fuel competition for mates by investing less energy in other physiological systems (Sheldon & Verhulst, 1996; Zuk & McKean, 1996). Freitak et al. (2005), for instance, showed that activation of the immune system in the pupal stage of a butterfly increased wing melanization in females but not in males. Importantly though, the potential existence of sexual differences should not confound any conclusion drawn here.

In summary, our results caution against any close relationship between pupal cuticular melanisation and the melanin-based immune response in L. tityrus, based on both the lack of genetic and phenotypic correlations (with one exception). Rather, both processes appear to operate at least in part independently. Nevertheless, a significant genotype by environment (i.e. altitude by temperature) interaction for the encapsulation response clearly indicated local adaptation to different thermal environments in the immune response, reflecting different environmental needs across altitudes.


Financial support was provided by the German Research Council (DFG grant no. Fi 846/1-4).