UV-reflectivity of parafocal eyespot elements on butterfly wings

in normal and abnormal specimens

Mouyu Yang, Ahti Pyörnilä & V. Benno Meyer-Rochow

Yang, M., Pyörnilä, A. & Meyer-Rochow, V. B. 2004: UV-reflectivity of para-focal eyespot elements on butterfly wings in normal and abnormal specimens. —Entomol. Fennica 15: 34–40.

An unusual specimen of Aglais urticae, lacking characteristic UV-reflectingparafocal eyespot elements along the margins of both fore and hind wings, iscompared with normal, wild-type specimens. Wing scales, responsible for gener-ating structural coloration, are missing in the abnormal individual and have beenreplaced with a type that is typical of pigment-based colours. Other modificationsseen in the abnormal specimen include firstly, a distal expansion of a uniformlybrown region, that otherwise occupies a proximal position on the hind wings ofthe wild type, and secondly, the lack of a characteristic orange cross-vein bandthat runs proximal to the parafocal eyespot elements on the hind wing. The differ-ences in coloration between abnormal and wild type are seen as evidence of aproximal-distal developmental axis (originally proposed by Nijhout 1991) andsupport a view recently aired by Beldade and Brakefield (2003). It is now clearthat studies on butterfly eyespot development must consider not only pigment-containing scales, but also the structurally modified scales responsible for physi-cal colours, i.e. UV reflectivity.

Mouyu Yang & V. Benno Meyer-Rochow, Faculty of Engineering & Sciences, In-

ternational University Bremen (IUB), P. O. Box 750 561, D-28725 Bremen, Ger-

many

Ahti Pyörnilä, Department of Biology, Oulu University, P.O. Box 3000, SF-

90014 Oulu, Finland

Received 7 October 2003, accepted 1 December 2003

1. Introduction

In recent years the formation of wing eyespots inbutterflies has received a considerable amount ofattention (Brakefield & French 1999, Keys et al.1999, McMillan et al. 2002, Beldade & Brake-field 2003). Yet, in spite of the many thoroughstudies on the way eyespots are generated, one as-pect appears to have been glossed over: all of thepatterns studied lay within the spectral range ofhuman vision, none covered the ultraviolet wave-lengths.

Does that matter? We believe it does, sincenumerous species in New Zealand (Meyer-Rochow 1991), Asia (Eguchi & Meyer-Rochow1983), and Europe (Brunton & Majerus 1995)possess colour patterns on their wings that areonly visible in the UV range and, therefore, inter-pretations of whether a “colour pattern” is or isnot present ought to include also observations inthe ultraviolet range of the spectrum. Most insectscan perceive UV-A light (wavelength 320–390nm) with their photoreceptors (Briscoe & Chittka2001) and at least some butterflies use the UV

© Entomologica Fennica. 29 April 2004

patterns on their wings to communicate with con-specifics (Obara & Hidaka 1968, Silberglied &Taylor 1973, Rutowski 1981, Meyer-Rochow1991, Meyer-Rochow & Järvilehto 1997).

Moreover, considering that eyespots incorpo-rating yellow, red, or black colours are based onpigments inside the butterfly’s scales (Köhler &Feldotto 1935, Ghiradella 1984), but UV-reflect-ing patches are the results of physical phenomenadepending on structural scale-surface modifica-tions (Lippert & Gentil 1959, Meyer-Rochow &Eguchi 1983, Ghiradella 1994, Vukusic et al.1999), one could question whether the geneticmechanisms, regarded responsible for the gener-ation of eyespots generally (Keys et al. 1999), areindeed also applicable to UV scales.

With the help of a mutant Small Tortoiseshellbutterfly (Aglais urticae), lacking UV-reflectingeyespots and exhibiting additional pattern abnor-malities, we hope to further stimulate researchinto these questions of butterfly wing colour de-velopment.

2. Material and methods

The Small Tortoiseshell nymphalid butterflyAglais urticae (Fig. 1a) was bred in the field un-der standard conditions in Oulu (ca. 66º N). Ofhundreds of normal specimens examined, one in-dividual caught our attention, because of its un-usual coloration (Fig. 1b). The condition must berare, for an internet request to Lepidopteran soci-eties to inform us of similar sightings remainedunanswered. The individual was killed and dried.Colour and UV photographs of this individualwere taken and compared with correspondingphotographs of specimens with normal color-ation. Because UV light consists of radiation ofshorter wavelengths, UV photographs tend not tobe in focus, as adjustments for distance are madein visible light. The UV-transmission filter had arange of 325–390 nm and a maximum transmit-tance (80%) to light of 360 nm, coincident withmaximum sensitivity of UV-photoreceptor cellsin the insect eye (Briscoe & Chittka 2001). Thecamera used was a Nikon F2, equipped with alens that, according to the suppliers, transmitted50% of all light of 360 nm (UV) and 81% of alllight of 380 nm wavelengths. Since the film used

(=Kodak TRI-X) was sensitive to wavelengthsranging from 300 nm wavelength to the near in-fra-red, our UV photographs were obtained withbright sunlight of 350–390 nm wavelengths.

Preparation for scanning electron microscopywas standard and involved one millimetre squaresamples of UV-reflecting and UV-absorbingwing regions. The chosen pieces, some of whichdorsal, some of which ventral side up, were stuckon regular SEM-aluminium stubs, coated undervacuum with 60/40 gold/palladium to a thickness

ENTOMOL. FENNICA Vol. 15 • Butterfly eyespots and UV-reflectivity 35

Fig. 1. – a. A normal individual of Aglais urticae, dorsal

side. Note bluish eyespots in blackish surroundings

along the edges of both fore- and hind-wings and the

sharp demarcation border between the reddish-brown

band on the hind wing, and the uniformly dark proxi-

mal region of the wing. – b. The mutant individual,

lacking the reddish-brown band of the hind wing (dor-

sal side). The uniformly dark areas have expanded

distally and the eyespots have been replaced by

patches of the reddish-brown band that also seems to

have shifted distally. The blackish surroundings of the

eyespots are completely missing, and bluish color-

ation is absent. The original photographs are in colour.

of 200 Ångström, and observed under a JeolJSM-6300F scanning electron microscope, oper-ated at 6 kV.

3. Results

Normal individuals of A. urticae are seen by a hu-man observer as shown in Fig. 1a. A number ofsmall and faintly blue parafocal eyespots, sur-rounded by blackish scales, are visible along themargins of both front and hind wings. On thehindwing there is a sharp colour transition from auniformly dark posterior region to a more dis-

tally-placed band of orange-brown coloration.Under UV illumination the blue scales of theparafocal eyespot elements become greatly morevisible as they are strongly reflecting the shorterwavelengths (Fig. 2a), while the hindwing de-marcation line between the more proximal darkregion and the lighter distal band appears more orless unchanged in appearance.

The abnormal individual, as seen by a humanobserver (Fig. 1b), possesses lighter, yellowish-brown spots in places of the bluish eyespots, andlacks the clear border between the darkly col-oured proximal wing region and the orange-brown cross-vein band so obvious in the normalindivdiduals. In fact, the characteristic hind wingband is absent and only remnants of it can be seenin places where the bluish eyespots used to be.The blackish scales appear to have altogether dis-appeared from the hindwing, but interestingly,the two black spots (probably true homologues ofeyespots) in the centre of the forewing have alsomoved distally, but the more proximal blackbands have remained in the same place. The ex-amination of the UV photograph of the abnormalspecimen (Fig. 2b) demonstrates that there is noUV reflectivity in the region of the parafocaleyespot elements. This suggests that the lack ofeyespot UV reflectivity appears to have been cou-pled with a distal expansion of the uniformly darkhind wing region and a replacement of the UV-re-flecting parafocal eyespot scales with pigment-containing scales.

To verify this notion, we examined scalesfrom the eyespot region of the aberrant individual(Fig. 3a) and a normal specimen (Fig. 3b). Thespacing of the longitudinal ribs was more or lessthe same in both individuals, but regarding cross-rib morphologies the two individuals were verydifferent. Wide spaces, allowing light to enter thescale, were developed between the cross ribs ofthe mutant. Such scales are typical of those in-volved in generating pigment-based wing color-ation. However, the presence of narrowly-spacedmicroridges, commonly associated with physicalcolours (Allyn & Downey 1977), was apparent inthe scales from the UV-reflecting region of thenormal individual. This seems to confirm thatscale modifications in the mutant A. urticae hadoccurred in places where UV reflectivity shouldhave taken place normally, although scales of dif-

36 Yang et al. • ENTOMOL. FENNICA Vol. 15

Fig. 2. – a. UV photograph of normal A. urticae indi-

vidual, demonstrating high reflectivity of eyespots, es-

pecially on the hind wings. Note the border between

the uniformly-coloured wing region and the reddish-

brown band. – b. UV photograph of the mutant A.

urticae, showing absence of eyespot UV reflectivity

and a distal expansion of UV absorbing uniformly-col-

oured wing areas.

ferent pigment-based colour can resemble thosetypical of the UV-reflecting wing areas (Janssenet al. 2001) and UV-reflectivity can be a conse-quence of pigmentation (Makino et al. 1952).

4. Discussion

Butterfly eyespots are considered serially homol-ogous pattern elements (Monteiro et al. 2003),probably derived through complex gene regulat-ing cascades. Galant et al. (1998) had earlierdemonstrated that initial cellular and molecular

processes in the formation of both neural andscale precursor cells were similar and that the spa-tial regulation of an AS-C gene was modified dur-ing Lepidopteran evolution. A distinction be-tween different colour scales, however, was notmade at that time. The question of how differentgenes are being expressed in association with dif-ferent colour scales was investigated by Brunettiet al. (2001) and it seems that signalling from thefocus of an eye spot induces the expression ofregulator genes that subsequently control the fi-nal colour patterns of the rings around the eye-spot. To what extent that also applies to structur-ally-produced and not pigment-based colourswas not explicitly studied, but one can probablyassume that for the UV scales similar controlmechanisms are in place. With the help of X-ray-induced mutants for eyespots in the nymphalidBicyclus anynana, Monteiro et al (2003) wereable to suggest that so-called ’focus regulatorygenes’are turned on in each wing cell “via the in-put of regional regulatory genes that bind to dis-crete modules on their cis-regulatory domains”.Their work focused on visible colour patterns andeyespots and was, thus, concerned with wingscales containing different pigments, but not nec-essarily differing morphologically.

In a separate paper it was suggested that alleyespots in a butterfly were “genetically inte-grated” and that there were correlations amongdifferent wing traits, all focusing on the same typeof pattern element, viz. the eye-spot (Beldade &Brakefield 2003). This is a departure from theearlier view that the pattern in its entirety, and notjust separate or isolated eyespots, ought to beconsidered “a single character” (Brakefield2001). Apparently single gene mutants in B.

anynana can cause large pattern changes in over-all eyespot size, shape, and number as well as col-our composition, but have “no effect on the otherpattern elements” (McMillan et al. 2002). Thisstatement is clearly at variance with our speci-men, in which lack of UV reflectivity of hindwing eyespots was combined with effects onother wing pattern elements, namely the cross-vein colour demarcation bands in the hind but notfore wings. In fact, Beldade et al. (2002 a, b) haveshown that even with serially-repeated elementssuch as eyespots, there is genetic variation en-abling developmental and evolutionary flexibil-

ENTOMOL. FENNICA Vol. 15 • Butterfly eyespots and UV-reflectivity 37

Fig. 3. – a. Scanning electron micrograph of scale de-

tail from eyespot region in the mutant A. urticae,

showing ladder-like arrangement with vast gaps be-

tween cross ribs, characteristic of pigment containing

scales. Only faint traces of microridges superimposed

on the longitudinal ribs are visible. The scale bar 1

µm. – b. Scanning electron micrograph of scale detail

from the eyespot region of a normal individual, show-

ing a pattern of densely-spaced narrow microridges

developed between adjacent longitudinal ribs, charac-

teristic of scales that are involved in UV-reflectivity on

the basis of structural features. The scale bar 1 µm.

ity for changes in individual elements.Nijhout (1991) originally dscribed a system

of homologies, termed the “nymphalid groundplan”, that linked all colour pattern elements withthe proximal/distal axis of the wing. What is dom-inating recent research, however, is the focus onthe anterior/posterior axis and the formation ofmany eyespots as the consequence of an ante-rior/posterior compartmentalization of sequentialgene expression. In our abnormal specimen, thelack of UV-reflecting marginal eyespots is com-bined with an absence of the hindwing colour de-marcation line (so prominent in normal individu-als) and a shift to a more distal location of the or-ange-yellow band. Lack of UV reflectivity, thus,appears to have resulted not actually in reducednumbers of parafocal eyespots or in a positionalchange of the eyespots, but in a pattern shift fromproximal (= mid-wing) to distal (= wing margin).How this finding would be compatible with theview of an anterior/posterior eyespot generationremains to be seen.

What could have caused the pattern change inour specimen? Was it a genetic mutation or did itrepresent a morphological phenotype, caused byextreme environmental conditions during rear-ing? Seasonal polyphenism with regard toeyespot sizes and numbers is, after all, known tooccur in a variety of butterfly species. It can betraced to certain environmental variables, e.g.dry/wet conditions and low/high temperatures(Nijhout 1991, Brakefield & French 1999).Moreover, in the swallowtail butterfly Papilio

xuthus, high titers of ecdysteroids shortly afterpupation apparently lead to increased ventraleyespot sizes, while hypodermal injections ofmolsin, an enzyme of Aspergillus saitoi, whichliberates tyrosine and phenylalanine, into 0–2 dayold pupae, also causes pattern perturbations, butchiefly in regard to (visible, i.e. pigment-based)coloration (Umebachi & Osanai 2003).

Since our aberrant specimen was bred to-gether with other perfectly normal ones underidentical conditions, we rule out the possibility ofpolyphenism, but cannot exclude the possibilitythat the one abnormal individual stemmed from apupa that had been frost-damaged (cf. illustra-tions 9 and 10 on Plate IV in Anonymous [1910]).Obviously, the best evidence of whether the ab-normal pattern in our interesting phenotype was

genetically-determined, as opposed to environ-mentally-induced, would have come from theprogeny of the abnormal individual. Alas, thespecimen was preserved before it had a chance tobreed. However, frost forms, lacking hindwingparafocal eyespots, may mate with normal indi-viduals, but according to experiments carried outby Max Standfuss (cited without proper referencein Anonymous [1910]), the offspring apparentlyresemble the frost form and not normal individu-als. We simply do not know whether the abnor-mal specimen was indeed a genetic mutant, per-haps caused by frost, or whether other mutagenswere involved in giving rise to the abnormal col-oration. All we can state for certain is that none ofthe experimentally-generated mutants or pheno-types examined by Brakefield et al. (1998) orBrakefield and French (1999) show eyespotchanges in combination with wing-band alte-rations that resemble those we reported in thispaper.

Our specimen, completely lacking the UV-re-flecting parafocal eyespot elements and exhibit-ing changes in other pattern elements, shows thatthere is a link between proximal and distal wingcoloration (very obvious with regard to thehindwing, but not so with regard to the forewing)and that, at least in our A. urticae, the pattern in itsentirety is not a “single character” producedalong the anterior/posterior axis. Consequently, itwould seem that in the evolution of parafocaleyespots on butterfly wings proximal/distal geneexpressions not only occur, but can be uncoupledfrom anterior/posterior associations.

Since our results depended to a considerableextent on the visualisation of eyespots containingor not containing UV-reflecting scales, we feelthat our recommendation to routinely use UVphotography in researches on butterfly colourpatterns is vindicated. The functional anatomy ofthe eye of A. urticae, incidentally, has been beau-tifully studied by Kolb (1985), who also pre-sented evidence for UV-sensitivity in that spe-cies.

Acknowledgements. VBM-R thanks Prof. M. Järvilehtoand the Academy of Finland for supporting his initial workin Finland through a visiting professorship in the Univer-sity of Oulu. MY thanks the International University Bre-men for financial support. The authors would like to ac-knowledge Dr. J. Itämies of the Zoological Museum of

38 Yang et al. • ENTOMOL. FENNICA Vol. 15

Oulu, the staff of the Electron Microscopy Unit of the Uni-versity of Oulu, and Dr. Doekele Stavenga of the Univer-sity of Groningen for providing the necessary “push” tocomplete the study.

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