Small Eyespots Research Articles

Larval growth rate is not a major determinant of adult wing shape and eyespot size in the seasonally polyphenic butterfly Melanitis leda.

Insects often show adaptive phenotypic plasticity where environmental cues during early stages are used to produce a phenotype that matches the environment experienced by adults. Many tropical satyrine butterflies (Nymphalidae: Satyrinae) are seasonally polyphenic and produce distinct wet- and dry-season form adults, providing tight environment-phenotype matching in seasonal environments. In studied Mycalesina butterflies, dry-season forms can be induced in the laboratory by growing larvae at low temperatures or on poor food quality. Since both these factors also tend to reduce larval growth rate, larval growth rate may be an internal cue that translates the environmental cues into the expression of phenotypes. If this is the case, we predict that slower-growing larvae would be more likely to develop a dry-season phenotype. We performed the first experimental study on seasonal polyphenism of a butterfly in the tribe Melanitini. We measured both larval growth rate and adult phenotype (eyespot size and wing shape) of common evening brown butterflies (Melanitis leda), reared at various temperatures and on various host-plant species. We constructed provisional reaction norms, and tested the hypothesis that growth rate mediates between external cues and adult phenotype. Reaction norms were similar to those found in Mycalesina butterflies. We found that both among and within treatments, larvae with lower growth rates (low temperature, particular host plants) were more likely to develop dry-season phenotypes (small eyespots, falcate wing tips). However, among temperature treatments, similar growth rates could lead to very different wing phenotypes, and within treatments the relationships were weak. Moreover, males and females responded differently, and eyespot size and wing shape were not strongly correlated with each other. Overall, larval growth rate seems to be weakly related to eyespot size and wing shape, indicating that seasonal plasticity in M. leda is primarily mediated by other mechanisms.

Thermal plasticity in protective wing pigmentation is modulated by genotype and food availability in an insect model of seasonal polyphenism

Abstract Phenotypic variation in natural populations results from complex interactions between organisms and their changing environments. The environment shapes both phenotypic frequencies (during adaptation) and organismal phenotypes (through phenotypic plasticity). Developmental plasticity, in particular, refers to the phenomenon whereby an organism's phenotype depends on the environmental conditions during development. It can match phenotype to ecological conditions and help organisms to cope with environmental heterogeneity, including differences between alternating seasons. Experimental studies of developmental plasticity often focus on the impact of individual environmental cues and do not take explicit account of genetic variation. In contrast, natural environments are complex, comprising multiple variables with combined effects that are poorly understood and may vary among genotypes. We investigated the effects of multifactorial environments on the development of the seasonally plastic eyespots of Bicyclus anynana butterflies. Eyespot size depends on developmental temperature and is involved in alternative seasonal strategies for predator avoidance. In nature, both temperature and food availability undergo seasonal fluctuations. However, our understanding of how thermal plasticity in eyespot size varies in response to food availability and across genotypes remains limited. To address this, we investigated the combined effects of temperature (T; two levels: 20°C and 27°C) and food availability (N; two levels: control and limited) during development. We examined their impact on wing and eyespot size in adult males and females from multiple genotypes (G; 28 families). We found evidence of thermal and nutritional plasticity and temperature‐by‐nutrition interactions (significant T × N) on the size of eyespots in both sexes. Food limitation resulted in relatively smaller eyespots and tempered the effects of temperature. Additionally, we found differences among families for thermal plasticity (significant G × T effects), but not for nutritional plasticity (non‐significant G × N effects) nor for the combined effects of temperature and food limitation (non‐significant G × T × N effects). Our results reveal the context dependence of thermal plasticity, with the slope of thermal reaction norms varying across genotypes and across nutritional environments. We discuss these results in the light of the ecological significance of pigmentation and the value of considering thermal plasticity in studies of the biological impact of climate change. Read the free Plain Language Summary for this article on the Journal blog.

Butterfly eyespots exhibit unique patterns of open chromatin.

Background: How the precise spatial regulation of genes is correlated with spatial variation in chromatin accessibilities is not yet clear. Previous studies that analysed chromatin from homogenates of whole-body parts of insects found little variation in chromatin accessibility across those parts, but single-cell studies of Drosophila brains showed extensive spatial variation in chromatin accessibility across that organ. In this work we studied the chromatin accessibility of butterfly wing tissue fated to differentiate distinct colors and patterns in pupal wings of Bicyclus anynana. Methods: We dissected small eyespot and adjacent control tissues from 3h pupae and performed ATAC-Seq to identify the chromatin accessibility differences between different sections of the wings. Results: We observed that three dissected wing regions showed unique chromatin accessibilities. Open chromatin regions specific to eyespot color patterns were highly enriched for binding motifs recognized by Suppressor of Hairless (Su(H)), Krüppel (Kr), Buttonhead (Btd) and Nubbin (Nub) transcription factors. Genes in the vicinity of the eyespot-specific open chromatin regions included those involved in wound healing and SMAD signal transduction pathways, previously proposed to be involved in eyespot development. Conclusions: We conclude that eyespot and non-eyespot tissue samples taken from the same wing have distinct patterns of chromatin accessibility, possibly driven by the eyespot-restricted expression of potential pioneer factors, such as Kr.

Aberrant light sensing and motility in the green alga Chlamydomonas priscuii from the ice-covered Antarctic Lake Bonney

ABSTRACT The Antarctic green alga Chlamydomonas priscuii is an obligate psychrophile and an emerging model for photosynthetic adaptation to extreme conditions. Endemic to the ice-covered Lake Bonney, this alga thrives at highly unusual light conditions characterized by very low light irradiance (<15 μmol m−2 s−1), a narrow wavelength spectrum enriched in blue light, and an extreme photoperiod. Genome sequencing of C. priscuii exposed an unusually large genome, with hundreds of highly similar gene duplicates and expanded gene families, some of which could be aiding its survival in extreme conditions. In contrast to the described expansion in the genetic repertoire in C. priscuii, here we suggest that the gene family encoding for photoreceptors is reduced when compared to related green algae. This alga also possesses a very small eyespot and exhibits an aberrant phototactic response, compared to the model Chlamydomonas reinhardtii. We also investigated the genome and behavior of the closely related psychrophilic alga Chlamydomonas sp. ICE-MDV, that is found throughout the photic zone of Lake Bonney and is naturally exposed to higher light levels. Our analyses revealed a photoreceptor gene family and a robust phototactic response similar to those in the model Chlamydomonas reinhardtii. These results suggest that the aberrant phototactic response in C. priscuii is a result of life under extreme shading rather than a common feature of all psychrophilic algae. We discuss the implications of these results on the evolution and survival of shade adapted polar algae.

Regeneration of the Eyespot and Flagellum in Euglena gracilis during Cell Division.

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26–97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis.

What Drives the Diversification of Eyespots in Morpho Butterflies? Disentangling Developmental and Selective Constraints From Neutral Evolution

Eyespots are wing colour pattern elements repeatedly observed in many butterflies, whose developmental genetics has been extensively documented. Nevertheless, the evolutionary forces shaping their diversification across species still remain largely unknown. Here we investigate the evolution of eyespots in the 30 species of the neotropical genus Morpho. Morpho butterflies display a series of eyespots located on the ventral side of their wings, highly variable among species, ranging from large, conspicuous structures to relictual spots. Applying geometric morphometrics to a large sample (N = 910) spanning all Morpho species and both sexes, we assess eyespots number, position, size and shape. We detect a divergence in eyespots position between understory and canopy species that opposes an L-shaped configuration in canopy species to a line pattern in understory species, where the eyespots tend to fuse and form a stripe in white and pale species. This effect is stronger than expected based on a Brownian motion model of phylogenetic divergence, suggesting an adaptation to the microhabitat and an influence of the wing colour on the evolution of pattern elements. Remarkably, this shift in colour pattern is strongly correlated with a shift in wing shape. However, using a thin-plate spline interpolation, we show that the shape change is insufficient to explain the evolution of eyespots position as a developmental side-effect of wing shape evolution, also pointing at an adaptive effect. Finally, we find a significant negative correlation between eyespot relative size and the within-species variation in eyespot number, position, size and shape, suggesting a relaxed or apostatic selection on small eyespots. We hypothesize that contrasted ecology may explain the observed differences between species: large and phenotypically stable eyespots might act as attack deflectors; small, variable faded eyespots might rather enhance crypsis; and pale species stripe pattern might disrupt the outline of the wing.

Lizards as Predators of Butterflies: Shape of Wing Damage and Effects of Eyespots

In warm climates, lizards may be important as predators of butterflies and thus exert selection pressure on butterfly colour patterns, including eyespots. However, this has received little attention. Two studies reported no evidence that butterfly eyespots deflect lizard attacks, while possible intimidating effects of butterfly eyespots on lizards have not been investigated. We exposed common evening brown butterflies (Melanitis leda) with a wide range of eyespot sizes (dry season forms with very small and faint eyespots, intermediate phenotypes with small eyespots, and wet season forms with large or very large eyespots) to house geckos, and recorded the location and shape of wing surface loss. We supplemented these data with direct observations of attacks of house geckos (Hemidactylus frenatus) and bronze skinks (Eutropis macularia) on common evening brown butterflies. We found that about three quarters of symmetrical wing surface loss, and about half of the non-symmetrical wing surface loss was rounded like the mouth of a gecko. Symmetrical as well as non-symmetrical wing surface loss was significantly more common in hindwings. We surmise that rounded wing surface loss and a bias in symmetrical wing surface loss to hindwings can be taken as evidence for failed lizard attacks. Rounded wing surface loss was more common in butterflies with small eyespots than in those with very small eyespots or with large eyespots. This suggests that small eyespots can deflect lizard attacks away from the head towards the wing margins, and that large eyespots can thwart lizard attacks. In four cases, skinks appeared to explore rather than attack butterfly eyespots. In contrast to earlier studies, our data provide evidence that butterfly eyespots can be an effective defence against lizards, and thus that predation by lizards can select for eyespots in butterflies.

Development of a Hibiscus syriacus ‘Danna’ with Small and Vivid Red Eye Spot on Bright Pink Petals

Development of a Hibiscus syriacus ‘Danna’ with Small and Vivid Red Eye Spot on Bright Pink Petals

Focusing on butterfly eyespot focus: uncoupling of white spots from eyespot bodies in nymphalid butterflies.

BackgroundDevelopmental studies on butterfly wing color patterns often focus on eyespots. A typical eyespot (such as that of Bicyclus anynana) has a few concentric rings of dark and light colors and a white spot (called a focus) at the center. The prospective eyespot center during the early pupal stage is known to act as an organizing center. It has often been assumed, according to gradient models for positional information, that a white spot in adult wings corresponds to an organizing center and that the size of the white spot indicates how active that organizing center was. However, there is no supporting evidence for these assumptions. To evaluate the feasibility of these assumptions in nymphalid butterflies, we studied the unique color patterns of Calisto tasajera (Nymphalidae, Satyrinae), which have not been analyzed before in the literature.ResultsIn the anterior forewing, one white spot was located at the center of an eyespot, but another white spot associated with either no or only a small eyespot was present in the adjacent compartment. The anterior hindwing contained two adjacent white spots not associated with eyespots, one of which showed a sparse pattern. The posterior hindwing contained two adjacent pear-shaped eyespots, and the white spots were located at the proximal side or even outside the eyespot bodies. The successive white spots within a single compartment along the midline in the posterior hindwing showed a possible trajectory of a positional determination process for the white spots. Several cases of focus-less eyespots in other nymphalid butterflies were also presented.ConclusionsThese results argue for the uncoupling of white spots from eyespot bodies, suggesting that an eyespot organizing center does not necessarily differentiate into a white spot and that a prospective white spot does not necessarily signify organizing activity for an eyespot. Incorporation of these results in future models for butterfly wing color pattern formation is encouraged.

Attack risk for butterflies changes with eyespot number and size.

Butterfly eyespots are known to function in predator deflection and predator intimidation, but it is still unclear what factors cause eyespots to serve one function over the other. Both functions have been demonstrated in different species that varied in eyespot size, eyespot number and wing size, leaving the contribution of each of these factors to butterfly survival unclear. Here, we study how each of these factors contributes to eyespot function by using paper butterfly models, where each factor is varied in turn, and exposing these models to predation in the field. We find that the presence of multiple, small eyespots results in high predation, whereas single large eyespots (larger than 6 mm in diameter) results in low predation. These data indicate that single large eyespots intimidate predators, whereas multiple small eyespots produce a conspicuous, but non-intimidating signal to predators. We propose that eyespots may gain an intimidation function by increasing in size. Our measurements of eyespot size in 255 nymphalid butterfly species show that large eyespots are relatively rare and occur predominantly on ventral wing surfaces. By mapping eyespot size on the phylogeny of the family Nymphalidae, we show that these large eyespots, with a potential intimidation function, are dispersed throughout multiple nymphalid lineages, indicating that phylogeny is not a strong predictor of eyespot size.

Body size affects the evolution of eyespots in caterpillars

Many caterpillars have conspicuous eye-like markings, called eyespots. Despite recent work demonstrating the efficacy of eyespots in deterring predator attack, a fundamental question remains: Given their protective benefits, why have eyespots not evolved in more caterpillars? Using a phylogenetically controlled analysis of hawkmoth caterpillars, we show that eyespots are associated with large body size. This relationship could arise because (i) large prey are innately conspicuous; (ii) large prey are more profitable, and thus face stronger selection to evolve such defenses; and/or (iii) eyespots are more effective on large-bodied prey. To evaluate these hypotheses, we exposed small and large caterpillar models with and without eyespots in a 2 × 2 factorial design to avian predators in the field. Overall, eyespots increased prey mortality, but the effect was particularly marked in small prey, and eyespots decreased mortality of large prey in some microhabitats. We then exposed artificial prey to naïve domestic chicks in a laboratory setting following a 2 × 3 design (small or large size × no, small, or large eyespots). Predators attacked small prey with eyespots more quickly, but were more wary of large caterpillars with large eyespots than those without eyespots or with small eyespots. Taken together, these data suggest that eyespots are effective deterrents only when both prey and eyespots are large, and that innate aversion toward eyespots is conditional. We conclude that the distribution of eyespots in nature likely results from selection against eyespots in small caterpillars and selection for eyespots in large caterpillars (at least in some microhabitats).

Deflective and intimidating eyespots: a comparative study of eyespot size and position in Junonia butterflies

Eyespots are conspicuous circular features found on the wings of several lepidopteran insects. Two prominent hypotheses have been put forth explaining their function in an antipredatory role. The deflection hypothesis posits that eyespots enhance survival in direct physical encounters with predators by deflecting attacks away from vital parts of the body, whereas the intimidation hypothesis posits that eyespots are advantageous by scaring away a potential predator before an attack. In the light of these two hypotheses, we investigated the evolution of eyespot size and its interaction with position and number within a phylogenetic context in a group of butterflies belonging to the genus Junonia. We found that larger eyespots tend to be found individually, rather than in serial dispositions. Larger size and conspicuousness make intimidating eyespots more effective, and thus, we suggest that our results support an intimidation function in some species of Junonia with solitary eyespots. Our results also show that smaller eyespots in Junonia are located closer to the wing margin, thus supporting predictions of the deflection hypothesis. The interplay between size, position, and arrangement of eyespots in relation to antipredation and possibly sexual selection, promises to be an interesting field of research in the future. Similarly, further comparative work on the evolution of absolute eyespot size in natural populations of other butterfly groups is needed.

Temporal Gene Expression Variation Associated with Eyespot Size Plasticity in Bicyclus anynana

Seasonal polyphenism demonstrates an organism's ability to respond to predictable environmental variation with alternative phenotypes, each presumably better suited to its respective environment. However, the molecular mechanisms linking environmental variation to alternative phenotypes via shifts in development remain relatively unknown. Here we investigate temporal gene expression variation in the seasonally polyphenic butterfly Bicyclus anynana. This species shows drastic changes in eyespot size depending on the temperature experienced during larval development. The wet season form (larvae reared over 24°C) has large ventral wing eyespots while the dry season form (larvae reared under 19°C) has much smaller eyespots. We compared the expression of three proteins, Notch, Engrailed, and Distal-less, in the future eyespot centers of the two forms to determine if eyespot size variation is associated with heterochronic shifts in the onset of their expression. For two of these proteins, Notch and Engrailed, expression in eyespot centers occurred earlier in dry season than in wet season larvae, while Distal-less showed no temporal difference between the two forms. These results suggest that differences between dry and wet season adult wings could be due to a delay in the onset of expression of these eyespot-associated genes. Early in eyespot development, Notch and Engrailed may be functioning as repressors rather than activators of the eyespot gene network. Alternatively, temporal variation in the onset of early expressed genes between forms may have no functional consequences to eyespot size regulation and may indicate the presence of an 'hourglass' model of development in butterfly eyespots.

Bird attacks on a butterfly with marginal eyespots and the role of prey concealment against the background

Small eyespots on butterflies have long been thought to deflect attacks and birds are presumptive drivers selecting for these patterns. However, evidence of this function is still ambiguous. Margin ...

Deflective effect and the effect of prey detectability on anti-predator function of eyespots

Eyespots (patterns of roughly concentric rings) are often thought to have an anti-predator function. Previous experiments have lent support for the intimidation hypothesis by demonstrating a deterring effect of eyespots, but so far there is little evidence for the deflective effect (direction of attacks toward less vital body parts). We studied predators’ responses towards large and small eyespots and towards prey with no, one, or a pair of eyespots and if this response is influenced by whether or not prey blend into background. In two experiments, we used artificial, triangular prey items and blue tits (Cyanistes caeruleus) as predators. In experiment 1, we found evidence for the deflective effect of small but not large eyespots, independent of whether the prey was presented on a concealing or exposing background. In experiment 2, we found that predators avoided the prey with a pair of small eyespots more than the prey without eyespots, but interestingly, we only found this deterring effect on the concealing background. There was no difference in attacks between the prey with one large and two small or one large and no eyespots. We conclude that deflective function may select for eyespots, and background may influence the deterring function of eyespots.

Surgical Manipulations on Pupal Wings from the African Butterfly Bicyclus anynana: Damage and Cauteries

INTRODUCTIONThis protocol describes cautery experiments with young pupal wings of the African butterfly Bicyclus anynana to examine the potential role of groups of cells in wing pattern determination. Experiments can be performed on either the left or right wing, leaving the other wing as a control. The pupal wing case overlies a stack of four sheets of epithelial cells that will form, in turn, the dorsal and ventral surfaces of the forewing and then the respective surfaces of the hindwing. The dorsal surface of the forewing is immediately underneath the cuticle. The trachea or wing veins of the forewing are visible through the pupal cuticle under a binocular microscope, providing landmarks. Raised “bumps” or irregularities on the pupal cuticle indicate where the eyespot organizers (or foci) are present on the underlying forewing. The wild-type pattern of the dorsal forewing consists of a small anterior eyespot and a large posterior eyespot; each eyespot has a central white pupil, a black inner ring, and an outer gold ring. Experiments that involve a short insertion of sharpened needles through the pupal cuticle can explore the effects of damage to groups of cells at different times from shortly after pupation until pattern determination has occurred (~24 h after pupation). This type of experiment originally helped to characterize the eyespot organizers because early focal damage to the location of these cells can result in shrinkage or complete elimination of the corresponding eyespot in the adult wing (relative to the control wing).

The deflection hypothesis: eyespots on the margins of butterfly wings do not influence predation by lizards

Feeding experiments with lizards are used to examine the function of small eyespot markings found along the wing margins of many butterfly species. Such eyespots are frequently suggested to function by deflecting the attacks of vertebrate predators away from the vulnerable body towards the wing margins, which can tear easily; the eyespots are considered to mislead predators and to act as targets for their attacks. Such misdirected attacks give the butterflies a chance to evade capture, albeit sometimes losing pieces of wing tissue. As a model prey species, we used fruit-feeding individuals of the tropical butterfly Bicyclus anynana that were attacked in a standard way in laboratory cages by the anolis lizard, Anolis carolinensis. We also manipulated the butterflies' wing patterns by pasting eyespots on different parts of the wings to examine the deflection hypothesis in more detail. Our results indicate no influence on the lizard attacks either of the presence of eyespots, or of their position on the wings. The lizards attacked butterflies in a highly stereotyped manner both when the prey were presented on matching or on contrasting backgrounds. We thus found no support for the deflection hypothesis for attacks by insectivorous lizards. Indeed, our only support to date has been obtained for naive flycatcher birds, but even this requires further corroboration. Although effective deflection may occur rather infrequently, except perhaps under certain ecological conditions such as high-density feeding of butterflies on fallen fruit, it may still be sufficiently consistent over time to have contributed to shaping the evolution of marginal eyespot patterns. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92, 661–667.

IDENTIFICATION OF A PSYCHROPHILIC GREEN ALGA FROM LAKE BONNEY ANTARCTICA: CHLAMYDOMONAS RAUDENSIS ETTL. (UWO 241) CHLOROPHYCEAE1

An unusual psychrophilic green alga was isolated from the deepest portion of the photic zone (&lt;0.1% of incident PAR) at a depth of 17 m in the permanently ice‐covered lake, Lake Bonney, Antarctica. Here we identify and report the first detailed morphological and molecular examination of this Antarctic green alga, which we refer to as strain UWO 241. To determine the taxonomic identity, UWO 241 was examined using LM and TEM and partial sequences of the small subunit (SSU), internal transcribed spacer (ITS) 1 and ITS2 regions (including the 5.8S) of the ribosomal operon. These data were compared with those of previously described taxa. We identified UWO 241 as a strain of Chlamydomonas raudensis Ettl (SAG 49.72). Chlamydomonas raudensis is closely related to C. noctigama Korshikov (UTEX 2289) as well as foraminifer symbionts such as C. hedleyi Lee, Crockett, Hagen et Stone (ATCC 50216). In addition, its morphology, pigment complement, and phototactic response to temperature are reported. Chlamydomonas raudensis (UWO 241) contains relatively high levels of lutein and low chl a/b ratios (1.6±0.15), and the phototactic response was temperature dependent. The Antarctic isolate (UWO 241) included the typical photosynthetic pigments found in all chl a/b containing green algae. It possesses a small eyespot and, interestingly, was positively phototactic only at higher nonpermissive growth temperatures. Comparison of SSU and ITS rDNA sequences confirms the identification of the strain UWO 241 as C. raudensis Ettl and contradicts the previous designation as C. subcaudata Wille.

Artificial selection on the shape of reaction norms for eyespot size in the butterfly Bicyclus anynana: direct and correlated responses

Abstract The tropical butterfly Bicyclus anynana shows phenotypic plasticity in its ventral wing pattern as an adaptive response to wet-dry seasonality. Wet season form individuals have large eyespots, whereas individuals of the dry season generation have small eyespots. In the laboratory these forms can be obtained by rearing larvae at high and low temperatures, respectively. To study the extent to which the shape of the nearly linear reaction norms for eyespot size can be changed we applied four generations of sib selection by rearing full-sib families across three temperatures. In addition, we measured ecdysteroid titres shortly after pupation in the final generation. Although phenotypic variation in shape was present initially, the experiment yielded lines with reaction norms with similar shapes but different elevations. High, positive genetic correlation across temperatures can explain this lack of response. Differences in ecdysteroid titres did not readily relate to differences in eyespot size.

The genetic basis of eyespot size in the butterfly Bicyclus anynana: an analysis of line crosses.

The tropical butterfly Bicyclus anynana shows wide variation in the size of the eyespots on the ventral side of its wings. Differences in the environmental temperature during late larval and early pupal stages are a major source of this variation, but variation also exists within temperatures. Using lines selected at a single temperature for large and small eyespots, and a number of crosses derived from these lines, we studied the genetic basis of eyespot size in B. anynana. We applied Lande's modification of the Castle-Wright (C-W) estimator to estimate the minimum number of genes contributing to the difference between the two lines. Estimates indicated that at least five genes are involved. As the C-W estimator is based on a number of simplifying assumptions, we tested to what extent one of these assumptions (additive gene action) is actually met. Joint-scaling tests indicated that the assumption of additivity is not satisfied and that dominance and probably epistasis play a role. Because reciprocal crosses were available we looked for evidence for sex-linkage and cytoplasmic effects. No evidence for cytoplasmic effects was found, but the data were consistent with the presence of one or more loci on the X-chromosome. The results are discussed in the context of the current model of eyespot formation.