Eyespot Development Research Articles

Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria

We investigated both the phenotypic and developmental integration of eyespots on the fore- and hindwings of speckled wood butterflies Pararge aegeria. Eyespots develop within a framework of wing veins, which may not only separate eyespots developmentally, but may at the same time also integrate them by virtue of being both signalling sources and barriers during eyespot development. We therefore specifically investigated the interaction between wing venation patterns and eyespot integration. Phenotypic covariation among eyespots was very high, but only eyespots in neighbouring wing cells and in homologous wing cells on different wing surfaces were developmentally integrated. This can be explained by the fact that the wing cells of these eyespots share one or more wing veins. The wing venation patterns of fore- and hindwings were highly integrated, both phenotypically and developmentally. This did not affect overall developmental integration of the eyespots. The adaptive significance of integration patterns is discussed and more specifically we stress the need to conduct studies on phenotypic plasticity of integration.

Cryptic variation in butterfly eyespot development: the importance of sample size in gene expression studies

Previous studies have shown that development can be robust to variation in parameters such as the timing or level of gene expression. This leads to the prediction that natural populations should be able to host developmental variation that has little phenotypic effect. Cryptic variation is of particular interest because it can result in selectable phenotypes when "released" by environmental or genetic factors. Currently, however, we have little idea of how variation is distributed between genes or over time in pattern formation processes. Here we survey expression of Notch (N), Spalt (Sal), and Engrailed (En) during butterfly eyespot determination to better understand how pattern formation may vary within a population. We observed substantial heterochronic variance in the progress of spatial expression patterns for all three proteins, suggesting some degree of developmental buffering in eyespot development. Peak variance for different proteins was found at both early and late stages of development, contrasting with previous models suggesting that the distribution of variance should be more temporally focused during pattern formation. We speculate that our observations are representative of a standing reservoir of cryptic variation that may contribute to phenotypic evolution under certain circumstances. Our results also provide a strong cautionary message that gene expression studies with limited sample sizes can be positively misleading in terms of inferring expression pattern time series, as well as for making cross-species phylogenetic comparisons.

Comparative insights into questions of lepidopteran wing pattern homology

BackgroundButterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occuring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-β signaling pathways as putative morphogens for eyespot development.ResultsSaturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development.ConclusionWingless and TGF-β ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution.

Efficacy of Hydrogen Peroxide versus Formalin Treatments to Control Mortality Associated with Saprolegniasis on Lake Trout Eggs

AbstractWe compared the efficacy of hydrogen peroxide versus formalin treatments to control fungal infections on eggs of lake trout Salvelinus namaycush incubated at a hatchery in Wisconsin. Four strains of lake trout eggs were incubated in six vertical‐flow Heath incubators; three replicate incubators for each chemical. Each incubator had 13 trays containing approximately 25,000–30,000 eggs/tray. Formalin (1,667 mg/L) or hydrogen peroxide (1,000 mg/L) treatments were administered once daily for 15 min up to the development of visible eye spots in the eggs (eyed egg stage). Eyed and dead eggs were separated using a photoelectric egg sorter, and the number of live and dead eggs was determined volumetrically. In the hydrogen peroxide test group, the bottom trays of each incubator had fungus present on the eggs, whereas no fungus was observed on eggs treated with formalin. The mean percentage of eyed eggs for an incubator treated with formalin (75%) was significantly greater than an incubator treated with hydrogen peroxide (70%). Formalin was the most efficacious therapeutant; however, both therapeutants were effective in increasing lake trout egg survival up to the eyed egg stage.

Butterfly Wing Pattern Evolution Is Associated with Changes in a Notch/Distal-less Temporal Pattern Formation Process

In butterflies there is a class of “intervein” wing patterns that have lines of symmetry halfway between wing veins. These patterns occur in a range of shapes, including eyespots, ellipses, and midlines, and were proposed to have evolved through developmental shifts along a midline-to-eyespot continuum. Here we show that Notch (N) upregulation, followed by activation of the transcription factor Distal-less (Dll), is an early event in the development of eyespot and intervein midline patterns across multiple species of butterflies. A relationship between eyespot phenotype and N and Dll expression is demonstrated in a loss-of-eyespot mutant in which N and Dll expression is reduced at missing eyespot sites. A phylogenetic comparison of expression time series from eight moth and butterfly species suggests that intervein N and Dll patterns are a derived characteristic of the butterfly lineage. Furthermore, prior to eyespot determination in eyespot-bearing butterflies, N and Dll are transiently expressed in a pattern that resembles ancestral intervein midline patterns. In this study we establish N upregulation as the earliest known event in eyespot determination, demonstrate gene expression associated with intervein midline color patterns, and provide molecular evidence that wing patterns evolved through addition to and truncation of a conserved midline-to-eyespot pattern formation sequence.

Efficacy of Aqui-S, formalin and praziquantel against the monogeneans, Benedenia seriolae and Zeuxapta seriolae, infecting yellowtail kingfish Seriola lalandi lalandi in New Zealand

This study assessed the efficacy of bath administrations of Aqui-S, formalin and praziquantel against adults and eggs of Benedenia seriolae and Zeuxapta seriolae, two monogenean ectoparasites of kingfish ( Seriola lalandi lalandi). Our results showed that 2.5 ppm praziquantel administered for either 24 or 48 h was the most effective treatment for removing B. seriolae (>99%). However, B. seriolae treated with praziquantel for 24 h deposited viable eggs. Formalin at 250 and 400 ppm for 1 h followed by a 5 min freshwater dip was less effective for removing B. seriolae (ca. 80%), but both treatments inhibited the production of eggs by treated parasites. However, a large proportion of B. seriolae eggs exposed to either formalin treatment remained viable. Praziquantel (2.5 ppm for either 24 or 48 h) and formalin (400 ppm for 1 h+5 min f/w dip) were the most effective treatments for removing Z. seriolae from the gills of kingfish (>99%) but the parasites thus removed continued to deposit viable eggs. Exposure to the anaesthetic Aqui-S did not significantly increase numbers of B. seriolae or Z. seriolae removed from the host above those of the control, but it was ineffective in preventing the production of eggs by treated parasites, and did not reduce egg viability. Eggs of B. seriolae were not prevented from hatching unless exposed to air for a period greater than 3 h, and Z. seriolae eggs were only prevented from hatching if exposed to air for periods greater than 5 h prior to the development of eyespots. These results indicate that both primary and secondary treatments will be required to successfully control B. seriolae and Z. seriolae infections in captive kingfish.

UV-reflectivity of parafocal eyespot elements on butterfly wings in normal and abnormal specimens

An unusual specimen of Aglais urticae, lacking characteristic UV-reflecting parafocal eyespot elements along the margins of both fore and hind wings, is compared with normal, wild-type specimens. Wing scales, responsible for generating structural coloration, aremissing in the abnormal individual and have been replaced with a type that is typical of pigment-based colours. Other modifications seen in the abnormal specimen include firstly, a distal expansion of a uniformly brown region, that otherwise occupies a proximal position on the hind wings of the wild type, and secondly, the lack of a characteristic orange cross-vein band that runs proximal to the parafocal eyespot elements on the hindwing. The differences in coloration between abnormal and wild type are seen as evidence of a proximal-distal developmental axis (originally proposed by Nijhout 1991) and support a view recently aired by Beldade and Brakefield (2003). It is now clear that studies on butterfly eyespot development must consider not only pigmentcontaining scales, but also the structurallymodified scales responsible for physical colours, i.e. UV reflectivity.

Female choice depends on size but not symmetry of dorsal eyespots in the butterfly Bicyclus anynana.

The eyespots on the ventral wings of Bicyclus anynana butterflies are exposed when at rest and interact with predators. Those on the dorsal surface are not exposed in this way, and may be involved in courtship and mate choice. In this study, we examined whether the size and fluctuating asymmetry (FA) of dorsal eyespots are reliable signals of male quality. High developmental stability is considered to result in low FA, and to be associated with high quality. Individuals of high quality are predicted to produce sexually selected traits that are large and symmetrical, at a relatively low cost. In this study, we manipulated eyespot development to uncouple eyespot size and FA in order to examine their independent roles in signalling to the female. Individual females in cages were given the choice between two or three males differing in eyespot traits. The results indicate that although size per se of the eyespots is used as a signal, FA and wing size are not. We discuss the use of FA in studies of sexual selection and aspects of sexual selection on dorsal eyespot size.

Structure of a character and the evolution of butterfly eyespot patterns

All butterfly eyespots appear to be formed by a similar developmental mechanism. Thus the great diversity of patterns observed across different species of butterfly has evolved through changes in the genetic modulation of this mechanism. Our descriptions of both the development of eyespots and the genetic variation for eyespot traits in an African butterfly, Bicyclus anynana, are used here to discuss the most appropriate ways of applying the character concept to the evolution of butterfly eyespot patterns. J. Exp. Zool. (Mol. Dev. Evol.) 291:93-104, 2001.

A dynamic model of the influence of rotation and crop management on the disease development of eyespot. Proposal of cropping systems with low disease risk

A dynamic model of the effect of rotation and crop management on the frequency of plants infected by eyespot (anamorph Pseudocercosporella herpotrichoides, teleomorph Tapesia yallundae) in a field is proposed and its parameters are estimated on a series of experiments in France during two years. A first equation estimates disease frequency as a function of thermal time and of two parameters associated to the primary (from infectious crop residues) and secondary (from living diseased plants) infection cycles. Two other equations are proposed to estimate the values of the two infection cycle parameters as a function of macro-environment and cropping system; interactions between cultivation techniques were integrated using multiplicative equations. The primary infection cycle parameter depended on crop rotation, soil tillage, sowing date, tiller number per plant and available nitrogen. The secondary infection cycle parameter depended on tiller number per plant. The macro-environment effect on the two parameters is consistent with epidemiological models. The proposed model allows to choose those cropping systems that minimise disease risk for a given set of environmental and technical constraints.

Influence of crop management on eyespot development and infection cycles of winter wheat

Wheat was assessed at four crop growth stages for eyespot (anamorph Pseudocercosporella herpotrichoides, teleomorph Tapesia yallundae) in a series of field trials that studied the effects on disease frequency of five wheat management techniques (sowing date and density, nitrogen fertiliser dose and form, removal/burial of cereal straw). An equation expressing disease level as a function of degree-days was fitted to the observed disease levels. This equation was based on eyespot epidemiology and depended on two parameters illustrating the importance of the primary and the secondary infection cycles respectively. Cultural practices were classified according to the importance of their effects on disease, and these effects could be related to infection cycles and host plant architecture. Sowing date had the earliest and strongest effect; early sowing always increased disease frequency through the primary infection cycle, and its influence on the secondary cycle was variable. Disease frequency was increased by high plant density and/or a low shoot number per plant through primary infection; the secondary cycle was, however, decreased by a low shoot number per plant, which reduced late disease development at high plant density. High nitrogen doses increased disease levels and the severity of both infection cycles, but this effect was partly hidden by a simultaneous stimulation of tillering and thus an indirect decrease of disease incidence. When significant, ammonium (vs ammonium nitrate) fertiliser decreased eyespot levels and infection cycles whereas straw treatment (burial vs removal of straw from the previous cereal crop) had no effect.

Effect of NPK Fertiliser Combinations on the Incidence and Severity of Eyespot

The effect of NPK fertiliser combinations on the development of eyespot was studied both in the greenhouse and in the field. Low levels of nitrogen fertilisers (urea) were found to reduce the severity of eyespot on wheat in comparison to controls without fertilisers. The severity of eyespot, however, increased as the dosage of nitogen fertiliser was increased above 60 kg/ha. Combinations of urea and phosphorus increased severity of eyespot while combinations with potassium reduced severity. A “recommendation” was made for such application: 90 kgN/ha or 90 kgN: 30 kgP/ha: 30 kgK/ha.

Development of Eyespot and Brown Foot Rot in Wheat Plants Inoculated with Mixtures of Pseudocercosporella herpotrichoides Types and Fusarium Species

AbstractThe effects of Fusarium avenaceum, F. culmorum and Microdochium nivale on eyespot development and of types of the eyespot fungus Pseudocercosporella herpotrichoides on brown foot rot caused by the Fusarium or Microdochium spp. were investigated by sequential inoculation of wheat plants grown in pots in a controlled environment. The W‐type of the eyespot fungus induced more severe disease than the R‐type, but its symptons were suppressed to a greater extent by Fusarium spp., especially F. avenaceum. Brown foot rot symptoms were sometimes suppressed by P. herpotrichoides but were occasionally more severe when the Fusarium inoculum was applied after the P. herpotrichoides. There, sults are discussed in relation to observations of natural infections.

Eyespot Development on Butterfly Wings: The Epidermal Response to Damage

Eyespot colour patterns decorate the wings of many butterfly species. The eyespot is specified in the early pupal epidermis by signals from a central "focus," and it has been suggested that the focus is the source of a diffusible morphogen gradient. We show that ectopic eyespots can be induced in nonfocal positions throughout the distal, but not the proximal, wing epidermis of Bicyclus anynana by mild epidermal damage inflicted at 12-18 hr (into a 6- to 7-day pupal period). Damage may lower, locally and transiently, the threshold for response to morphogen. Here, we have tested two predictions of the gradient model. As predicted, mild damage close to the focus (parafocal) locally extends the eyespot, making it markedly asymmetrical, even after early operations (1 or 6 hr), when remote epidermis is nonresponsive. Early parafocal operations also have an unexpected result, however, reducing the extent of the eyespot in other directions, perhaps through a long-range "wound effect" on the signaling activity of the focal cells. The model also correctly predicts that increasing the severity of a nonfocal operation will prolong the transient damage and hence give ectopic eyespots after early (1- or 6-hr) operations. We do not, however, find the expected size increase in ectopics induced by the later (12 or 18 hr) severe damage. Similarly, we demonstrate the predicted effect of early (1 or 6 hr) mild damage in increasing the response to a subsequent (18 hr) operation at the same position. The gradient model is therefore supported by most aspects of eyespot induction in response to epidermal damage.

Eyespot Development on Butterfly Wings: The Focal Signal

The eyespot colour pattern on butterfly wings is specified in the early pupal epidermis by signals from a central "focus." In Bicyclus anynana we show that a small square of focal epidermis, grafted to a range of distal wing sites, induces eyespot formation in surrounding host tissue. Signaling is limited to the focus, and even an adjacent (parafocal) graft does not maintain its normal fate (of contributing to the eyespot) and does not influence its surroundings. Along the wing, there is an abrupt change in the epidermis, as a focus grafted to a proximal site provokes no host response. The results of several grafting experiments demonstrate that their different response properties are autonomous to small areas of the distal and proximal epidermis and that the nonresponding proximal tissue can nonetheless transmit the focal signal. The Bicyclus dorsal forewing has a small anterior and a large posterior eyespot, and we show that this results mainly from a difference in focal signals, not in the epidermal response. A grafted posterior focus induces a large eyespot, whereas an anterior focus induces a small eyespot Furthermore, the anterior and posterior eyespots differ in proportions, and this difference also depends on the identity of the focus, not on the responding epidermis. Eyespots are specified over many cell diameters from the focus by a mechanism which could consist of one long-range signal, such as a morphogen gradient or of a cascade of short-range interactions initiated by the focus. Focal control of the difference in size and proportion between the anterior and posterior eyespot is more readily compatible with a gradient mechanism. Neither model, however, readily explains why the pattern induced by a grafted focus is smaller, but its peripheral gold annulus is broader than in the corresponding control eyespot. Also, there is no direct evidence for long-range gradients, in the butterfly wing or any other insect epithelium.

The development of eyespot patterns on butterfly wings: morphogen sources or sinks?

ABSTRACT We have studied the development of the eyespot colour pattern on the adult dorsal forewing of the nymphalid butterflies, Bicyclus safitza and B. anynana, by cauterising the presumptive eyespot centres (the foci) on the pupal wing. The effects on pattern depended on age at cautery. Early focal cautery (at 1-12 hours after pupation) usually reduced or eliminated the eyespot, while cautery at a non-focal site usually had no effect. These results resemble those of a previous study on another species but, in addition, we find that a later cautery (at 12-24 hours) had the converse effect of generating pattern, so that focal cautery enlarged the anterior eyespot (but usually not the large posterior eyespot) and nonfocal cautery induced a new ectopic eyespot. The effects of cautery on patterning are more extensive by an order of magnitude than the cell death which is caused, so implicating a long-range mechanism, such as a morphogen gradient, in eyespot development. The focus clearly acts to establish the normal eyespot pattern, but a simple source/diffusion model is not supported by the response to late cautery. We suggest two alternative forms of gradient model in which late damage can mimic and augment the action of a focus. In the Source/Threshold model, the focus is a morphogen source, and cautery can remove the focus but also transiently lowers the response threshold in surrounding cells. In the Sink model, the focus generates the gradient by removing morphogen, and cautery can eliminate the focus but it also causes a transient destruction or leakage of morphogen. These models can explain most features of the results of cautery.

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt ?

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt (-) were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500∶440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 μM), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.

The Development of Eyespot on Seedling Leaf Sheaths in Winter Wheat and Winter Barley Crops Inoculated with W‐type or R‐type Isolates of Pseudocercosporella herpotrichoides

AbstractIn 1986–88 the development of eyespot lesions in winter wheat or winter barley differed plots inoculated with W‐type isolates of Pseudocercosporella herpotrichoides and plots inoculated with R‐type isolates. In the spring of 1986, after a cold winter, the incidence (%shoots infected) and severity (number of leaf sheaths penetrated) of eyespot lesions in wheat before GS 30/31 were greater in plots inoculated with R‐type isolates than in those inoculated with W‐type isolates. In 1987, after amild winter, eyespot incidence and severity in both wheat and barley were initially greater in W‐type plots than in R‐type plots. However, by GS 30/31 or 1987. In 1988, when the crop was October‐sown, eyespot incidence and severity were greater in W‐type than in R‐type plots at GS 30/31. Differences in eyespot incidence and severity between W‐type and R‐type plots were smaller in barley than in wheat. Both the incidence and severity of eyespot were greater in early‐sown than in late‐sown plots. Seed rate, had little effect on the rate of lesion development in 1987, but in 1988 the rate of penetration was less at the low seed rate for both wheat and barley.

Competence and delay of metamorphosis in the Pacific oysterCrassostrea gigas

The temporal relationship between the onset of behavioral and morphogenetic competence was determined inCrassostrea gigas (Thunberg) larvae by exposure to appropriate chemical inducers during development. Larvae exhibited settlement behavior in response to L-3,4-dihydroxyphenylalanine (L-DOPA) before becoming competent to metamorphose in response to epinephrine. For larvae past the onset of competence, the apparent stimulus-threshold decreased and they were increasingly likely to metamorphose subsequent to induced settlement behavior. In the absence of chemical stimulation, cultured oyster larvae were able to delay metamorphosisand maintain competence for at least 30 d. Competence was correlated with, but not dependent upon, larval size and eyespot development. A mechanistic model of oyster settlement and metamorphosis is proposed which incorporates these new data.

The influence of food concentration and temperature on growth and morphological differentiation of blue mussel Mytilus edulis L. larvae

The relationship between growth rate, rate of morphological development, and length of larval life, was examined for larvae of the blue mussel Mytilus edulis L. The larvae were reared at phytoplankton ( Isochrysis galbana, clone T-ISO) concentrations between 0.5 × 10 4 and 30 × 10 4 cells · ml −1 at either 12 or 16°C. Growth rates generally increased with increasing food concentration and were highest at the higher temperature; maximum shell growth rate was ≈ 8 μm · day −1. The number of days required for larvae to develop recognizable eyespots sometimes varied proportionately with changes in growth rate, but often did not; temperature was especially effective at uncoupling rate of growth from rate of morphological development. Maintained in clean glass dishes at 16°C, many eyespotted larvae survived until the end of the study, as long as 8 wk after they first developed eyespots (≈80 days after fertilization). Nearly 30% of those larvae that survived under these conditions eventually metamorphosed in the absence of filamentous substratum, suggesting that metamorphosis can be postponed at least 45 days at 16°C. Food concentration had no effect on 1. (1)the time elapsed between eyespot development and noninduced attachment; 2. (2) the average shell length of individuals that did attach; or 3. (3) the mean size of larvae remaining at the end of the study.