Long-Range Effects of Wing Physical Damage and Distortion on Eyespot Color Patterns in the Hindwing of the Blue Pansy Butterfly

Article Long-Range Effects of Wing Physical Damage and Distortion on Eyespot Color Patterns in the Hindwing of the Blue Pansy Butterﬂy Junonia orithya

Joji M. Otaki

The BCPH Unit of Molecular Physiology, Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan; [email protected]; Tel.: +81-98-895-8557 Received: 27 November 2018; Accepted: 17 December 2018; Published: 19 December 2018

Abstract: Butterfly eyespot color patterns have been studied using several different approaches, including applications of physical damage to the forewing. Here, damage and distortion experiments were performed, focusing on the hindwing eyespots of the blue pansy butterfly Junonia orithya. Physical puncture damage with a needle at the center of the eyespot reduced the eyespot size. Damage at the eyespot outer rings not only deformed the entire eyespot, but also diminished the eyespot core disk size, despite the distance from the damage site to the core disk. When damage was inflicted near the eyespot, the eyespot was drawn toward the damage site. The induction of an ectopic eyespot-like structure and its fusion with the innate eyespots were observed when damage was inflicted in the background area. When a small stainless ball was placed in close proximity to the eyespot using the forewing-lift method, the eyespot deformed toward the ball. Taken together, physical damage and distortion elicited long-range inhibitory, drawing (attracting), and inducing effects, suggesting that the innate and induced morphogenic signals travel long distances and interact with each other. These results are consistent with the distortion hypothesis, positing that physical distortions of wing tissue contribute to color pattern determination in butterfly wings.

Keywords: butterﬂy wing; color pattern; damage; distortion; eyespot; hindwing; long-range signal; morphogenic signal

1. Introduction An important research topic in developmental biology is the characterization of physiological functions and mechanisms of organizers for developmental fate determination. An organizer, discovered first in amphibian embryos using surgical manipulations, releases a signal called a morphogen to determine the developmental fate of immature cells [1–4]. Since the original surgical study of Spemann and Mangold (1924) [1], at least four regions of embryos have been identified as organizers in vertebrates [4]. Surgical manipulations have played an important role in butterfly biology. Cautery or puncture damage studies and surgical transplantation studies on nymphalid butterfly wings have demonstrated that the center of the prospective eyespot in a pupal forewing tissue functions as an organizer; an application of cautery or simple physical puncture damage to the center of the prospective eyespot reduced the eyespot size in adult wings [5–11], and transplantations of the center of the prospective eyespot induced an ectopic eyespot around the transplant [5,6,12]. These two lines of evidence established that the center of an eyespot functions as an organizer and a source of morphogen for color pattern determination. Additionally, damage responses in the background area (where no color pattern element is located) [6,10,11,13,14] and physiologically induced color pattern changes [6,15–20] have

Insects 2018, 9, 195; doi:10.3390/insects9040195 www.mdpi.com/journal/insects Insects 2018, 9, 195 2 of 15 been investigated to understand the nature of the putative morphogenic signals and the immature scale cells that receive and interpret the signals. As an experimental system, the butterfly wing system has several technical advantages. Butterfly wings are mostly two-dimensional, although they are three-dimensional in a rigorous sense [21], and thus, suitable for identifying normal and induced fates. Eyespot organizers are easily identifiable at the pupal stage, because they are located immediately beneath the pupal cuticle spots [9,21]. Live organizing cells have been observed in vivo as nadir cells at the bottom of the focal indentation in the ventral forewing [22]. Butterflies have right and left wings, and their color patterns are almost always identical, which means that the internal control is always available when only the right or left wing is subjected to experimental manipulation [23]. The results are visually compelling, as the color pattern changes, and thus, easy to interpret after experimental manipulation. Color fates are limited in a small number of colors, and there is the one-cell one-scale rule, meaning that a given scale cell produces just a single scale of a discrete color [6,24], which makes the interpretation at the cellular level simple. However, the mechanism for color pattern determination has been enigmatic. Classically, the putative morphogen for eyespot color patterns was thought to establish a static concentration gradient of the morphogen, and based on the local concentration, each immature cell determines the cell’s fate [6,7,25–27] according to the concentration gradient model for positional information [28–30]. However, it has been noted that the classical gradient model cannot explain a variety of real nymphalid eyespots as opposed to the ideal concentric eyespot [31]. Importantly, parafocal elements (PFEs) have been shown to be remote, outermost eyespot rings [31–33]; eyespots and PFEs are collectively called the border symmetry system in the nymphalid ground plan [6,24,34,35]. Accordingly, a model that explains both eyespots and PFEs has been proposed [24,36–38]. Experimentally, physical damage to the forewings of the peacock pansy butterfly Junonia almana revealed that two adjacent eyespots interact with each other in an inhibitory or activating manner and that damage to the background area induces an ectopic organizer for eyespot-like structures [10,11]. These experimental results have been incorporated into the induction model, which proposes dynamic morphogenic signal interactions for color pattern determination [24,31,37]. However, information on the damage response has been accumulated mostly from forewings, and information from hindwings is limited. There is a possibility that hindwing responses to physical damage are different from those of the forewing in Junonia coenia [6], although this was not the case with J. almana [11]. In addition, the previous damage experiment was performed mostly to test the function of the central cells at the prospective eyespot as an organizer but not to examine the possible long-range effects between the damage-induced signals and the innate eyespot signals. Because physical distortions may play an important role in color pattern formation in butterfly wings, according to the distortion hypothesis for morphogenic signals [24,39], hindwing responses are expected not only from simple physical damage but also from physical distortions. Here, I performed damage and distortion experiments, focusing on the hindwing of the blue pansy butterfly Junonia orithya. The hindwing of this species has two large eyespots against the background area without any other complicated patterns except the peripheral bands (i.e., PFEs, submarginal bands, and marginal bands) (Figure1A). Thus, this system is ideal for observing interactions between experimental stimuli and innate eyespot signals.

2. Materials and Methods

2.1. Butterﬂies In this study, J. orithya (Linnaeus, 1758) was used as an experimental model. Only female individuals were examined after treatment because of sexual dimorphism in this species; females have large anterior and posterior eyespots on their hindwings with a brown or blue background area Insects 2018, 9, 195 3 of 15

(Figure1A), whereas males have a compromised anterior eyespot (or black spot) and a relatively small Insects 2018, 9, x FOR PEER REVIEW 3 of 15 posterior eyespot on their hindwings with a metallic blue background area. Females of thisthis speciesspecies werewere field-collectedfield-collected fromfrom thethe NishiharaNishihara CampusCampus ofof UniversityUniversity ofof thethe Ryukyus, Okinawa,Okinawa, Japan in 2015–2018.2015–2018. Eggs were harvested from these females on the surfacesurface ofof host plantplant leavesleaves inin aa 300300 mmmm ×× 300 mmmm ×× 300300 mm frameless glass tank called a Crystal Cube 300300 ◦ (Kotobuki(Kotobuki Kogei,Kogei, Tenri, Tenri, Nara, Nara, Japan) Japan) at at ambient ambient temperatures temperatures of of approximately approximately 27 27C. °C. They They were were fed fed ad libitumad libitum with awith commercially a commercially available available “ion supply “ion drink”, supply POCARI drink”, SWEAT POCARI (Otsuka SWEAT Pharmaceutical, (Otsuka Tokyo,Pharmaceutical, Japan). Tokyo, Japan). The larvaelarvae were were reared reared with with natural natural host host plant pl leavesant leaves native native to Okinawa-jima to Okinawa-jima Island, PhylaIsland, nodiflora Phyla ornodifloraPlantago or Plantago asiatica. asiatica In addition,. In addition,Hemigraphis Hemigraphis alternata alternatawas occasionally was occasionally given given when when the former the former two plantstwo plants were were not available, not available, because because the blue the blue pansy pansy butterfly butterfly on Okinawa-jima on Okinawa-jima Island Island appeared appeared to use to thisuse plantthis plant in the in wild the inwild my in observation. my observation. These hostThese plant host leaves plant were leaves wild were harvested wild harvested mainly from mainly the Nishiharafrom the Nishihara Campus ofCampus the University. of the University.

Figure 1. TheThe hindwing hindwing of of JunoniaJunonia orithya orithya. (.(A) NomenclatureA) Nomenclature of the of hindwing the hindwing elements elements and sub- and sub-elements.elements. (B) Treatment (B) Treatment modes. modes. The Thephysical physical puncture puncture dama damagege sites sites (Modes (Modes 1–7 in 1–7 asterisks) in asterisks) and andball ballplacement placement sites sites(Modes (Modes 8 and 8 9 and in white 9 in whitecircles) circles) are indicated are indicated by numbers by numbers of treatment of treatment modes. modes.Modes Modes7 and 9 7include and 9 include the broader the broader background background area. area.

2.2. Experimental Operations Two treatmenttreatment modalitiesmodalities were were performed: performed: physical physical puncture puncture damage damage with with a a needle needle (Modes (Modes 1–7) 1– and7) and ball ball placement placement on on the the hindwings hindwings after after the the forewing-lift forewing-lift operation operation (Modes (Modes 8 and 8 and 9). 9). The The effect effect of theof the transient transient forewing-lift forewing-lift procedure procedure (no (no ball ball placement placement between between the the wings) wings) was was also also tested tested (Mode (Mode 10). The10). The treatment treatment was was performed performed only only on on the the right right wing. wing. The The left left wing wing was was left left intactintact forfor comparison. Each individualindividual waswas treatedtreated byby aa singlesingle operationoperation throughoutthroughout thisthis study.study. For thethe physicalphysical puncturepuncture damagedamage experimentsexperiments (Modes(Modes 1–7), a stainless needle of 0.50 mm inin diameter (Shiga Konchu, Tokyo, Japan)Japan) was used to damagedamage the wing tissue within 6 hourshours after pupation.pupation. The site to be damaged was visually navigatednavigated and identiﬁedidentified based on the surface patterns of thethe pupalpupal wing wing cuticle cuticle (i.e., (i.e., pupal pupal cuticle cuticle spots). spots). The The needle needle was was pushed pushed down down approximately approximately 3 mm 3 inmm depth in depth and movedand moved up and up downand down slowly slowly 10 times. 10 times. The treatmenttreatment modesmodes thatthat werewere performedperformed areare summarizedsummarized asas followsfollows (Figure(Figure1 1B):B): physical physical damage at the centercenter ofof thethe posteriorposterior eyespoteyespot (Mode 1), physicalphysical damage at the posterior side of thethe posteriorposterior eyespoteyespot (Mode 2), physical damage at the proximal side of the posterior eyespot (Mode 3), physicalphysical damagedamage atat thethe centercenter ofof the anterior eyespoteyespot (Mode 4),4), physical damage at the posterior side of thethe anterioranterior eyespoteyespot (Mode(Mode 5), physicalphysical damage at the backgroundbackground area betweenbetween thethe anterioranterior andand posteriorposterior eyespotseyespots (Mode(Mode 6),6), andand physicalphysical damagedamage atat thethe proximalproximal backgroundbackground areaarea (Mode(Mode 7).7). For thethe ball ball placement placement (distortion) (distortion) experiments experiment (Modess (Modes 8 and 8 9), and a stainless 9), a stainless ball (Tsubaki ball Precision(Tsubaki Balls)Precision of 0.50 Balls) mm of in 0.50 diameter mm in (Tsubaki diameter Nakashima, (Tsubaki Katsuragi,Nakashima, Nara, Katsuragi, Japan) wasNara, used Japan) to produce was used wing to tissueproduce distortion wing tissue within distortion 30 min postpupation.within 30 min Apost ballpupation. was placed A ball at the was anterior placed side at the (Mode anterior 8) of side the right(Mode hindwing 8) of the right after thehindwing forewing-lift after the procedure, forewing-l andift procedure, the lifted forewingand the lifted was placedforewing back was into placed the back into the original position. The ball was thus sandwiched between the forewing and hindwing. A ball was also similarly placed at the proximal side of the hindwing where no color pattern element was located (Mode 9). For comparison, the forewing was lifted transiently, and within 1 min, the lifted forewing was placed back into the original position (Mode 10).

Insects 2018, 9, 195 4 of 15 original position. The ball was thus sandwiched between the forewing and hindwing. A ball was also similarly placed at the proximal side of the hindwing where no color pattern element was located (Mode 9). For comparison, the forewing was lifted transiently, and within 1 min, the lifted forewing was placed back into the original position (Mode 10). These treatment modes are summarized as follows (Figure1B): ball placement at the proximal side of the anterior eyespot (Mode 8), ball placement at the proximal background area (Mode 9), forewing-lift control (no ball placement) (Mode 10), and no treatment (Mode 11).

2.3. Data Analysis Because the color patterns of this species are highly variable among individuals, even in females, the color patterns of the treated right hindwing were always compared with those of the non-operated left hindwing of a given individual. The eyespot size change was visually evaluated based on the core disk size of the treated right hindwing in comparison with that of the left hindwing without treatment. The outer black ring and orange ring were not considered for eyespot size evaluation, because they were easily drawn to neighboring damage or distortion, whereas the black core disk appeared to be less sensitive. Similarly, minor deformation that did not contribute to the size change was not considered to be size modiﬁcation. However, phenotypic traits that were different from the left hindwing in an individual were descriptively recorded. Statistical analysis (χ2 test or Fisher’s exact test) was performed using JSTAT (Yokohama, Japan) when necessary. For χ2 tests, statistical values after Yates correction were reported.

3. Results

3.1. Physical Puncture Damage at the Posterior Eyespot In response to physical damage at the center of the posterior eyespot (Mode 1), the treated individuals showed a reduction in the size of the treated eyespot without exception (n = 4) (Figure2A–C). The anterior eyespots showed no change. Interestingly, in a treated individual, the PFE in the proximity of the treated eyespot moved toward the center of the reduced eyespot (Figure2B), although this compromised PFE (cPFE) was not observed in other individuals (Figure2A and C). In response to physical damage at the posterior side of the posterior eyespot (Mode 2), the eyespot slightly reduced (n = 7) or slightly enlarged (n = 3) (Figure2D–F). The reduction (Figure2D, E) may have been because the damage operation wrongly damaged the organizing cells at the center of the prospective eyespot. However, the noncentral damage might have inﬂuenced the eyespot size without physically damaging the organizing cells. The slight enlargement (Figure2F) may have been because of fusion with the ectopic eyespot induced by the treatment. In response to physical damage at the proximal portion of the posterior eyespot (Mode 3), the majority of the treated individuals showed a reduction of the posterior eyespot core disk (n = 23) (Figure2G–K) despite a notable distance from the damage site to the core disk. In some of these individuals, the longitudinal direction of the eyespot changed toward the damaged site. Some individuals showed eyespot enlargement (n = 3) (Figure2L) or no change ( n = 9). In 3 cases, the black area induced by the treatment fused with the outer black ring of the anterior eyespot (Figure2L), in which case the outer black ring of the anterior eyespot was thickened. However, in the other 20 cases, the anterior eyespot showed no change. Insects 2018, 9, 195 5 of 15 Insects 2018, 9, x FOR PEER REVIEW 5 of 15

FigureFigure 2.2. ColorColor pattern pattern modifications modifications induced induced by physicalby physical damage damage at the at posterior the posterior eyespot eyespot in J. orithya in J.. Inorithya each. individual,In each individual, the right the wing right was wing operated was operated on, and the on, left and wing the wasleft wing left intact was left for controlintact for purposes. control Thepurposes. green arrowsThe green indicate arrows the indicate sites of thethe puncturesites of the damage puncture and thedamage induced and patterns the induced around patterns those sites.around The those insets sites. are The enlargements insets are enlargements of the modified of the eyespots. modified (A eyespots.–C) Damage (A–C at) Damage the center at the (Mode center 1). The(Mode eyespot 1). The was eyespot reduced was in size. reduced In B, thein parafocalsize. In B, element the parafocal (PFE) in element the operated (PFE) compartment in the operated was alsocompartment reduced in was size also and reduced dislocated in si towardze and thedislocated center oftoward the eyespot, the cent indicateder of the aseyespot, compromised indicated PFE as (cPFE).compromised (D–F) DamagePFE (cPFE). at the (D posterior–F) Damage side at (Mode the posterior 2). In (D ,sideE), the (Mode posterior 2). In eyespot D and becameE, the posterior smaller. Ineyespot (E), the became outer blacksmaller. ring In was E, the open. outer In (blackF), an ring ectopic was eyespot open. In was F, an produced, ectopic eyespot and the was innate produced, eyespot wasand drawnthe innate to and eyespot fused with was thedrawn ectopic to eyespot.and fused (G –withL) Damage the ectopic at the proximaleyespot. ( sideG–L (Mode) Damage 3). In at most the individuals,proximal side the (Mode eyespot 3). wasIn most deformed individuals, and reduced the eyespot in size. was The deformed entire eyespot and reduced was drawn in size. to The the damageentire eyespot site despite was drawn the size to reduction. the damage In contrast,site despite in (theL), thesize entire reduction. posterior In contrast, eyespot in was L, elongatedthe entire towardposterior the eyespot damage was site, elongated resulting toward in a slight the damage increase si inte, the resulting eyespot in size.a slight The increase pink arrow in the indicates eyespot ansize. additional The pink modified arrow indicates site of thean innateadditional eyespots modified (the fusionsite of ofthe the inna outerte eyespots black ring (the of fusion the anterior of the eyespotouter black with ring the of induced the anterior black ey area).espot with the induced black area). 3.2. Physical Puncture Damage at the Anterior Eyespot 3.2. Physical Puncture Damage at the Anterior Eyespot In response to physical damage at the center of the anterior eyespot (Mode 4), the treated In response to physical damage at the center of the anterior eyespot (Mode 4), the treated individuals showed eyespot reduction in size without exception (n = 7) (Figure3A–C). Interestingly, individuals showed eyespot reduction in size without exception (n = 7) (Figure 3A–C). Interestingly, the posterior eyespot responded by thickening the outer black ring in 4 individuals with treated the posterior eyespot responded by thickening the outer black ring in 4 individuals with treated wings (Figure3A–C). Among these, posterior eyespot enlargement together with the outer black ring wings (Figure 3A–C). Among these, posterior eyespot enlargement together with the outer black ring was observed (n = 1) (Figure3A). However, in 3 individuals, the posterior eyespot core disk slightly was observed (n = 1) (Figure 3A). However, in 3 individuals, the posterior eyespot core disk slightly reduced in size despite the expansion of the outer black ring (Figure3B, C). reduced in size despite the expansion of the outer black ring (Figure 3B, C). In response to physical damage at the posterior side of the anterior eyespot (Mode 5), the majority In response to physical damage at the posterior side of the anterior eyespot (Mode 5), the of the treated individuals showed eyespot reduction (n = 9) (Figure3D). However, enlargement ( n = 1) majority of the treated individuals showed eyespot reduction (n = 9) (Figure 3D). However, enlargement (n = 1) (Figure 3E) and no size change but with deformation (n = 2) were observed (Figure

Insects 2018, 9, 195 6 of 15

Insects 2018, 9, x FOR PEER REVIEW 6 of 15 (Figure3E) and no size change but with deformation ( n = 2) were observed (Figure3F). Among these 123F). individuals, Among these the 12 posterior individuals, eyespot the posterior responded eyes bypot thickening responded the by outer thickening black ring the inouter 8 individuals black ring despitein 8 individuals a notable despite distance a notabl from thee distance center offrom the the eyespot center (Figure of the3 D–F).eyespot (Figure 3D–F). To confirmconfirm thatthat thisthis asymmetricasymmetric thickeningthickening (i.e.,(i.e., drawingdrawing oror attracting)attracting) effect diddid notnot originateoriginate from naturalnatural asymmetric asymmetric variation, variation, hindwings hindwings of no-treatmentof no-treatment individuals individuals (Mode (Mode 11; see 11; below) see below) were examinedwere examined (n = 51). (n = Only 51). Only one individual one individual showed showed an asymmetric an asymmetric enlargement enlargement of the of outer the outer black black ring ofring the of posteriorthe posterior eyespot eyespot that that was was somewhat somewhat similar similar to to the the experimental experimental results. results.The The experimentalexperimental and no-treatment frequencies of of such such modifications modifications were were significantly significantly different different (Fisher’s (Fisher’s exact exact test, test, p p< <0.0001). 0.0001). This This means means that that the the drawing drawing effects effects were were likely likely caused caused by by the the physical puncture damage at the posterior sideside ofof thethe anterioranterior eyespot.eyespot.

Figure 3. Color pattern modifications modifications induced by phys physicalical damage of the anterior eyespot in J.J. orithya orithya.. InIn eacheach individual, individual, the the right right wing wing was was operated operated on, and on, the and left the wing left was wing left was intact left for intact control for purposes. control Thepurposes. green The arrows green indicate arrows sites indicate for puncture sites for damage puncture and damage the induced and the patterns induced around patterns those around sites. Thethose pink sites. arrows The pink indicate arrows additional indicate modified additional sites modified of the innate sites eyespots.of the innate The eyespots. insets are The enlargements insets are ofenlargements the modified of eyespots. the modified (A–C eyespots.) Damage ( atA– theC) Damage center (Mode at the 4). center The eyespots(Mode 4). were The reduced eyespots in were size. Inreduced response in size. to this In treatment,response to the this posterior treatment, eyespot the posterior core disk eyespo was enlargedt core disk (A )was or slightly enlarged reduced (A) or (slightlyB,C). However, reduced in(B,C). all theHowever, cases shown, in all the the cases outer sh blackown, ringthe outer of the black posterior ring of eyespot the posterior was thickened eyespot atwas the thickened side facing at the anteriorside facing eyespot. the anterior (D–F) Damageeyespot. ( atD the–F) posteriorDamage at side the (Mode posterior 5). Inside all (Mode the cases 5). shown,In all the the cases anterior shown, eyespot the anterior was deformed, eyespot and was the de outerformed, black and ring the of outer the posterior black ring eyespot of the facingposterior the anterioreyespot facing eyespot the was anterior thickened eyespot (pink was arrows). thickened (pink arrows).

3.3. Physical Puncture Damage at the Background AreaArea Physical damage of the background area between the anterior and posterior eyespot (Mode 6) inducedinduced a ringring breakbreak ((nn == 4) (Figure4 4A),A),an anectopic ectopiceyespot-like eyespot-like structurestructure fusedfused withwith twotwoinnate innate eyespots (n = 4) (Figure 4 4B,B, C)C) oror aa blackblack areaarea( (n = 2). Physical damagedamage to to the the proximal proximal background background area area (Mode (Mode 7) induced 7) induced a small a orange small areaorange enclosed area byenclosed a black by area a black (n = area 5) (Figure (n = 5) 4(FigureD,E) or 4D,E) a relatively or a relatively large black large area black with area or with without or without an orange an orange area (arean = 3)(n (Figure = 3) (Figure4F). Among 4F). Among these, fusion these, or fusion a drawing or a drawing effect ofthe effect outer of blackthe outer ring ofblack either ring anterior of either or posterioranterior or eyespot posterior was eyespot observed was in observed 6 cases despite in 6 ca ases notable despite distance a notable between distance the between damage the site damage and the eyespotsite and (Figurethe eyespot4D–F). (Figure 4D–F).

Insects 2018, 9, 195 7 of 15 Insects 2018, 9, x FOR PEER REVIEW 7 of 15

FigureFigure 4. 4. ColorColor pattern pattern modifications modifications induced induced by by phys physicalical damage damage to tothe the background background area area in J. in orithyaJ. orithya. In . eachIn each individual, individual, the right the rightwing wingwas operated was operated on, and on, the andleft wing the left was wing left intact was leftfor control. intact for The control. green arrowsThe green indicate arrows sites indicate of puncture sites damage of puncture and the damage induced and patterns the induced around patternsthose sites. around The pink those arrows sites. indicateThe pink additional arrows indicate modified additional sites of the modified innate eyespots. sites ofthe The innate insets eyespots.are enlargements The insets of the are modified enlargements sites. (ofA– theC) Damage modified between sites. (A the–C )two Damage eyespots between (Mode the 6). twoIn A, eyespots the outer (Mode black ring 6). In was (A ),broken, the outer and blackthe entire ring anteriorwas broken, eyespot and was the deformed. entire anterior In B and eyespot C, ectopic was eyespot-like deformed. Instructures (B,C), ectopic were produced eyespot-like and fused structures with thewere anterior produced and posterior and fused eyespots. with the (D anterior–F) Damage and at posterior the proximal eyespots. background (D–F) area Damage (Mode at 7). the In proximal addition tobackground the induced area elements, (Mode the 7). outer In addition black ring to of the the induced innate eyespots elements, is drawn the outer toward black the ring damage of the site. innate eyespots is drawn toward the damage site. 3.4. Ball Placement 3.4. Ball Placement Ball placement at the proximal side of the anterior eyespot (Mode 8) was performed. In this Ball placement at the proximal side of the anterior eyespot (Mode 8) was performed. In this treatment (Mode 8), the ball placement site was close to or inside the anterior eyespot. Among the 10 treatment (Mode 8), the ball placement site was close to or inside the anterior eyespot. Among individuals tested, no change was observed in 5 individuals. The other 5 individuals had some the 10 individuals tested, no change was observed in 5 individuals. The other 5 individuals had changes in either or both eyespots (Figure 5A–C) including anterior eyespot enlargement and some changes in either or both eyespots (Figure5A–C) including anterior eyespot enlargement and deformation (n = 1) (Figure 5A), reduction and deformation (n = 1) (Figure 5B), and deformation deformation (n = 1) (Figure5A), reduction and deformation ( n = 1) (Figure5B), and deformation without a size change (n = 3) (Figure 5C). In these cases, the anterior eyespot deformation toward the without a size change (n = 3) (Figure5C). In these cases, the anterior eyespot deformation toward ball was characteristic pf this treatment. Among these, one individual showed both anterior and the ball was characteristic pf this treatment. Among these, one individual showed both anterior and posterior eyespot enlargement (Figure 5A) and another individual showed a reduced and deformed posterior eyespot enlargement (Figure5A) and another individual showed a reduced and deformed anterior eyespot and an enlarged outer black ring of the posterior eyespot (Figure 5B). In these 2 anterior eyespot and an enlarged outer black ring of the posterior eyespot (Figure5B). In these individuals, the outermost black ring was thickened, possibly toward the ball insertion point. The 2 individuals, the outermost black ring was thickened, possibly toward the ball insertion point. posterior eyespots showed no change in 3 cases that had minor changes in the anterior eyespot The posterior eyespots showed no change in 3 cases that had minor changes in the anterior eyespot (Figure 5C). (Figure5C). To confirm that these asymmetric modifications induced by the ball placement above did not To confirm that these asymmetric modifications induced by the ball placement above did not originate from natural asymmetric variation, the hindwings of the forewing-lift individuals (Mode originate from natural asymmetric variation, the hindwings of the forewing-lift individuals (Mode 10; 10; see below) were examined (n = 30). No individuals showed a similar asymmetric modification, see below) were examined (n = 30). No individuals showed a similar asymmetric modification, and and the results of these two modes were significantly different (Fisher’s exact test; p = 0.0004). This the results of these two modes were significantly different (Fisher’s exact test; p = 0.0004). This means means that the ball placement likely caused the color pattern changes. that the ball placement likely caused the color pattern changes. Ball placement on the background area (the proximal portion of the hindwing) (Mode 9) was Ball placement on the background area (the proximal portion of the hindwing) (Mode 9) was then then performed. In this treatment (Mode 9), the ball placement site was relatively far from the performed. In this treatment (Mode 9), the ball placement site was relatively far from the eyespots, eyespots, being different from the treatment above (Mode 8). No change was observed throughout being different from the treatment above (Mode 8). No change was observed throughout the wings the wings of 27 individuals, but some were modified (n = 22) (Figure 5D-I), resulting in a modification of 27 individuals, but some were modified (n = 22) (Figure5D-I), resulting in a modification rate of rate of 44.9%. However, the modification profiles were as follows: enlargement (n = 6), reduction (n 44.9%. However, the modification profiles were as follows: enlargement (n = 6), reduction (n = 2), and = 2), and no change (n = 14) in the anterior eyespot; and enlargement (n = 17), reduction (n = 1), and no change (n = 14) in the anterior eyespot; and enlargement (n = 17), reduction (n = 1), and no change no change (n = 4) in the posterior eyespot. Five individuals showed some changes in both anterior (n = 4) in the posterior eyespot. Five individuals showed some changes in both anterior and posterior and posterior eyespots (Figure 5D, E, I). Among them, both eyespots were enlarged (Figure 5D,E), eyespots (Figure5D, E, I). Among them, both eyespots were enlarged (Figure5D,E), except in one except in one individual (Figure 5I), where the large innate fusion eyespot was reduced, probably because the most anterior eyespot organizer was inhibited due to the operation.

Insects 2018, 9, 195 8 of 15 individual (Figure5I), where the large innate fusion eyespot was reduced, probably because the most anteriorInsects 2018 eyespot, 9, x FOR organizer PEER REVIEW was inhibited due to the operation. 8 of 15

FigureFigure 5. Color 5. Color pattern pattern modifications modifications induced induced by ball by ballplacement placement and anda transient a transient forewing-lift forewing-lift operation operation in J. orithyain. J.In orithya each individual,. In each individual, the right wing the was right operated wing was on, operatedand the left on, wing and was the left left intact wing for was control left intact purposes. for The greencontrol arrows purposes. indicate The sites green for punc arrowsture indicate damage sites or ball for placement. puncture The damage pink orarrows ball placement.indicate modified The pink sites. (A–Carrows) Ball placement indicate modifiedat the proximal sites. side (A– Cof) the Ball anterior placement eyespot at the(Mode proximal 8). The side insets of are the enlargements anterior eyespot of the modified(Mode eyespots. 8). The ( insetsD–I) Ball are placement enlargements at the of proximal the modified background eyespots. area (D (Mode–I) Ball 9). placement at the proximal background area (Mode 9). 3.5. Forewing-Lift Control 3.5. Forewing-Lift Control The wing-wide changes that were observed in the ball placement experiments above were tested to determineThe wing-wide if they changes were induce that wered by observed the transient in the forewing-lift ball placement procedure experiments without above ball were placement tested to(Mode determine 10). In if theytotal, were 30 indivi inducedduals by thewere transient examined. forewing-lift Surprisingly, procedure 12 individuals without ball showed placement some (Modemodifications 10). In (Figure total, 30 6A–F), individuals and no weremodifications examined. were Surprisingly, observed in 12either individuals the anterior showed or posterior some modificationseyespots in 18 (Figure individuals6A–F), (Figure and no 6G–I), modifications resulting were in a observed modification in either rate theof 40.0%. anterior No or individuals posterior eyespotshad reduced in 18 anterior individuals eyespots, (Figure but6G–I), anterior resulting eyespots in a modification were enlarged rate in of 2 40.0%. individuals No individuals (Figure 6A,D). had reducedOn the other anterior hand, eyespots, 6 individuals but anterior showed eyespots enlarged were posterior enlarged eyespots in 2 individuals (Figure (Figure 6B–D),6 A,D).and 2 Onshowed the otherreduced hand, posterior 6 individuals eyespots showed (Figure enlarged 6E,F). posterior eyespots (Figure6B–D), and 2 showed reduced posteriorThe eyespotshindwings (Figure of individuals6E,F). with no treatment were also examined for comparison (Mode 11). AmongThe hindwings51 individuals, of individuals only 3 individuals with no treatmenthad color werepattern also differences examined between for comparison the right (Mode hindwing 11). Amongand the 51 left individuals, hindwing (Figure only 3 individuals6J–L), resulting had in color a modification pattern differences rate of 5.6%. between the right hindwing and theEach left modification hindwing (Figure should6J–L), be evaluated resulting inin acomparis modificationon to ratethe ofcontralateral, 5.6%. nontreated left wing due Eachto individual modification variation, should bebut evaluated frequencies in comparison of modifications to the contralateral,can be tested nontreated statistically left to wing evaluate due todifferent individual modes variation, of operation. but frequencies The possible of modifications difference can between be tested the statistically modified to evaluateand nonmodified different modesindividuals of operation. (between TheModes possible 9 and 10) difference was compared between using the the modified χ2 test, andresulting nonmodified in no significance individuals (df = 2 (between1, t = 0.037, Modes p = 0.32). 9 and This 10) means was compared that the ball using placement the χ test, at the resulting proximal in nobackground significance area (df did= 1, nott = induce 0.037, pany= 0.32). change. This When means Modes that the10 and ball 11 placement were compared, at the proximalthe difference background was significant area did (df not = 1, induce t = 13.3, any p = change.0.0003). This When means Modes that 10 the and forewing-lift 11 were compared,procedure itse thelf differenceinduced changes. was significant When Modes (df = 9 and 1, t =11 13.3,were pcompared,= 0.0003). Thisthe difference means that was the also forewing-lift significant procedure (df = 1, t =itself 19.5, inducedp <0.0001). changes. Together When with Modes the previous 9 and 11results, were compared,this also means the differencethat the forewing-lift was also significant procedure ( dfitself= 1, causedt = 19.5, thep color<0.0001). pattern Together changes. with the previous results, this also means that the forewing-lift procedure itself caused the color pattern changes.

Insects 2018, 9, 195 9 of 15 Insects 2018, 9, x FOR PEER REVIEW 9 of 15

Figure 6. Color pattern modifications induced by the forewing lift and no treatment controls in J. orithya. Figure 6. Color pattern modifications induced by the forewing lift and no treatment controls in J. orithya. In eachIn each individual, individual, the theright right wing wing was was operated operated on, on,and and the theleft leftwing wing was was left leftintact intact for forcontrol control purposes. purposes. TheThe pink pink arrows arrows indicate indicate modified modified sites. sites. (A– (AF)– FForewing-lift) Forewing-lift operation operation (Mode (Mode 10). 10). Only Only modified modified individualsindividuals are areshown shown here. here. (G–I ()G Forewing-lift–I) Forewing-lift operation operation (Mode (Mode 10). Only 10). nonmodified Only nonmodified individuals individuals are shownare shownhere. (J– here.L) No (J –treatmentL) No treatment (Mode 11). (Mode Rare 11). individuals Rare individuals with asymmetric with asymmetric color patterns color are patterns shown are here.shown here.

4. Discussion4. Discussion

4.1.4.1. Hindwing Hindwing Damage Damage Experiments Experiments ThisThis study study focused focused on onthe the hindwing hindwing eyespots eyespots of ofthe the blue blue pansy pansy butterfly, butterfly, J. orithyaJ. orithya. Previous. Previous damagedamage studies studies have have focused focused on onthe the forewing forewing [5–10,13,14], [5–10,13,14 except], except a brief a brief description description of J. of coeniaJ. coenia [6] [6] andand a recent a recent study study of J. of almanaJ. almana [11],[11 probably], probably because because the the forewing forewing is located is located on onthe the surface surface of a of pupa a pupa and,and, thus, thus, easy easy to manipulate. to manipulate. However, However, at least at least in J. in orithyaJ. orithya, the, the hindwing hindwing color color pattern pattern is simpler is simpler thanthan that that of the of the forewing, forewing, and and therefore, therefore, the the hind hindwingwing deserves deserves the the attention attention of ofdamage damage studies. studies. Previously,Previously, Nijhout Nijhout (1991) (1991) [6] [briefly6] briefly descri describedbed a hindwing a hindwing damage damage experiment experiment using using J. coeniaJ. coenia withoutwithout a rigorous a rigorous method method and and data data presentation, presentation, me mentioningntioning that that the the eyespot eyespot damage damage did did not not elicit elicit anyany changes changes but but the the background background damage damage induced induced an anectopic ectopic eyespot. eyespot. It was It was not not until until Iwasaki Iwasaki and and OtakiOtaki (2017) (2017) [11] [11 that] that the the hindwing hindwing damage damage response response was was systematically systematically studied studied using using J. almanaJ. almana, , demonstratingdemonstrating that that the the hindwing hindwing eyespot eyespot responded responded to damage to damage similarly similarly to the to theforewing forewing eyespot. eyespot. However,However, the the major major hindwing hindwing eyespot eyespot of J. of almanaJ. almana is ais large a large fusion fusion of two of two eyespots, eyespots, which which makes makes it it unsuitableunsuitable for forstudying studying damage damage resp responseonse profiles profiles of simple of simple eyespots. eyespots. In this In this sense, sense, the the present present study study is theis the first first damage damage study study to focus to focus on onsimple simple hindwing hindwing eyespots eyespots in inbutterflies. butterflies. The central damage to the eyespot reduced the eyespot size in both anterior and posterior eyespots in the hindwing of J. orithya (Figures 2A–C, 3A–C), suggesting that organizing cells are located at the center of the hindwing eyespot, which functions as a morphogen source, consistent

Insects 2018, 9, 195 10 of 15

The central damage to the eyespot reduced the eyespot size in both anterior and posterior eyespots in the hindwing of J. orithya (Figure2A–C and Figure3A–C), suggesting that organizing cells are located at the center of the hindwing eyespot, which functions as a morphogen source, consistent with Iwasaki and Otaki (2017) [11]. The present results are in contrast to Nijhout (1991) [6], who suggested a sink (instead of source) of morphogen at the center of an eyespot using J. coenia. If so, this discrepancy may originate from fundamental mechanistic differences in having a morphogen source or sink between the two species. An alternative explanation is that the discrepancy stemmed from developmental heterochrony. In J. almana, late damage at the center of a forewing or hindwing eyespot enlarged the eyespot [10,11] as if the center of the prospective eyespot functioned as a morphogen sink. The late damage response in J. almana can be explained as a summation of the innate and experimentally induced signals, because the organizer is no longer active at the time of damage [10,11]. The J. coenia hindwing may have a developmental time course different from that of the J. almana or J. orithya hindwing. If so, any damage after pupation may be too late to induce any changes in eyespots. However, another explanation is that the cautery treatment in Nijhout (1991) [6] was simply misplaced, for inﬂicting precise damage at the organizer is difﬁcult in the hindwing. In the case of the J. almana damage experiment, some individuals who were subjected to central eyespot damage did not show any changes, probably because of a misplacement of damage [11].

4.2. Long-Range Effects Damage at the eyespot outer black ring or its proximity not only deformed the entire eyespot toward the damage site, but also diminished the core disk despite a notable distance to the central area; the eyespot signal was drawn toward the damage site, and simultaneously, the core disk signal was inhibited (Figure2D,E,G–L and Figure3D–F). Thus, noncentral damage to an eyespot may be able to cause dysfunction of the existing organizing cells in J. orithya. The damage-induced inhibitory signals that traveled long distances likely repressed the function of the organizing cells. However, because the damage site was relatively close to the central area, the possibility that the core disk area was directly damaged in error during the damage operation for the outer rings cannot be excluded completely. Interestingly, when damage was inﬂicted to the posterior side of the anterior eyespot, the outer black ring of the posterior eyespot was thickened and drawn toward the damage site (Figure3D–F). In this case, the damage site was far apart from the posterior eyespot, excluding the possibility that the posterior eyespots were directly damaged in error. Indeed, the posterior eyespot core disk size did not change. Similarly, damage at the middle of the two eyespots induced fusion of the two innate and one induced eyespots (Figure4A–C), and the damage to the proximal background area drew the anterior and posterior eyespots toward the damage site (Figure4D–F). These results showed that not only the induced signals, but also the innate signals are long ranging. The different behavior of the outer parts (the outer black and orange rings) and the core parts (the core disk) of an eyespot may be an example of the uncoupling rule, in which different sub-elements may behave independently [10,24,40].

4.3. Three Essential Damage Effects The effects of damage on color pattern modiﬁcations that were observed in the J. orithya hindwing in the present study may be classiﬁed as one of the following three types: (1) an inhibitory effect on the entire eyespot or the eyespot core disk (when damage was made at the core disk or the outer rings), (2) a drawing (attracting) effect on the outer black ring (when damage was made outside or in proximity to the eyespot), and (3) an inducing effect for ectopic elements on the background area (when damage was made at the background area) (Figure7A). Additionally, fusion was observed between the induced element and the innate eyespots (especially when damage was made at the background area between the two eyespots); however, fusion is not likely a phenomenon that was directly elicited by damage but a secondary phenomenon in which the induced and innate elements interacted with each other. Additionally, fusion is probably mechanistically similar to the drawing effect. Insects 2018, 9, 195 11 of 15

Among the three damage effects, the most fundamental response may be the inducing effect because it is observed in the background area without any interactions with elemental signals. Whether the inhibitory or drawing effect occurs may be dependent on the distance between the induced and innateInsects signals.2018, 9, x WhenFOR PEER the REVIEW two are relatively close, they inhibit each other, and when the two are relatively11 of 15 far away, they attract each other, resulting in the drawing effect or elemental fusion. Homophilic two are relatively far away, they attract each other, resulting in the drawing effect or elemental fusion. interactions (drawing effect) and heterophobic interactions (inhibitory effect) may be two principles of Homophilic interactions (drawing effect) and heterophobic interactions (inhibitory effect) may be signal interactions [10,24], but they may also depend on distance. two principles of signal interactions [10,24], but they may also depend on distance. Interestingly, the three essential effects can be combined in a single case (Figure7B), suggesting Interestingly, the three essential effects can be combined in a single case (Figure 7B), suggesting that the three effects are elicited from a coherent morphogenic signal but act independently. Other that the three effects are elicited from a coherent morphogenic signal but act independently. Other combinations are also observed when two eyespots are considered simultaneously (Figure7C); when combinations are also observed when two eyespots are considered simultaneously (Figure 7C); when the anterior eyespot was damaged and thus reduced in size, the posterior eyespot expressed the the anterior eyespot was damaged and thus reduced in size, the posterior eyespot expressed the drawing effect. Additionally, when an ectopic eyespot was induced in the middle of the two eyespots, drawing effect. Additionally, when an ectopic eyespot was induced in the middle of the two eyespots, the induced eyespot fused smoothly with the innate eyespots, indicating that the inducing effect and the induced eyespot fused smoothly with the innate eyespots, indicating that the inducing effect and fusion occurred simultaneously. fusion occurred simultaneously.

FigureFigure 7. Summary 7. Summary of the ofdamage-induced the damage-induced color pattern color modi patternfications. modiﬁcations. The damage The sites damage are indicated sites are by blueindicated stars. (A by) blueThree stars. essential (A) Three effects: essential inhibitory, effects: draw inhibitory,ing (attracting), drawing (attracting),and inducing and effects. inducing (B) Modificationseffects. (B) Modiﬁcationswith the three with essential the three effects essential combined. effects combined.(C) Responses (C) Responsesof the anterior of the anteriorand posterior and eyespotsposterior to a eyespotssingle damage. to a single damage.

4.4.4.4. Compromised Compromised PFE PFE after after Damage Damage InIn this this study, study, cPFE cPFE was was obtainedobtained (Figure(Figure2 2B).B). ThisThis isis an important example example of of direct direct evidence evidence for forthe the unity unity of the of the border border symmetry symmetry system, system, meaning meaning that PFEs that PFEs and eyespots and eyespots belong belong to the tosame the system. same system.That is, ThatPFE is is, the PFE remote is the “outermost remote “outermost ring” of an ring” eyespot of an [32,33]. eyespot It [should32,33]. be It shouldnoted that be notedPFE did that not PFErespond did not to respondeyespot todamage eyespot in damage previous in studies previous de studiesspite the despite facts that the factsPFE thatwas PFEdislocated was dislocated and even andabsorbed even absorbed to the nearest to the eyespot nearest eyespotin temperature-shoc in temperature-shockk or chemical or chemical treatments treatments [6,15–20] [6 and,15– 20that] and PFE thathas PFE been has considered been considered a part aof part the ofborder the border symmetr symmetryy system system together together with witheyespot eyespot based based on local on localtungstate tungstate treatment treatment [32] [ 32and] and color color pattern pattern analysis analysis [31,33]. [31,33 This]. This PFE PFE paradox paradox has has been been explained explained by bythe the heterochronic heterochronic nature nature of of signal signal release; release; the the PFE signal isis releasedreleased beforebefore thethe eyespot eyespot signal signal is is released,released, and and the the signals signals have have a wave-like a wave-like self-progressing self-progressing nature nature once releasedonce released [24,31 ,33[24,31,33,36].,36]. The cPFE The thatcPFE responded that responded to the physical to the physical damage damage in the present in the present study is study thus surprising.is thus surprising. Although Although this is just this a is just a single case, it probably means that the release timing of the PFE signal is somewhat variable among individuals, and this case supports the current explanation for the PFE paradox, confirming the status of the PFE as a part of the border symmetry system.

4.5. Distortion-Induced Changes

Insects 2018, 9, 195 12 of 15 single case, it probably means that the release timing of the PFE signal is somewhat variable among individuals, and this case supports the current explanation for the PFE paradox, conﬁrming the status of the PFE as a part of the border symmetry system.

4.5. Distortion-Induced Changes A small stainless ball was placed proximally to the anterior eyespot using the forewing-lift method, and a drawing effect toward the ball placement site was observed (Figure5A–C), which was fundamentally similar to the damage results. The fact that the ball placement and needle damage resulted in similar phenotypes is important, because it suggests that tissue distortion (instead of puncture damage) is sufficient to influence eyespot size and shape. The ball placement results here are consistent with those of J. almana [39]. When the ball was placed more proximally, eyespots were enlarged with or without deformation in many cases (Figure5D–I). Surprisingly, wing-wide modifications of both anterior and posterior eyespots were also observed by transient forewing-lift operations without ball placement (Figure6A–F). Thus, it can be concluded that the ball placed at the proximal background area did not contribute significantly to eyespot modification. However, this conclusion does not necessarily mean that distortion does not work at all. Rather, the J. orithya wing tissue is so sensitive to distortion that the forewing-lift operation alone can elicit eyespot changes, and the ball could not overcome this “background” level. In J. almana, a transient forewing-lift operation did not induce any modification [39]. The reason for this species difference is not clear, but the hindwing of J. orithya is likely more sensitive to subtle physical changes.

4.6. Possible Mechanism of Physical Damage and Candidates for Long-Range Morphogenic Signals Because physical damage can produce an eyespot-like ectopic element in the background area and because the induced eyespot can fuse smoothly with the innate eyespots (Figure4), damage-induced signals may be similar to, or even identical to, the innate morphogenic signals in their characteristics. Damage probably not only kills cells, but also introduces distortions in the wing tissue. Hence, ball placement likely causes similar outcomes. In butterfly wing color pattern systems, some candidate morphogens or their related molecules have been identified [41–52]; for example, Wnt has been studied intensively. The spatial mRNA distribution patterns of Wnt family genes in the pupal wing tissues match nicely to many color pattern elements expressed in adult wings [48,51]. This means that Wnt proteins do not travel a long distance before specifying colors, which makes them unlikely candidates for long-range signals that were posited in this study, although they certainly play an important role in color pattern formation. It has been demonstrated that physical damage induces calcium oscillation and waves around the damage site in the pupal wing tissue [53] and that physical damage upregulates at least a few genes that are likely responsible for eyespot formation [41]. Moreover, genes for wound healing and calcium signaling have been detected in prospective eyespot tissues in transcriptome analysis [42]. Similarly, spontaneous calcium waves have been detected that propagate from organizers in the pupal wing tissue [53]. Calcium signals travel long distances from the prospective eyespot centers and from damage sites in butterfly wings [53], and calcium signaling genes are expressed in the prospective eyespot [42]. In the distortion hypothesis, the morphogenic signals are physical distortions that induce calcium waves, followed by gene expression changes [24,39], although a causal contribution of calcium signals to color pattern determination has not been demonstrated yet. Contributions of mechanical force and the extracellular matrix to morphogenic signal propagation in butterfly wings have been proposed based on the contact-mediated eyespot color pattern changes in J. almana [39]. Synergistic interactions between two eyespots or between two innate or damage-induced signals have been revealed in J. almana [11]. The present results further establish the long-range interactive nature of eyespot morphogenic signals in butterfly wings. Insects 2018, 9, 195 13 of 15

However, other possibilities have not been excluded experimentally. For example, damage may activate hemocytes that eliminate cellular debris. A tissue repair process including cell migration and division may also be involved. These cellular responses may inﬂuence ectopic color patterns at a damage site.

5. Conclusions The present study examined color pattern responses to physical puncture damage and distortion on the hindwing of J. orithya. Inducing, drawing, and inhibitory effects were observed, suggesting that morphogenic signals are long-ranging and interactive. The present results are consistent with the distortion hypothesis, but further studies are necessary to reveal the nature of the morphogenic signals for eyespot color pattern determination in butterﬂies.

Author Contributions: J.M.O. conceived of, designed, and conducted the experiments, analyzed and interpreted the data, and wrote the manuscript. Funding: This study was supported by the basic research fund from the University of the Ryukyus and by JSPS KAKENHI, Grant-in-Aid for Scientific Research (C), grant number JP16K07425. Acknowledgments: The author would like to acknowledge the members of the BCPH Unit of Molecular Physiology for their technical assistance and discussions. Conflicts of Interest: The author declares no conflict of interest.

References

1. Spemann, H.M.; Mangold, H. Über Induktion von Embryonalanlagen durch Implantation artfremder Organisatoren. Arch. Mikrosk. Anat. Entwicklungsmechank 2001, 100, 599–638. [CrossRef] 2. Sander, K.; Faessler, P.E. Introducing the Spemann-Mangold organizer: Experiments and insights that generated a key concept in developmental biology. Int. J. Dev. Biol. 2001, 45, 1–11. 3. De Robertis, E.M. Spemann’s organizer and self-regulation in amphibian embryos. Nat. Rev. Mol. Cell Biol. 2006, 7, 296–302. [CrossRef][PubMed] 4. Anderson, C.; Stern, C.D. Organizers in development. Curr. Top. Dev. Biol. 2016, 117, 435–454. [CrossRef] [PubMed] 5. Nijhout, H.F. Pattern formation on lepidopteran wings: Determination of an eyespot. Dev. Biol. 1980, 80, 267–274. [CrossRef] 6. Nijhout, H.F. The Development and Evolution of Butterfly Wing Patterns; Smithsonian Institution Press: Washington, DC, USA, 1991; ISBN 0-87474-917-4. 7. French, V.; Brakefield, P.M. The development of eyespot patterns on butterfly wings: Morphogen sources or sink? Development 1992, 116, 103–109. 8. French, V.; Brakefield, P.M. Eyespot development on butterfly wings: The focal signal. Dev. Biol. 1995, 168, 112–123. [CrossRef] 9. Otaki, J.M.; Ogasawara, T.; Yamamoto, H. Morphological comparison of pupal wing cuticle patterns in butterflies. Zool. Sci. 2005, 22, 21–34. [CrossRef] 10. Otaki, J.M. Artificially induced changes of butterfly wing colour patterns: Dynamic signal interactions in eyespot development. Sci. Rep. 2011, 1, 111. [CrossRef] 11. Iwasaki, M.; Otaki, J.M. Synergistic damage response of the double-focus eyespot in the hindwing of the peacock pansy butterfly. In Lepidoptera; Perveen, F.K., Ed.; InTech: Rijeka, Croatia, 2017; pp. 65–82. ISBN 978-953-51-3659-0. [CrossRef] 12. Brakefield, P.M.; Gates, J.; Keys, D.; Kesbeke, F.; Wijngaarden, P.J.; Monteiro, A.; French, V.; Carroll, S.B. Development, plasticity and evolution of butterfly eyespot patterns. Nature 1996, 384, 236–242. [CrossRef] [PubMed] 13. Nijhout, H.F. Cautery-induced colour patterns in Precis coenia (Lepidoptera: Nymphalidae). J. Embryol. Exp. Morphol. 1985, 86, 191–203. [PubMed] 14. Brakefield, P.M.; French, V. Eyespot development on butterfly wings: The epidermal response to damage. Dev. Biol. 1995, 168, 98–111. [CrossRef][PubMed] Insects 2018, 9, 195 14 of 15

15. Nijhout, H.F. Colour pattern modification by cold-shock in Lepidoptera. J. Embryol. Exp. Morphol. 1984, 81, 287–305. [PubMed] 16. Otaki, J.M. Colour-pattern modifications of butterfly wings induced by transfusion and oxyanions. J. Insect Physiol. 1998, 44, 1181–1190. [CrossRef] 17. Otaki, J.M. Reversed type of color-pattern modifications of butterfly wings: A physiological mechanism of wing-wide color-pattern determination. J. Insect Physiol. 2007, 53, 256–537. [CrossRef][PubMed] 18. Otaki, J.M. Physiologically induced colour-pattern changes in butterfly wings: Mechanistic and evolutionary implications. J. Insect Physiol. 2008, 54, 1099–1112. [CrossRef][PubMed] 19. Otaki, J.M.; Ogasawara, T.; Yamamoto, H. Tungstate-induced color-pattern modifications of butterfly wings are independent of stress response and ecdysteroid effect. Zool. Sci. 2005, 22, 635–644. [CrossRef][PubMed] 20. Serfas, M.S.; Carroll, S.B. Pharmacologic approaches to butterfly wing patterning: Sulfated polysaccharides mimic or antagonize cold shock and alter the interpretation of gradients of positional information. Dev. Biol. 2005, 287, 416–424. [CrossRef] 21. Taira, W.; Otaki, J.M. Butterfly wings are three-dimensional: Pupal cuticle focal spots and their associated structures in Junonia butterflies. PLoS ONE 2016, 11, e0146348. [CrossRef] 22. Iwasaki, M.; Ohno, Y.; Otaki, J.M. Butterfly eyespot organiser: In vivo imaging of the prospective focal cells in pupal wing tissues. Sci. Rep. 2017, 7, 40705. [CrossRef][PubMed] 23. Adhikari, K.; Otaki, J.M. A single-wing removal method to assess correspondence between gene expression and phenotype in butterflies. The case of Distal-less. Zool. Sci. 2016, 33, 13–20. [CrossRef][PubMed] 24. Otaki, J.M. Self-similarity, distortion waves, and the essence of morphogenesis: A generalized view of color pattern formation in butterfly wings. In Diversity and Evolution of Butterfly Wing Patterns: An Integrated Approach; Sekimura, T., Nijhout, H.F., Eds.; Springer: Singapore, 2017; pp. 119–152. ISBN 978-981-10-4955-2. [CrossRef] 25. Nijhout, H.F. The color patterns of butterflies and moths. Sci. Am. 1981, 245, 104–115. [CrossRef] 26. Monteiro, A.; French, V.; Smit, G.; Brakefield, P.M.; Metz, J.A. Butterfly eyespot patterns: Evidence for specification by a morphogen diffusion gradient. Acta Biotheor. 2001, 49, 77–88. [CrossRef][PubMed] 27. Gilbert, S.F. Developmental Biology, 3rd ed.; Sinauer Associates: Sunderland, MA, USA, 1991; ISBN 0-87893-245-3. 28. Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 1969, 25, 1–47. [CrossRef] 29. Wolpert, L. One hundred years of positional information. Trends Genet. 1996, 12, 359–364. [CrossRef] 30. Wolpert, L.; Tickle, C.; Arias, A.M.; Lawrence, P.; Lumsden, A.; Robertson, E.; Meyerowitz, E.; Smith, J.; Lawrence, E.; McClements, M. Principles of Development, 5th ed.; Oxford University Press: New York, NY, USA, 2015; ISBN 978-0-19-870988-6. 31. Otaki, J.M. Color-pattern analysis of eyespots in butterfly wings: A critical examination of morphogen gradient models. Zool. Sci. 2011, 28, 403–413. [CrossRef][PubMed] 32. Dhungel, B.; Otaki, J.M. Local pharmacological effects of tungstate on the color-pattern determination of butterfly wings: A possible relationship between the eyespot and parafocal element. Zool. Sci. 2009, 26, 758–764. [CrossRef][PubMed] 33. Otaki, J.M. Color-pattern analysis of parafocal elements in butterfly wings. Entomol. Sci. 2009, 12, 74–83. [CrossRef] 34. Nijhout, H.F. Elements of butterfly wing patterns. J. Exp. Zool. 2001, 291, 213–225. [CrossRef][PubMed] 35. Otaki, J.M. Color pattern analysis of nymphalid butterfly wings: Revision of the nymphalid groundplan. Zool. Sci. 2012, 29, 568–576. [CrossRef][PubMed] 36. Otaki, J.M. Generation of butterfly wing eyespot patterns: A model for morphological determination of eyespot and parafocal element. Zool. Sci. 2011, 28, 817–827. [CrossRef][PubMed] 37. Otaki, J.M. Structural analysis of eyespots: Dynamics of morphogenic signals that govern elemental positions in butterfly wings. BMC Syst. Biol. 2012, 6, 17. [CrossRef][PubMed] 38. Nijhout, H.F. The common developmental origin of eyespots and parafocal elements and a new model mechanism for color pattern formation. In Diversity and Evolution of Butterfly Wing Patterns: An Integrated Approach; Sekimura, T., Nijhout, H.F., Eds.; Springer: Singapore, 2017; pp. 3–19, ISBN 978-981-10-4955-2. [CrossRef] Insects 2018, 9, 195 15 of 15

39. Otaki, J.M. Contact-mediated eyespot color-pattern changes in the peacock pansy butterfly: Contributions of mechanical force and extracellular matrix to morphogenic signal propagation. In Lepidoptera; Perveen, F.K., Ed.; InTech: Rijeka, Croatia, 2017; pp. 83–102, ISBN 978-953-51-3659-0. [CrossRef] 40. Iwata, M.; Otaki, J.M. Focusing on butterfly eyespot focus: Uncoupling of white spots from eyespot bodies in nymphalid butterflies. SpringerPlus 2016, 5, 1287. [CrossRef][PubMed] 41. Monteiro, A.; Glaser, G.; Stockslager, S.; Glansdorp, N.; Ramos, D. Comparative insights into questions of lepidopteran wing pattern homology. BMC Dev. Biol. 2006, 6, 52. [CrossRef][PubMed] 42. Özsu, N.; Monteiro, A. Wound healing, calcium signaling, and other novel pathways are associated with the formation of butterfly eyespots. BMC Genom. 2017, 18, 788. [CrossRef][PubMed] 43. Carroll, S.B.; Gates, J.; Keys, D.N.; Paddock, S.W.; Panganiban, G.E.; Selegue, J.E.; Williams, J.A. Pattern formation and eyespots determination in butterfly wings. Science 1994, 265, 109–114. [CrossRef][PubMed] 44. Keys, D.N.; Lewis, D.L.; Selegue, J.E.; Pearson, B.J.; Goodrich, L.V.; Johnson, R.L.; Gates, J.; Scott, M.P.; Carroll, S.B. Recruitment of a hedgehog regulatory circuit in butterfly eyespot evolution. Science 1999, 283, 532–534. [CrossRef][PubMed] 45. Monteiro, A.; Chen, B.; Ramos, D.M.; Oliver, J.C.; Tong, X.; Guo, M.; Wang, W.-K.; Fazzino, L.; Kamal, F. Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies. J. Exp. Zool. B Mol. Dev. Evol. 2013, 320, 321–331. [CrossRef][PubMed] 46. Martin, A.; Papa, R.; Nadeau, N.J.; Hill, R.I.; Counterman, B.A.; Halder, G.; Jiggins, C.D.; Kronforst, M.R.; Long, A.D.; McMillan, W.O.; et al. Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand. Proc. Natl. Acad. Sci. USA 2012, 109, 12632–12637. [CrossRef] 47. Martin, A.; Reed, R.D. Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems. Dev. Biol. 2014, 395, 367–378. [CrossRef] 48. Dhungel, B.; Ohno, Y.; Matayoshi, R.; Iwasaki, M.; Taira, W.; Adhikari, K.; Gurung, R.; Otaki, J.M. Distal-less induces elemental color patterns in Junonia butterfly wings. Zool. Lett. 2016, 2, 4. [CrossRef][PubMed] 49. Zhang, L.; Reed, R.D. Genome editing in butterflies reveals that spalt promotes and Distal-less represses eyespot colour patterns. Nat. Commun. 2016, 7, 11769. [CrossRef][PubMed] 50. Mazo-Vargas, A.; Concha, C.; Livraghi, L.; Massardo, D.; Wallbank, R.W.R.; Zhang, L.; Papador, J.D.; Martinez-Najera, D.; Jiggins, C.D.; Kronforst, M.R.; et al. Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity. Proc. Natl. Acad. Sci. USA 2017, 114, 10701–10706. [CrossRef] [PubMed] 51. Özsu, N.; Chan, Q.Y.; Chen, B.; Gupta, M.D.; Monteiro, A. Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies. Dev. Biol. 2017, 429, 177–185. [CrossRef][PubMed] 52. Zhang, L.; Mazo-Vargas, A.; Reed, R.D. Single master regulatory gene coordinates the evolution and development of butterfly colour and iridescence. Proc. Natl. Acad. Sci. USA 2017, 114, 10707–10712. [CrossRef][PubMed] 53. Ohno, Y.; Otaki, J.M. Spontaneous long-range calcium waves in developing butterfly wings. BMC Dev. Biol. 2015, 15, 17. [CrossRef][PubMed]

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