Abstract
Eyespot color patterns in butterfly wings are determined by the putative morphogenic signals from organizers. Previous experiments using physical damage to the forewing eyespots of the peacock pansy butterfly, Junonia almana (Linnaeus, 1758), suggested that the morphogenic signals dynamically interact with each other, involving enhancement of activation signals and interactions between activation and inhibitory signals. Here, we focused on the large double-focus fusion eyespot on the hindwing of J. almana to test the involvement of the proposed signal interactions. Early damage at a single focus of the prospective double-focus eyespot produced a smaller but circular eyespot, suggesting the existence of synergistic interactions between the signals from two sources. Late damage at a single focus reduced the size of the inner core disk but simultaneously enlarged the outermost black ring. Damage at two nearby sites in the background induced an extensive black area, possibly as a result of the synergistic enhancement of the two induced signals. These results confirmed the previous forewing results and provided further evidence for the long-range and synergistic interactive nature of the morphogenic signals that may be explained by a reaction-diffusion mechanism as a part of the induction model for color-pattern formation in butterfly wings.
Keywords
- butterfly wing
- color-pattern formation
- eyespot
- induction model
- Junonia almana
- physical damage
- reaction-diffusion model
1. Introduction
Animal bodies often have conspicuous color patterns such as stripes, dots, and eyespots. For example, various color patterns are notable in shells and fishes, and at least some of them have been explained well by some types of reaction-diffusion (RD) models [1–3]. In such models, activation and inhibitory signals interact based on local self-activation and lateral inhibition [4–6]. A patterning process is initiated randomly, but some of the final outputs, such as zebrafish stripes, are stably constructed. Thus, there is no specific organizer or its associated pre-pattern that is required to construct the final pattern.
In contrast, eyespot patterns that emerge consistently at particular locations in some fish or other species may require organizers, or something similar, that initiate the determination process at particular locations. Although careful adjustment of boundary conditions for RD equations may be able to computationally specify eyespot locations consistently, such a model may not be robust enough to reproduce a given eyespot pattern in every individual under different environmental and genetic conditions during development. As a compromise, a developmental system that involves both predetermined classical organizers (i.e., sources of the putative morphogenic signals) and RD mechanisms might be more realistic. A potential example of such a system is the spotted mandarin fish,
Another developmental system that may require both classical organizers and RD mechanisms is the butterfly color-pattern determination system. Butterfly color patterns are constructed based on three major symmetry systems, two peripheral systems, and other accessory systems [8–14]. Each symmetry system is composed of a collection of color-pattern elements. Among these elements, eyespots that belong to the border symmetry system are probably most conspicuous, at least to human eyes, and developmental mechanisms of eyespots have been studied relatively well. The initial specification of the central location of an eyespot has been successfully described by an RD model based on signals from wing veins in developing wing disk [8], although this model may be too fine-tuned to explain the developmental robustness of actual eyespots [15]. Interestingly, the subsequent determination process of an eyespot after the determination of its central location has been explained by a concentration gradient model, a non-RD model [8, 16, 17]. One of the reasons that the butterfly eyespot formation (except for the initial specification) has been considered a non-RD system may be that the center of the prospective eyespot has been known to behave as an organizer, as demonstrated by the following experiments. Cautery-based damage at the center of the prospective eyespot reduces or completely abolishes the prospective eyespot [18, 19], and transplantation of the central cells produces an ectopic eyespot at the transplanted site [18, 20, 21].
However, it has been noted that gradient models cannot explain the extreme diversity of eyespot morphology in nymphalid butterflies [22]. Moreover, gradient models cannot explain the morphological diversity of serial eyespots on the identical wing surface [23]. Furthermore, the status of parafocal elements as a part of the border symmetry system [10, 11, 22, 23] has not been explained by the previous models. Nijhout [15] recently proposed the grass fire model, in which parafocal elements can be produced together with eyespots by a simple RD system. It would not be surprising for the entire process of the butterfly eyespot determination to be solely based on RD mechanisms. However, another way of thinking about the system is that because an RD model in general does not require the existence of organizers, the butterfly color-pattern formation system may be something more than a simple RD system.
Accordingly, a model that includes both an organizer and the essence of an RD system has been proposed, and it is called the induction model [22–24]. For convenience, the induction model can be divided into two stages: the early and late stages. The early stage involves signal expansion and settlement from an organizer, and the late stage involves short-range activation and long-range inhibition, the essence of an RD model. In this model, the activation signals activate themselves, and the activation signals and inhibitory signals interact with each other. When two activation signals from different sources meet, synergistic enhancement may occur.
It is important to stress that the induction model is based on “inductive reasoning,” meaning that it is based on collective analysis of many actual butterfly eyespot patterns and physiologically induced color patterns [22, 23, 25]. Thus, the induction model can be applied to “non-typical” distorted eyespots and damage-induced changes, which are not explained well by the gradient models [22, 23]. The induction model is essentially a formal model based on observations, experimental results, and integrative logics, and it is not a computational model that introduces many unknown assumptions. It is true that the induction model proposes unknown mechanisms such as mechanistic waves [13], but these unknowns should be tested and replaced, if necessary, with alternative ideas.
Among the data supporting the induction model is that when a prospective large eyespot is damaged, an adjacent small eyespot becomes larger [26]. This result suggests an inhibitory effect from the prospective large eyespot to the small one. In the induction model, the inhibitory signal is upregulated in the edge of the activation signal, based on the principle of the local (short-range) self-activation and lateral (long-range) inhibition [5]. This inhibition signal works on activation signals not only from its own eyespot but also from other eyespot. Because both activation and inhibition signals behave autonomously once released from organizing cells, the inhibitory signal does not have to affect the signal source to make an eyespot smaller.
Another finding supporting the induction model is that the outermost black ring can be uncoupled from its inner core disk when a prospective eyespot is damaged late [25]. This is also explained by autonomous nature of signals that the induction model proposes. An alternative explanation is that two different chemical morphogens are released. This is not compatible with the conventional gradient model [22], and autonomous behavior of parafocal elements, an equivalent element to “eyespot ring,” prefers the induction model [23, 24].
To be sure, this approach is not intended to undermine computational models. Computational models can propose mathematically defined assumption that may be tested systematically, whereas the collective color-pattern analyses were mostly descriptive. However, both approaches are necessary to understand the complexity of butterfly color patterns. A novel and important way to distinguish between the induction model and the gradient model is to examine a fusion eyespot that has two signal sources. A fusion of two eyespots can be explained either by the conventional gradient model or by the induction model. However, the synergistic enhancement by activation signals from two different sources could occur if the induction model (or a similar model) operates (Figure 1A). The synergistic enhancement in the induction model can be achieved if activation signals merge together before the upregulation of inhibitory signals around the activation signals. In other words, the final size of a fusion eyespot is determined not by a simple summation of the two independent sources but by a synergistic enhancement process. Importantly, the synergistic enhancement is most active at the boundary between the two sources, and the resultant fused eyespot would thus tend to become nearly a true circle or slightly vertically elongated (Figure 1A). In contrast, a simple fusion process will often result in a laterally elongated fused eyespot (Figure 1A).
However, it is difficult to distinguish these two mechanistic possibilities simply based on the final morphology of the fusion eyespot alone, given that there may be conditions under which the typical morphology is not attained. For example, when two sources are positioned closely or when two signals are very strong, a simple fusion of the two would produce a near true circle. When the self-activation and synergistic enhancement processes failed to occur for some unexpected kinetic reasons before the upregulation of inhibitory signals, a laterally elongated fusion eyespot may result. Moreover, an essentially indistinguishable morphology will be exhibited by either mechanism at early fusing stages of a pair of eyespots (Figure 1A).
Nonetheless, physical damage at a single focus of a double-focus eyespot may resolve these two possibilities. Damage at a single focus would produce two circular eyespots, a large one and a small one, when a single gradient model is operating (Figure 1B). In contrast, damage at a single focus would produce a smaller but circular fusion eyespot with two foci if the induction model (or something similar) is operating, because of the synergistic enhancement and the global adjustment of the activation signals (Figure 1B). In other words, a double-focus eyespot would behave as if both foci were damaged. Therefore, characterization of the damage response of a double-focus eyespot that is constructed by fusion of two eyespots would test whether the induction model, or something similar, that involves the synergistic signal enhancement is more reasonable than the gradient model.
The best system to test this hypothesis is probably the large dorsal hindwing eyespot of the peacock pansy butterfly,
If the large size of this hindwing eyespot is a product of the synergistic enhancement of the signals from two organizers, mechanical damage at a single organizer could reduce the size of the entire eyespot. That is, when one organizer is debilitated by damage, the other intact organizer would “help” to restore the entire eyespot, although small, from the merged center. The eyespot would be relatively resistant to damage because of the synergistic enhancement process. In the line of this argument, it is possible to test whether the prediction of the induction model is consistent with the damage response of the double-focus eyespots.
Here, the damage response of the dorsal hindwing major eyespot of
2. Materials and methods
2.1. Butterflies
The peacock pansy butterfly,
2.2. Damage applications and image analysis
After prepupation, pupation time was checked repeatedly at intervals of a few hours, and pupae were categorized into three groups based on time post-pupation: 3–6 h (early), 6–12 h (middle), and 12–18 h (or 12–20 h) (late). Mechanical damage was made at specific positions on the right pupal wings (without a forewing lift) using a stainless needle of 0.50 mm in diameter (Shiga Konchu, Tokyo, Japan). A needle was inserted down to approximately 3 mm in depth and moved up and down five or more times before being removed entirely. The contralateral (left) wing was not damaged because it served as an internal control. The damage sites of the hindwing were determined in advance using a different set of pupae by the forewing-lift method performed in this species [29, 31]. The damaged pupae were kept at ambient temperature until eclosion. The adults that eclosed were frozen immediately after pupation. Wing images were obtained using a Canon MG5730 scanner (Tokyo, Japan). Color-pattern changes of the treated wings were evaluated in reference to the normal color patterns of the non-treated wings of the same individuals.
2.3. Definition of focus
In this paper, an eyespot “focus” was defined as a white spot at the central region of an eyespot in a compartment. The white spots do not necessarily correspond to locations of organizers in this species [31] and also in other species [32]. However, because a white spot indicates an approximate location of an organizer in this species, the white spot is conventionally called the focus in this paper.
3. Results
3.1. Anterior damage to the major eyespot
The anterior focus of the major eyespot was damaged at 3–6 h post-pupation (
Similarly, the anterior focus of the major eyespot was damaged, but much later, at 12–20 h post-pupation (
3.2. Posterior damage to the major eyespot
The posterior focus of the major eyespot was damaged at 3–6 h post-pupation (
Then, the core disk was damaged in the posterior side, avoiding the posterior focus, at 6–12 h post-pupation (
3.3. Damage to the outermost black ring of the major eyespot or in its close vicinity
The outermost black ring of the major eyespot was damaged at 6–12 h post-pupation. Because eyespot size and shape were slightly different from individual to individual, damage was made without distinction at the outermost black ring, at the yellow ring, or at the background immediately close to the outermost black ring (
In addition to damage at the distal side of the major eyespot, the proximal side of the major eyespot was damaged at the outermost black ring at 12–18 h post-pupation (
3.4. Damage to the background between the major and minor eyespots
To understand the reactivity of the background, the background between the major and minor eyespots was damaged at 12–18 h post-pupation (
3.5. Double background damage
In the experiments described above, a single site per individual was damaged. Here, to understand possible interactions between damage-induced signals, two sites in the background were damaged. First, two distant sites in the background were damaged, one between the major and minor eyespots and one between the major eyespot and parafocal elements, at 12–18 h post-pupation (
Then, two closely positioned sites in a wing between the major and minor eyespots were damaged at 12–18 h post-pupation (
When two closely positioned sites around the minor eyespots were damaged at 12–18 h post-pupation (
4. Discussion
4.1. Hindwing eyespot response
In the present study, response profiles of the double-focus eyespot and its surrounding wing surface in the hindwing of
4.2. Synergistic response to focal damage
The double-focus eyespot of
Additionally, the late anterior focal damage enlarged the outermost black ring but reduced the size of the inner core disk. The enhancement of the outermost black ring was not restricted to the anterior side; the enlargement was in all directions. This uncoupling behavior between the outermost black ring and the inner core disk within the same eyespot is indeed consistent with the late damage results of the forewing eyespot [26]. This uncoupling response can be explained if the normal signals are wave-like (which means that the signals behave independently from their source once released) and if the induced signal was added to the normal signal for the outermost black ring. The normal signal for the inner core disk was being released at the time of damage, and because some of the organizing cells were destroyed by physical damage, the inner core disk became smaller. This uncoupling response is an indication of independence of the signal for each sub-element (i.e., the outer black ring and the inner core disk). The wave-like nature of signals is also highlighted in these results. These results are not explainable by the gradient model.
4.3. Response to other types of damage
Semi-focal damage at the anterior side produced a yellow area inside the inner core disk, which is also consistent with the forewing results [26]. This result is also difficult to explain using a gradient model. A threshold increase in response to damage may be a remedy, but a threshold decrease should also be introduced to explain the induced black area in the background. Furthermore, the induced double-ring structures in the background require multiple threshold sets to be explained by the gradient model. These damage-induced rings have been shown to have scale structures that are similar to those of normal eyespots [31]. These complicated threshold arrangements are too complex to accept as a theoretical framework for color-pattern determination in butterfly wings.
Interestingly, the white focal area was elongated toward the damage site in the semi-focal damage. Notably, developmental signals for the white “focal” spot and the eyespot body to which that white spot belongs do not have to be identical [31, 32]. Indeed, the white focal spot is likely uncoupled from the rest of the sub-elements [32].
Damage at or around the outer black ring produced various results. A small black ring was produced in the yellow ring in some cases, but in other cases, the inner core disk, the yellow ring, and the outer black ring were often “pinched off” from the normal shape of the eyespot, suggesting that the ectopically induced signals are able to merge with natural signals locally. In other words, spontaneous and artificially induced signals are indistinguishable to developing scale cells. Furthermore, the extrusion of both the outermost black ring and the inner core disk toward a damaged site suggests that serial lateral interactions keep their shapes, which is reminiscent of the eyespots of the spotted mandarin fish [7]. In addition to these local effects, overall shape changes of the treated eyespots were often observed, although not extensively in response to this manipulation.
4.4. Synergistic response to double background damage
Double-damage experiments that produced extensive black areas confirmed that the induced signals at two sites can be combined to produce strong effects. It is likely that when two sites of damage were close enough, the induced area was more than a simple summation of two areas induced independently by two single damage treatments. These results can be interpreted as evidence for synergistic enhancement of two artificially upregulated signals in the hindwing of this species. This synergistic enhancement process may also occur spontaneously in the double-focus eyespot during development.
In some cases, the black signals induced by double damage merged with the outermost black ring of the natural eyespot, resulting in the rupture of the eyespot. This result again demonstrates the indistinguishability of the natural and induced signals. Somewhat surprisingly, the large black area highlighted one of the “inhibitory areas” that are usually invisible but associated with parafocal elements and the minor eyespot. That is, in an individual lacking the distinct minor eyespot, the induced black area could not invade the area surrounding the minor eyespot. This invisible area might have arisen if the inhibitory signal became stronger and larger in that area than the activation signal for black areas during development of the minor eyespot. Similarly, the induced black signal could not make contact with parafocal elements, suggesting that the inhibitory signals are present along parafocal elements. The similar inhibitory area that surrounds an eyespot has generally been termed the imaginary ring [13, 22]. The reason that the imaginary ring was not observed around the major eyespot is not well understood, but development of an imaginary ring around the major eyespot may require additional time before the end of the pattern determination period.
4.5. Possible mechanisms
Overall, these results strongly suggest that the signals that determine the final scale color of a given scale cell are highly dynamic. It is likely that a reaction-diffusion mechanism, as a part of the induction model, operates in the butterfly color-pattern determination system. The induction model consists of two stages. The early stage is a dissipation of signals from their source, and the late stage is essentially a reaction-diffusion mechanism that involves short-range activation and long-range inhibition. It is speculated that calcium signals play an important role in color-pattern determination in the late stage of the induction model; calcium signals traveling on the developing pupal wings have been observed [33]. On the basis of a linear relationship between scale size and cell size [34–36] and a relationship between scale color and scale size [31], it has been proposed that the putative morphogenic signals from organizers are ploidy signals that determine cellular size via polyploidization [31]. Calcium signals may play an important role in polyploidization.
The early stage of the induction model proposes that a signal moving slowly from its source is the original morphogen that subsequently triggers calcium waves as an activation signal. It is speculated that this slow-moving signal is waves of mechanical distortion [25]. Importantly, organizing centers are present as physical bumps or indentations [32, 37]. These organizing centers can be identified as the pupal cuticle spots in pupae [38, 39]. Based on this fact and other observations, the distortion hypothesis has been proposed, in which physical distortion of the wing tissue functions as the primary morphogenic signal [13]. Physical distortion of the wing epithelial sheet will be created when cells at the organizers selectively increase their sizes via an increase in cell number or polyploidization. Ecdysone receptor is expressed in focal cells in
In physical damage experiments, mechanical distortion of the wing epithelial sheet is probably introduced, nicely mimicking the natural developmental process that involves physical distortion waves. Although there are some classical histological studies on developing wing tissues of butterflies and moths [34, 43, 44], real-time live imaging studies have just begun on developing wing tissues and organizing cells [30, 37, 45]. The distortion hypothesis should be tested in the future in light of the importance of mechanical forces in development [46, 47]. Compatibility of these proposed mechanisms with other related mathematical models for eyespot focus determination [48–50] is also to be investigated in the future.
5. Conclusions
The present study provided experimental evidence that morphogenic signals for eyespot color patterns are able to synergistically interact with each other, focusing on the damage-induced color-pattern changes of the double-focus eyespot in the hindwing of the peacock pansy butterfly,
Acknowledgments
The authors greatly thank Masaki Iwata and other members of the BCPH Unit of Molecular Physiology for technical help and discussion. This project was supported by the basic research fund from University of the Ryukyus and by JSPS KAKENHI, Grant-in-Aid for Scientific Research (C), Grant number 16K07425. MI conducted the experiments and analyzed the data. JMO designed and conducted the experiments, analyzed the data, and wrote the paper. The authors declare that they have no competing interests.
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