Cell death and wing reduction during the metamorphosis of sex‐specific flightless morphs in winter geometrid moths

Winter geometrid moths show striking sexual dimorphism by having female‐specific flightless morphs. The evolutionary grades of wing reduction in winter geometrid moths vary and range from having short wings, vestigial wings, to being wingless. Although the ontogenetic processes underlying the development of the wingless or short‐wing morphs in Lepidoptera has been well studied, the mechanisms underlying the development of vestigial wing morphs in winter geometrid moths during metamorphosis are poorly understood. In the winter geometrid moth Sebastosema bubonaria Warren, 1896, the males have functional wings, but the females have vestigial wings. Here, we studied the ontogenetic processes affecting wing reduction in the winter geometrid moth S. bubonaria using light microscopy and transmission electron microscopy, and compared the ontogenetic process of wing reduction in this species with that in another species of the wingless‐female winter moth that we investigated previously. Our results showed that, in the vestigial‐wing morphs, the loss of pupal wing epithelium was terminated in the middle of the wing degeneration process, whereas in the wingless morph, the pupal wing epithelium disappeared almost completely and the final appearance of the wings differed slightly among flightless morphs. We propose that the extent and location of cell death in the pupal wing play an important role in the various patterns of reduced wings that are observed in flightless moths.


| INTRODUCTION
The study of the evolution of flightless and wingless insects, which began as an investigation into rudimentary organs by Darwin (1859), has become a central research theme in evolutionary biology. This adaptation is particularly striking and is linked to a wide variety of physiological and behavioral changes that have important ecological/ evolutionary implications. In terms of the evolutionary histories of insects, wingless, brachypterous, and short-winged morphs are considered to be secondarily derived from winged morphs, and the development of these morphs has been interpreted to be an evolutionary adaptation to increasing fecundity (Roff, 1990;Wagner & Liebherr, 1992).
Among insects, members of the order Lepidoptera are well suited for studying the evolution of flightlessness. Female-specific wing degeneration occurs in many groups, and morphs with either short or vestigial wings, and even wingless morphs, have been reported in 26 families (Sattler, 1991). In particular, female-specific wing reduction is known to occur in the family Geometridae (Hackman, 1966;Heppner, 1991;Hunter, 1995;Sattler, 1991), and this phenomenon has been observed in the three recognized subfamilies of Alsophilinae, Larentiinae, and Ennominae, all of which have adults that are active in the winter season (Yamamoto & Sota, 2007). Among these lineages of winter geometrid moths, the evolutionary grades of wing reduction range from having short wings, vestigial wings to being wingless (Nakajima, 1998).
Most winter geometrid moth species are nocturnal, but in some species, adult males actively search for mates on sunny days (Nakajima, 1998). In Sebastosema bubonaria Warren, for example, adult males have functional wings and fly only on sunny days, whereas the wings of the females are vestigial. This species is univoltine and the adults typically emerge from late February to early April in Japan. The larvae feed on the leaves of the limited number of plant species that are available and then pupate just below the ground surface from late April to early May (Nakajima et al., 2017). The pupae enter into a short diapause after pupation after which pupal-adult metamorphosis is initiated.
It has been known that programmed cell death is essential for tissue atrophy and remodeling during molting and metamorphosis of insects. In particular, ecdysone-mediated larval or pupal tissue degradation is spatiotemporally regulated and involves both apoptotic and autophagydependent cell death (Xu et al., 2020). We have previously shown that programmed cell death plays an important role in pupal wing degeneration in some lepidopteran moths (Niitsu, 2001;Niitsu & Kamito, 2021Niitsu & Kobayashi, 2008;Niitsu et al., , 2011Niitsu et al., , 2014. Another species of winter-geometrid moth, Nyssiodes lefuarius, is also diurnal and the females are wingless. In this species, the process of wing degeneration and its physiological mechanisms have been studied in detail (Niitsu, 2001;Niitsu & Kamito, 2022;Niitsu et al., 2014). Briefly, the wing discs of the larvae develop until pupation, but the pupal wing then decreases rapidly in size due to apoptotic and autophagic cell death, and is finally lost during the pupal-adult transition (Niitsu & Kamito, 2022;Niitsu et al., 2014).
Although the ontogenetic process of wing reduction in lepidopteran insects has been studied extensively (Lobbia et al., 2003;Nardi et al., 1991;Niitsu, 2001Niitsu, , 2003Niitsu & Kamito, 2021Niitsu & Kobayashi, 2008;Niitsu et al., , 2011Niitsu et al., , 2017Niitsu et al., , 2014, the detailed mechanisms of how vestigial wings are formed during metamorphosis in winter geometrid moths remain poorly understood. To clarify the ontogenetic process of wing reduction in the vestigial-winged morph, we used the diurnal winter moth S. bubonaria as a model insect and examined the cellular events that occur during pupal-adult development by light microscopy (LM) and transmission electron microscopy (TEM). We compared the ontogenetic process of wing reduction in this species with that in another winter geometrid moth species which we investigated previously. Based on the obtained results, we discuss the possible evolutionary implications of flightless morphs in winter geometrid moths.

| Insects
The study organism, S. bubonaria Warren, 1896, is regarded as the rarest winter geometrid species in Japan and is currently listed as critically endangered in the Japanese Red List compiled by the Ministry of Environment (MOE) (Sakamoto & Jinbo, 2022). Despite the endangered status of the species, our field studies did not require permission from the MOE to collect specimens. Adult females of S. bubonaria were captured in Yanagida-cho, Utsunomiya City, Tochigi Prefecture, Japan, and their offspring were used for the experiments.
Newly hatched larvae were reared under long photoperiod conditions (16L-8D) at 20°C on fresh leaves of Celastrus orbiculatus Thunb.
Under these conditions, pupae entered a short period of diapause immediately after pupation. After 2 weeks, the pupae began to undergo pupal-adult development.

| Observations of adult wings
The forewings and hindwings were removed from a dried male specimen and photographed (TG-6; Olympus). The forewings and hindwings of female adult specimens were removed from the wing base under a stereomicroscope (S9i; Leica). Images were taken with the digital camera built into the same microscope.

| Measurement of pupal length
At 3 months after pupation, the longitudinal dimension of each pupa was measured with digital calipers and images were taken (TG-6; Olympus). Line drawings were prepared on tracing paper using drafting pen (Rotring).

| Preparation of wing tissues
The pupal wings were dissected in a dissection dish (Bull Precision Inc.) and any attached tissues were carefully removed in cold phosphatebuffered saline (PBS; 137 mmol L −1 NaCl, 8.10 mmol L −1 Na 2 HPO 4 , 2.68 mmol L −1 KCl, 1.47 mmol L −1 KH 2 PO 4 , pH 7.4). The pupal wings were observed under a stereomicroscope and photographed (TG-6; Olympus). Three pupae of both sexes were examined at each stage.
Next, the dissected wings were fixed in Karnovsky's fixative (2% paraformaldehyde + 2.5% glutaraldehyde) followed by fixing in 1% osmium tetroxide. After dehydrating the samples through an ethanol series and propylene oxide, the tissues were embedded in Epon 812 (TAAB). Semi-thin sections (1-μm thick) were sectioned perpendicularly to the wing surface along the longitudinal axis of the wing and stained with Azure B. These sections were observed under a light microscope (DM 750; Leica).
For the TEM examinations, the female pupal wings were sectioned (90-nm thick) with an ultramicrotome equipped with a diamond knife.
Sections were stained with 4% uranium acetate for 20 min and then with 0.4% lead citrate for 10 min. The sections were observed with an electron microscope (JEM 1400 Plus; JEOL Inc.) at 80 kV.

| Color patterns of wings in male and female adults
The wings of S. bubonaria exhibited conspicuous sexual dimorphism

| Morphological observations of developing pupal wings
To clarify the ontogenetic process of wing reduction in the pupal wings of both sexes, we dissected the developing wings during the early pupal stage and observed the wing epithelia of each sex. At Day 5 after pupation, compared to males, the wing tracheal pattern of the females was difficult to distinguish due to strong similarity ( Figure

| LM observations of early pupal wings
Histological observations were performed. At Day 5 after pupation, the pupal cuticle of the forewing in both sexes was still thick and the wing epithelia were tightly attached to the pupal cuticle (Figure 5a,b).
The cells of the dorsal wing epithelium were comprised of a single layer that formed a cylindrical structure. No sign of adult development was observed and histological differences between the sexes were not yet apparent. At Day 24 after pupation, the wing epithelium became detached from the pupal cuticle (Figure 5c,d). In males, the epithelial monolayer thickened, but typical morphogenesis was not visible (Figure 5d). In females, columnar wing epithelial cells are transformed into a rather thin single layer of cells. A number of phagocytes were observed engulfing cellular debris (Figure 5c). Our LM observations showed that the regression of wing epithelial tissue begins immediately after the wing epithelial cells detach from the pupal epithelium at the onset of adult development.

| TEM observations of developing pupal wings
To examine the cellular changes in the pupal wings just before scale formation in both sexes, ultrastructural observations were performed using TEM. At Day 24 after pupation, cellular debris was visible in females ( Figure 6a). However, despite cellular degeneration, the condensation of chromatin was not observed. Clumps of rough endoplasmic reticulum (rer) were also abundant near the nuclei of the wing epithelial cells in both sexes (Figure 6a,b). In addition, the arrangement of nuclei in the wing epithelium of males appeared normal

| DISCUSSION
In this study, we used the winter geometrid moths, S. bubonaria, as a model insect, focusing specifically on the vestigial wing morphology exhibited by females. Our investigation has provided us with several novel viewpoints that have not been previously discussed. Based on the results obtained from both LM and TEM observations, we detected numerous autophagic vacuoles and some autophagosomes. Furthermore, we also observed phagocytosis in the degenerated wing epithelial cells. Ultrastructural observations also revealed the existence of cellular debris derived from the wing epithelium and abnormal mitochondria in the pupal wings of females during wing degeneration. These unique structures were also observed in degenerating wing epithelia in the wingless-female winter moth N. lefuarius (Niitsu & Kamito, 2022).
Throughout this study, we did not detect any of the morphological features typically associated with apoptosis, for example, condensed chromatin structures in the degenerated epithelial cells of females during the wing degeneration process. However, our observations clearly showed that evidence of autophagy. Based on the LM and TEM observations, we considered that programmed cell death caused rapid cellular degeneration of the wing epithelium of female S. bubonaria during the pupal-adult transition. Our histological findings also showed that the cell degeneration process in the vestigial-winged morph of the female winter moth S. bubonaria is similar to that observed in the wingless morph of the female winter moth N. lefuarius (Niitsu, 2001;Niitsu & Kamito, 2022;Niitsu et al., 2014) and the wingless-female tussock moth Orgyia leucostigma (Nardi et al., 1991). Our findings also showed that the loss of the pupal wing epithelium in S. bubonaria was terminated in the middle of the wing degeneration process, whereas in F I G U R E 3 Sebastosema bubonaria, lateral view of pupae. Left: female, right: male.
F I G U R E 4 Sebastosema bubonaria, optical micrographs of the pupal forewings at various stages of development. The pattern of the pupal wing trachea in the female (a) is the same as that in the male (f) at Day 5 after pupation. After adult development was initiated, the pupal wing of females degenerated (b-e). Immediately before adult emergence (e, j). The pupal wing of males formed normally from Day 5 (f) to 9 months (j) after pupation. The sections a-aʹ, b-bʹ, f-fʹ, and g-gʹ are shown in N. lefuarius, the pupal wing epithelium disappeared almost completely and the final wing shape differed between the species. Furthermore, previous studies showed that degeneration of the pupal wing in wingless female moths is a result of massive cell death occurring in a short period in various species (Lobbia et al., 2003;Nardi et al., 1991;Niitsu, 2001;Niitsu & Kamito, 2022;Niitsu et al., 2014). Niitsu and Kamito (2022) reported that in addition to apoptotic and autophagic cell death, abnormal mitochondria were visible during wing reduction in the wingless female winter moth N. lefuarius. Although the adults of these flightless moths typically have wings that are reduced or absent, they have fully developed pupal wings. This characteristic implies that the ancestral female had well-developed adult wings. Once developmental plans are acquired over the course of evolution, they cannot be easily Unlike the short-winged female adults of Protalcis concinnata (Wileman), the moths N. lefuarius and S. bubonaria have long been considered to be phylogenetically analogous due to similarities in their morphological and ecological characteristics. However, it is not known whether S. bubonaria and N. lefuarius share a common ancestor or whether they are phylogenetically distinct lineages. Whether the similarities in these morphs (i.e., wingless or vestigial) arose via similar evolutionary pathways of wing loss, as a result of parallel evolution, or as steps in a continuous transformation series can be discussed only once a robust phylogeny of the family is derived. In most winter geometrid moths, females have small or vestigial wings and their color patterns are similar to those of males. However, in S. bubonaria, the wing color pattern of females does not resemble that of males. We speculate that the evolutionary processes affecting sexual differentiation of the wing color pattern in S. bubonaria arose first, and that this was then followed by sex-specific wing degeneration. Future studies involving molecular phylogenetic analyses are therefore needed to better clarify the evolutionary history of these groups.
The shape of the future adult wing is defined by the position of the BL, which runs parallel to the entire periphery of the pupal wings. The cells in the region distal to the BL degenerate and disappear during early pupal development (Dohrmann & Nijhout, 1988;Fujiwara & Ogai, 2001;Kodama et al., 1995), and surviving cells in the region proximal to the BL form the adult wing. While the peripheral degeneration of developing adult wings is specific to Lepidoptera and is widely observed in numerous butterflies and moths, several exceptions exist among flightless female moths. In adults of the winter moth P. concinnata (Wileman), females have short, but not vestigial, wings, and males have fully developed wings. In this study, the shape of the sexually dimorphic  Autophagosome and abnormally shaped mitochondria are present (f). The dorsal surface is at the top in (a-f). Scale bars = 5 µm for (a, d), 2 µm for (b, e), 1 µm for (c), and 500 nm for (f). a, autophagic vacuole; am, abnormally shaped mitochondria; ap, autophagosome; bl, basal lamina; l, lysosome; n, nuclei; p, phagocyte; rer, rough endoplasmic reticulum; to, tracheole. wings is determined by the position of the BL, which is positioned farther inward from the distal edge in developing adult females than in males. In a previous study, we found that (1) the cells outside the BL region undergo massive cell death in females, and (2) the region proximal to the BL forms the short wing (Niitsu & Kamito, 2021). In this study, we classified the female morphs showing this pattern of wing development as being short-winged morphs. In the vestigial-winged females of S. bubonaria, massive cell death of the pupal wing epithelium occurs both within and outside of the BL region during pupal-adult metamorphosis, and the loss of pupal wing epithelium was terminated in the middle of the wing degeneration process. We classified the female morphs showing this pattern of wing development as being vestigial-winged morphs. In the wingless female morphs of N. lefuarius, massive cell death of the pupal wing epithelium occurs within and outside the BL region during pupal-adult metamorphosis, and the pupal wing epithelium disappeared almost completely. We classified female morphs showing this pattern of wing development as being wingless morphs. We, therefore, propose that three patterns of wing reduction exist in winter geometrid moths, all of which arise due to massive cell death occurring in different wing locations (Figure 7). Based on these findings, we propose the following hypothesis. The three patterns of wing reduction in females, that is, short-winged morphs, vestigial-winged morphs and wingless morphs, share the same basic BL pattern as the full-winged morphs. These basic BL patterns represent the ancestral BL pattern before the occurrence of wing reduction over evolutionary time. If wing reduction can be explained as a consequence of gradual evolution, then it is assumed that wings regressed from short wings to either vestigial or no wings (wingless).
However, explaining how the development and spatial characteristics of the BL affect the evolutionary transition toward the various flightless forms is difficult. Therefore, we speculate that the massive cell death observed in the female wings basal to the BL might be caused by a deviation in the developmental program and that this deviation affects the BL position of the pupal wings during pupal-adult metamorphosis. It has been reported that the transcription factor Cut and the messenger RNA of the signaling molecule wingless (wg) are coexpressed in the BL region and in the wing margin during wing development in Lepidoptera (Macdonald et al., 2010). It is assumed that elucidation of the expression patterns of Cut and the wg gene during massive cell death in pupal wings might provide a better understanding of the genetic background of female-specific wing degeneration in the Lepidoptera.

| CONCLUSION
In this study, our results showed that a third pattern of wing degeneration exists in females of the winter geometrid moths; we refer to females showing this pattern of wing reduction as vestigial wing morphs. This study showed, for the first time, that, the loss of pupal wing epithelium in vestigial morphs was terminated in the middle of the wing degeneration process, whereas in the wingless morph, the pupal wing epithelium disappeared almost entirely and the final wing shape differed among each flightless morph. We propose that the extent and location of cell death in the pupal wing play significant roles in the various patterns of wing degeneration that are observed in flightless moths. Our findings might provide new insights into understanding the evolution and diversity of flightless morphs with reduced wings in insects.  The BL indicates the boundary of the future adult wing. Peripheral tissues in the region degenerating due to programmed cell death are indicated by black plus signs (a-d). In the short-winged morph (b), the BL is positioned farther inward in the female than in the male, and the cells outside the BL region undergo massive cell death in both sexes. In the vestigial-winged and wingless morphs (c, d), massive cell death not only occurs outside the BL region but also within the BL region. Red arrows indicate the location of wing formation. Black arrows indicate the location of wing degeneration. The blue dotted lines (b-d) indicate the ancestral BL patterns. BL, bordering lacuna; t, trachea. experimental procedures. This work was supported by a Grant-in-Aid from JSPS KAKENHI (Grant No. JP19K06791 to SN and JP21H02215 to MY).

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in miyakodori at https://tokyo-metro-u.repo.nii.ac.jp/. The data that support the findings of this study are available from the corresponding author upon request.

PEER REVIEW
The peer review history for this article is available at https://www. webofscience.com/api/gateway/wos/peer-review/10.1002/jmor.