Comprehensive comparative morphology and developmental staging of final instar larvae toward metamorphosis in the insect order Odonata

The order Odonata (dragonflies and damselflies) is among the most ancestral groups of winged insects with drastic morphological changes upon metamorphosis, and thus important for understanding evo-devo aspects of insects. However, basic developmental descriptions of Odonata have been scarce. In an attempt to establish the foundation of developmental and experimental biology of Odonata, we present an unprecedentedly comprehensive survey of dragonflies and damselflies, in total 158 larvae representing 49 species and 14 families, wherein morphological changes of all the final and/or penultimate instar larvae were photographed and monitored everyday. Although their morphology and development were diverse, we consistently identified two visually recognizable morphogenetic events in the final larval instar, namely start of wing expansion and onset of melanization on the wing sheaths, thereby categorizing the final instar into three stages. While the duration of the first stage ranged 4–66 days across diverse Odonata species, the second or third stages exhibited relatively small variation ranging 3–22 days or 1–8 days, respectively, probably reflecting the steady and irreversible metamorphosis process after stage 2. We also described other characteristic morphological changes during the larval development, although they were observed only in some Odonata species and lineages.

www.nature.com/scientificreports/ Hence, in an attempt to establish the foundation of developmental and experimental biology of Odonata, we performed an unprecedentedly comprehensive morphological survey of dragonflies and damselflies. We everyday recorded the morphological changes during final and/or penultimate larval instar using 158 individuals of 49 Japanese Odonata species representing 14 families. From the enormous amount of morphological data based on approximately 5000 photos, the following two morphological changes, namely "the start of wing expansion" and "the onset of melanization on the wing sheaths", emerged as easily and commonly recognizable developmental indices across the diverse Odonata species. Based on these indicators, we propose that the final larval instar of Odonata could be generally classified into three developmental stages. Other morphological changes in the compound eyes, heads, larval labia, and wing sheaths were also described in detail, although they were observed in more specific Odonata lineages and species.

Methods
Terminology. Following the majority of previous publications, we described the final larval instar as F−0 (= Final) and the penultimate larval instar as F−1 (= F minus 1), because the number of larval instars is not fixed in many Odonata species 3,12 . We started daily photographing of each larva on the day of ecdysis unless otherwise mentioned (Table S1), where the "day 1" denotes the day on which photographing started for each instar.

Melligomphus viridicostus
Gomphidae Gomphidae www.nature.com/scientificreports/ The larvae were individually kept in petri dishes with water in the laboratory. The damselfly larvae (suborder Zygoptera) were fed with Artemia brine shrimps, whereas dragonfly larvae (suborders Anisozygoptera and Anisoptera) were fed with Chironomus midge larvae. Soon after feeding cessation of the F−0 instar larvae prior to the melanization on the wing sheaths, we transferred them individually to a plastic cage for adult emergence as described previously 12 .

Species identification.
Most Japanese Odonata species were identified by their morphological characters 8,9 , while some species were identified by their nuclear DNA sequences as described below. We extracted DNA from a caudal gill of Zygopteran larvae or a pair of forelegs of newly-emerged Anisopteran adults by using QIAamp DNA Mini Kit (Qiagen). Nuclear ITS1 region was PCR-amplified from the DNA samples using the primers ITS-F0 (5′-GGA AAG ATG GCC AAA CTT GA-3′) and 5.8S-AS1 (5′-GCC GGC CCT CAG CCA G-3′) 21 . PCR-amplified products were subjected to DNA sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130xl Genetic Analyzer (Applied Biosystems). Nuclear ITS sequences of all the Japanese Odonata species have been previously deposited in the public database 22 .
Photographing. F−0 and/or F−1 instar larvae were photographed everyday using a stereoscopic microscope S8APO (Leica Microsystems) with a digital high definition microscope camera MC120HD (Leica), a stereoscopic microscope S9D (Leica Microsystems) with a digital high definition microscope camera MC190HD (Leica), or a stereoscopic microscope SZ-6850T (Relyon) with a digital high definition microscope camera TrueChrome Metrics (Relyon). Most larvae were photographed from the day of ecdysis, whereas some larvae were photographed from the day of collection, when the insects were already F−0 instar (See Table S1). The obtained photographs were adjusted by using software GIMP (GNU Image Manipulation Program) 2.10.6.

Species no. Family Species
The number of individuals Chlorogomphus brunneus

Results and discussion
Three developmental stages during F−0 instar commonly observed among diverse Odonata species. We took photos of 158 Odonata larvae (150 F−0 instar and/or 22 F−1 instar) of 49 species everyday ( Fig. 1; Tables 1, S1), and all the 4979 photos were aligned for comparison of morphological characters within and between the species (Figs. 2; S1, S2, S3). First, we compared daily morphological changes of F−0 instar larvae thoroughly. The morphological indicators used for larval staging were selected on account of the following requirements: (i) the morphological changes can be observed without damaging the larvae, (ii) the changes are discontinuous rather than gradual so that the specific timing of entry to the next stage can be clearly defined, and (iii) the indicators should be applicable to as many species as possible. The most prominent morphological changes commonly observed in almost all the examined species were "the start of wing expansion" and "the onset of melanization on the wing sheaths". Based on these indicators, the F−0 larval instar could be classified into three developmental stages for most of the examined Odonata species (Figs. 3, S4; Table 1). In stage 1, the wing sheaths are flat without noticeable structures and pigmentation. The tips of the forewings are hidden under the hindwings (Figs. 3B, S4). In stage 2, the wing sheaths begin to expand. We can distinguish stage 2 from stage 1 by the criteria that the tips of the forewings appear from under the hindwings (Figs. 3B, S4, white arrowheads). In stage 3, black dots appear around the anterior edges of the wing sheaths, and black pigmentation also appears at the base of the wing sheaths (Figs. 3B, S4, black arrowheads). To help readers verify our judgment, we provide daily photographs and the duration of each stage for all individuals (Figs. S1, S2, S3; Table S1; higher quality photos are deposited in the Dryad Digital Repository: https ://doi.org/10.5061/dryad .tht76 hdz6). Among the 49 species we examined, stage 3 was difficult to define for only two species (Epiophlebia superstes and Sinogomphus flavolimbatus), because their entire body is very dark in color (Tables 1, S1; Fig. S1). It should be noted that two species of the genus Lestes (Lestidae) did not exhibit black dots on the wings but could be classified as stage 3 by melanization at the base of the wing sheaths (Tables 1, S1; Fig. S1) While a previous study partly mentioned these criteria for developmental staging of Pyrrhosoma nymphula 11 , here we demonstrate that these developmental criteria are applicable to most of the diverse Odonata species (Table 1). Next, we examined morphological changes during F−1 (penultimate) larval instar of Odonata, which were described in a few previous studies for only three species: Pachydiplax longipennis 18 , Aeshna viridis 16 and Ischnura senegalensis 12 . For all the 22 F−1 instar larvae representing 12 species (Tables 1, S1), the compound eyes gradually tilted during F−1 instar, and the wing sheaths began to expand a few days before ecdysis to F−0 instar (Figs. 2C, S1).

Variation in the duration of developmental stages during F−0 instar within and between Odonata species.
We noticed significant intra-specific variation in the total duration of the F−0 larval instar ( Fig. 3D; Table S1). We compared the durations of these developmental stages and identified large variation in the durations of stage 1 within and between the Odonata species (Fig. 3D,E). By contrast, the durations of stage 2 and stage 3 were less variable between and within the species (Figs. 3D,E, S5). Many river-dwelling Odonata species (e.g. Calopterygidae, Gomphidae, Cordulegastridae, Chlorogomphidae, and Macromiidae) are known to overwinter as F−0 instar larvae 14,17 . In these species, notably, while the total durations of F−0 larval instar were generally and conspicuously long (Table S1), the durations of stage 2 and stage 3 were relatively short and similar between species, 3-21 days and 1-8 days respectively (Figs. 3E, S5). The duration of larval instar (especially before entering stage 2 of F−0 instar) under natural conditions may differ from our results because only a limited type of food was supplied in this study. However, we confirmed that the larvae can become adults without food after entering stage 2 of F−0 instar. Overall, the durations after the onset of morphological change (i.e., stage 2 and on) exhibited relatively small variation, as reported in I. senegalensis 12 , suggesting that the developmental processes for metamorphosis after stage 2 proceed steadily and irreversibly.
Morphological changes in compound eyes of damselfly larvae. To our knowledge, Grieve 9 first mentioned the expansion of the compound eyes during F−0 larval instar in the damselfly Ischnura verticalis (Coenagronidae). Recently, in I. senegalensis (Coenagrionidae), we categorized F−0 larval instar into five developmental stages that entail the following two-step expansions of the compound eyes: the dorsal expansion (Fig. 4B,a black arrowhead) in the 2nd stage (stage 12) and the posterior expansion (Fig. 4C,D, white arrowheads) in the 4th stage (stage 17) after Okude et al. 12 (Fig. 4Y). Our comparative survey of diverse damselfly species (Zygoptera) revealed that the two-step expansions of the compound eyes were recognized only in Coenagrionidae species (Fig. 4; Table 1), and the two-step expansions are sometimes observed simultaneously during stage 2 in this study (ex. Enallagma circulatum; Fig. 4E-H). We confirmed that stage 1 of Okude et al. 12 is consistent with stage 1 of this study, whereas stage 19 of Okude et al. 12 is the same as stage 3 of this study (Fig. 4Y).
In Lestidae (Fig. 4I-L) and Calopterygidae species (Fig. 4M-P), the dorsal expansion of the compound eyes was certainly recognizable, but this expansion proceeded gradually from stage 1 to stage 2 and thus was not suitable as an indicator for developmental staging, whereas the posterior expansion of the compound eyes was faintly observed before stage 3 (Fig. 4L,P, white arrowheads). In Platycnemididae and Rhipidolestes (family status is uncertain) species, the expansion of the compound eyes was observed less obviously, which was partly due to the melanization on their body surface and thus not suitable as an indicator for developmental staging (Fig. 4Q-X). Hence, we concluded that the developmental stages 12, 15 and 17 defined by Okude et al. 12 (Fig. 4Y) are only applicable to some Coenagrionidae species.
Morphological changes in compound eyes of dragonfly larvae. Among dragonfly species (Anisozygoptera and Anisoptera), Aeshnidae larvae exhibited the relatively complicated expansion patterns in their compound eyes as previously described [14][15][16] . During stage 1, the compound eyes expanded gradually toward the midline (Fig. 5A-D). During stage 2, apolysis-like phenomenon was observed from the anterior side to the www.nature.com/scientificreports/ posterior side of the compound eyes ( Fig. 5E-L, black arrowheads; Fig. 5Y). This apolysis-like phenomenon was also observed, though faintly, in the compound eyes of some Libellulidae species (e.g. C. servilia) (Figs. 5O, S1; Table 1). The expansion of the compound eyes was also observed commonly in dragonfly species other than Aeshnidae species (Fig. 5M-X). In some Libellulidae species, newly-formed regions of the compound eyes were clearly distinguishable morphologically ( Fig. 5N-P,R-T, white arrowheads, Fig. 5Z). These regions gradually expanded from mid-stage 1 to stage 3 ( Fig. 5M-T,Y,Z). Meanwhile, the boundary of the newly formed regions was less obvious in several dragonfly species including Libellulidae (ex. Rhyothemis fuliginosa), and thus cannot be regarded as common developmental indicators (Fig. 5U-X; Table 1).
In Gomphidae, Petaluridae, Chlorogomphidae, Cordulegastridae, Macromiidae and Corduliidae species, the posterior expansion patterns of the compound eyes were similar to those in Libellulidae species (Fig. 6). It should be noted that the morphological changes of the compound eyes were difficult to observe in species and/or individuals with dark-colored heads (ex. Epiophlebia superstes, Fig. 6S-U), as reported for Pseudothemis zonata (Libellulidae) 19 . On the day before adult emergence, the color of the compound eyes resembled that of adult (Fig. 6U).
Other morphological changes observed in F−0 instar larvae. We also summarized other morphological changes that were constantly observed within some specific Odonata lineages ( Fig. 7; Table 1). In most damselfly species, specific markings appeared after stage 2 on their heads, prothoraces, around ocelli and mandibles (Fig. 7A,B; Table 1, white arrowheads), and thinning of antennae was observed during stage 2 (Fig. 7A,B, white arrowhead), as reported 12 .   www.nature.com/scientificreports/ Previous studies regarded the wrinkles on the wing sheaths (described as "accordion-like" 15 ) as an indicator of metamorphosis in Odonata 10,11,15,18 . In general, the wrinkles appeared a few days after the onset of stage 2 (Fig. 7C). While the wrinkles on the wings were clearly visible in many Odonata species (Fig. 7D,G, white arrowheads, Table 1), they were less obvious in some species (ex. Orthetrum albistylum: Fig. 7I-J). Just before adult emergence at the end of stage 3, the bristles on the body surface were melanized (Fig. 7K-L), although this change was often masked by dark pigmentation of the body surface.
The degradation of larval labium, or shrinkage of tissues within the larval prementum, has been also used as an indicator of metamorphosis in Odonata 10,11,14,16 . We photographed the daily changes in the larval labium of 18 larvae representing 7 species (Table 1; Fig. S3). The degradation of larval labium was more prominent in dragonfly species (Anisoptera) (Fig. 7Q-Y) than in damselfly species (Zygoptera) ( Fig. 7O-P,Z-b). We confirmed that the degradation of larval labium occurred from late stage 2 to stage 3 ( Fig. 7Q-c, white arrowheads). It should be noted that the changes in larval labium were not prominent in some species like Tanypteryx pryeri (Petaluridae, Fig. 7M-N) and I. senegalensis (Coenagrionidae, Fig. 7O-P). www.nature.com/scientificreports/

Conclusion and perspective
In this study, we described the daily morphological changes of F−0 and/or F−1 instar larvae, using 158 individuals of 49 Odonata species. Based on the morphology of wing sheaths, we propose that F−0 larval instar could be classified into three developmental stages for most of the examined Odonata species. The order Odonata is among the most ancestral insects with metamorphosis and are important in studying the origin of insect metamorphosis. However, even the dynamics of insect hormone titers (e.g. ecdysteroids) during F−0 instar has not been described in detail 23,24 , which is ascribed to, at least partly, the lack of developmental descriptions in Odonata. The comprehensive and detailed information provided in this study will enable the precise developmental staging of larval-adult metamorphosis across diverse Odonata species and will advance the comparative analysis of the physiological and molecular mechanisms of metamorphosis with other insects toward understanding the evolution of metamorphosis.
Recently, we established a gene knockdown method by electroporation-mediated RNAi in Odonata 25,26 . Considering that precise larval staging is critical for effective gene knockdown 25,26 , this study will facilitate the identification of appropriate developmental stages for RNAi, thereby facilitating the molecular genetic studies on various topics in Odonata.