{"paper_id":"4a4e5544-4975-45b3-82ee-bef649bca9b0","body_text":"Comparisons of developmental processes of air- breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparisons of developmental processes of air- breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins Naoto Inui, Toru Miura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4023002/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2024 Read the published version in Developmental Biology Advances → Version 1 posted 8 You are reading this latest preprint version Abstract Background The acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be suitable models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., 'uncovered lungs' in Nagurus okinawaensis (Trachelipodidae), 'dorsal respiratory fields' in Alloniscus balssi (Alloniscidae), and pleopods without respiratory structures in Armadilloniscus cf. ellipticus (Detonidae). Results In N. okinawaensis with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in Al. balssi with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in Ar. cf. ellipticus , only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis. Conclusions This study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the timing and mode of development vary among species with different respiratory structures. The positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields, suggesting that these organs derive from different origins despite the similar location of their functional organs. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs. appendage dorsal respiratory fields hemolymph homology pleopodal lung terrestrialization uncovered lungs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Terrestrialization is one of the key innovations in the evolutionary history of animals. Terrestrial species are known to have acquired several novel traits that are not found in ancestral aquatic species, such as hard skeletal systems that resist gravity, protective shells or skins that protect against dryness and ultraviolet light, and reproductive systems that do not require water [ 1 , 2 ]. One of the most important changes is in the respiratory system [ 3 ]. Major animal phyla colonizing land, such as vertebrates, arthropods and molluscs, independently acquired air-breathing organs [ 3 – 7 ]. However, in many cases, the detailed evolutionary processes of these traits remain unclear because of the extinction of species that had the ancestral or transitional traits [ 8 – 10 ]. Especially in arthropods, which are the most successful land colonizers, many lineages lack aquatic or semiterrestrial species with ancestral traits [ 6 , 10 ]. To study the evolution of respiratory organs, terrestrial isopods (Crustacea, Isopoda, Oniscidea) can be suitable materials. Terrestrial isopods are a unique group in that they have extant species that represent each evolutionary stage of the water-to-land transitions [ 11 ]. Isopod species inhabiting arid terrestrial environments have acquired novel respiratory structures called the pseudotrachea/lung inside their abdominal appendages, pleopods, for air breathing [ 11 , 12 ]. The morphology of respiratory structures in isopods are diversified in the most-derived terrestrial isopod lineage, i.e., Crinocheta. The isopod respiratory structures can be classified into three types: dorsal respiratory fields, uncovered lungs, and covered lungs [ 12 , 13 ]. Simple dorsal respiratory fields are found in species of relatively basal lineages inhabiting wet environments, while species with complex covered lungs are known in derived lineages inhabiting arid terrestrial environments [ 13 , 14 ]. Therefore, it was suggested that the three types of isopod respiratory structures probably share the same origins, which evolved to dorsal respiratory fields, and then to uncovered lungs, and finally to complex, desiccation-resistant covered lungs [ 15 ]. However, the same type of respiratory organs is seen in several different isopod families [ 16 , 17 ], so that the evolutionary processes and the homology between organs in different isopods have remained controversial [ 14 , 15 , 17 , 18 ]. Understanding developmental mechanisms is crucial for studying morphological evolution [ 19 ]. However, despite the importance and diversity of the morphologies of respiratory structures in terrestrial isopods, few developmental studies of these structures have been conducted [ 20 ]. Although postembryonic development of covered lungs in Porcellio scaber was described [ 18 , 20 ], there are only limited descriptions of uncovered lung development with respect to external morphology [ 15 , 21 ], and nothing is known about development of dorsal respiratory fields. In this study, therefore, to gain insights into the evolutionary trajectories of these respiratory structures, we performed histomorphological studies on the development of these latter two types of respiratory structures. Uncovered lungs in Nagurus okinawaensis Nunomura, 1992 (Trachelipodidae) and dorsal respiratory fields in Alloniscus balssi (Verhoeff, 1928) (Alloniscidae) were observed during postembryonic development (Fig. 1 ). In addition, to assess possible ancestral states, the pleopods of Armadilloniscus cf. ellipticus (Harger, 1878), which belongs to the basal family Detonidae in Crinocheta, were also observed (Fig. 1 ). Based on these observations, together with previous findings, the evolutionary relationships of these respiratory organs are discussed. Results Uncovered lungs in Nagurus okinawaensis Adult structure Scanning electron microscopy (SEM) showed that, in N. okinawaensis , adults had highly wrinkled respiratory structures on exopods of all the pleopods (Fig. 2 A-B, white arrowheads). All of the respiratory structures were located on the proximal lateral side of each pleopod. On the third to fifth pleopods, these structures were adjacent to swollen ridges (Fig. 2 B, yellow arrowheads). In addition, histological observations on transverse sections of the pleopods were carried out. The dorsal side of the exopods was covered with thicker cuticle compared to the ventral side, but the respiratory structures closer to the dorsal sides were covered with a finely furrowed epithelium and thin cuticle (Fig. 2 C). Some of these furrows in the structures were deeply invaginated and formed tubular structures (Fig. 2 C, black arrowheads). Inside these structures, hemolymph sinuses were observed and sinuses were partially stained with eosin (Fig. 2 C). In the first and second pleopods, some parts of the respiratory structures were covered by the dorsal epithelium, whereas in the third to fifth pleopods they were not covered and were continuous with swollen ridges (Fig. 2 C). The location of the hemolymph sinuses corresponded to the ridges (Fig. 1 B). Developmental process The morphological structures of the first, second and third pleopods were observed during postembryonic development, from the first manca stage to the post-manca juvenile stages. SEM observations on the dorsal surface of the pleopods showed that no respiratory structures were present in the second and third pleopods during the manca stages (Fig. 3 ). Also, no structures were observed in the first pleopod at manca 3 stage, when the first pleopod appeared (Fig. 3 A). In the post-manca juvenile stage, a swollen ridge was formed on the proximal lateral side only in the third pleopod (Fig. 3 , yellow arrowheads). After these stages, wrinkled surface structures appeared on the proximal side of all pleopods, followed by the appearance of respiratory structures with surface grooves (Fig. 3 , white arrowheads). At the stages when they appeared, these structures consisted of a few grooves (Fig. 3 ). In later stages, the number of grooves increased and the structures became more complex (Fig. 3 ). In transverse sections, a cell-dense region was observed at the proximal side of the pleopods in the first and second pleopods during the manca stages (Fig. 4 A-B, white arrowheads). Later, in the post-manca juvenile stages, hemolymph sinuses were observed at the same location, around which the epithelium was partially invaginated (Fig. 4 A-B, yellow arrowheads). In contrast, in the third pleopod, the hemolymph sinus was already observed in the manca stages. During development, the surrounding epithelium gradually changed to structures with numerous grooves on the dorsal surface (Fig. 4 C, yellow arrowheads). The thickness of the cuticle was the same between the dorsal and ventral surfaces during the manca stages. However, during the post-manca juvenile stages, the cuticle was highly thickened on the dorsal surface, except for the respiratory structures (Fig. 4 ). Dorsal respiratory fields in Alloniscus balssi Adult structure SEM observations on pleopods revealed that adults had slightly wrinkled respiratory structures on the exopods of all pleopods (Fig. 2 A, white arrowheads). All structures were located on the proximal lateral side of the pleopods with swollen ridges in the structures (Fig. 2 B black arrowheads). In addition, the third to fifth pleopods had distinct swollen ridges at the borders between the structures and the dorsal surface of the pleopods (Fig. 2 B, yellow arrowheads). Observations on transverse sections showed that the dorsal sides of the pleopods were covered by a thick cuticle, while the respiratory structures were covered by a very thin cuticle. Within the structures, three internal hemolymph sinuses were observed (Fig. 5 C). All pleopods had similar tissues and the locations of hemolymph sinuses corresponded to the swollen ridges (Fig. 5 , yellow and black arrowheads). The ventral side of the pleopods was composed of columnar epithelial cells (Fig. 5 C). Developmental processes The first, second and third pleopods were morphologically observed during the manca stages and the post-manca juvenile stages. SEM observation on the dorsal surfaces of pleopods revealed that primordial regions where the respiratory structure is formed were already present in the manca stages (Fig. 6 , white arrowheads). These regions were separated from the dorsal surface by the lateral sides of the pleopods and became enlarged and wrinkled during development (Fig. 6 ). As the regions expanded, swollen ridges developed on the lateral side and in the center of the regions (Fig. 6 , black arrowheads). In addition, another swollen ridge was observed near the proximal side of the third pleopod in the manca stages (Fig. 6 C, yellow arrowheads). Histological observations showed that the ventral epithelium of the pleopods protruded laterally in the manca stages (Fig. 7 , white arrowheads). The protrusions were composed mainly of columnar epithelial cells and corresponded to the primordial respiratory region on the surface (Fig. 6 ). These protruding epithelial tissues expanded with growth (Fig. 7 ). In the first pleopods, proximal hemolymph sinuses were observed during the post-manca juvenile stages (Fig. 7 A, yellow arrowheads). In the second and third pleopods, the proximal hemolymph sinuses were observed during the manca stages (Fig. 7 B-C, yellow arrowheads), and the epithelium around these hemolymph sinuses did not cause major structural changes. As the protruding ventral epithelium expanded and increased in size, additional hemolymph sinuses were observed in the structures (Fig. 7 , black arrowheads). Pleopods of Armadillidium cf. ellipticus Finally, to compare the previous results with the structures in a species lacking special respiratory structures, the pleopods of Ar. cf. ellipticus were examined in an adult and at manca 1 stage (Fig. 8 ). Although no respiratory structures developed on the proximal lateral side of the pleopods, fine scale-like surface structures were observed on the third, fourth and fifth pleopods (Fig. 8 B). In addition, swollen ridges were observed on the third to fifth pleopods (Fig. 8 B, yellow arrowhead). Observations of histological sections revealed that hemolymph sinuses were located in the proximal sides of all pleopods (Fig. 8 C, yellow arrowhead). The cuticle was thicker in the dorsal epithelium of all pleopods (Fig. 8 C). At manca 1 stage, no obvious surface structures were observed (Fig. 8 D-E), but a cell-dense regions was observed (Fig. 8 F) where hemolymph sinuses occur in adults (Fig. 8 C). No differences in thickness between dorsal and ventral cuticles were observed at this stage. Discussion Comparison of developmental processes between uncovered lungs and dorsal respiratory structures In previous studies, adult morphologies have been described mainly in species of the genus Trachelipus (Trachelipodidae) for uncovered lungs [ 15 , 21 ] and in the genus Oniscus (Oniscidae) for dorsal respiratory fields [ 14 , 15 , 22 ]. In this study, the detailed developmental processes were described focusing on uncovered lungs in N. okinawaensis and on dorsal respiratory fields in Al. balssi , in comparison with pleopod structures of the basal lineage Ar. cf. ellipticus . In N. okinawaensis , the location of the respiratory structures and the hemolymph sinuses also corresponded to those of Trachelipus [ 14 , 15 ]. Although the proportion of covered area was smaller than in Trachelipus [ 14 , 15 ], the adult respiratory structures in N. okinawaensis were generally consistent with the features of ‘uncovered lungs’ in Trachelipus species. Uncovered lungs of N. okinawaensis developed mainly by modification of the lateral epithelium around the proximal hemolymph sinus (Fig. 9 A). Histological observations suggested that the wrinkled respiratory surfaces and tubular structures in the groove are formed by epithelial invagination (Fig. 3 ). The adult respiratory structures in Al. balssi revealed in this study agreed with the previous descriptions of dorsal respiratory fields in the genus Oniscus , except for the number of hemolymph sinuses [ 14 , 15 , 22 ]. In this species, the regions that would become the respiratory structure were observed at manca 1 stage, immediately after hatching. These regions were formed by the lateral protrusion of the ventral epithelium, which gradually became surface wrinkles (Fig. 9 B). Furthermore, observations in Ar. cf. ellipticus suggested that in the species lacking respiratory structures, only the proximal hemolymph sinuses and the dorsal cuticle developed during postembryonic development (Fig. 8 ; Fig. 9 C). The formation of a proximal hemolymph sinus and a thickened dorsal cuticle were shared processes in all species (Fig. 3 ; Fig. 6 ; Fig. 8 ). In addition, swollen ridges developed on the third through fifth pleopods with the formation of the hemolymph sinus. The previous studies suggested that these swollen structures may play a role in maintaining moisture and fluids around the endopods [ 15 , 17 ]. For uncovered lungs in Nagurus , this study showed that the lateral margin/semilunar area, a region homologous to the respiratory structure proposed in previous studies [ 14 , 15 , 17 ], is transformed into the respiratory structure during its development. On the other hand, for the dorsal respiratory fields in Alloniscus , the developmental origin of the respiratory structure is not the lateral epithelium of the hemolymph sinus, but a laterally protruding ventral epithelium (Fig. 9 B-C). It is known that the hemolymph flow around the respiratory structures largely differs between dorsal respiratory fields in Oniscus and uncovered lungs in Trachelipus [ 18 ]. Although the species examined were different, these differences were also supported by the observations in the present study. Evolutionary origins of respiratory structures in terrestrial isopods Among Crinocheta lineages with different types of air-breathing organs, the Olibrinidae or Detonidae are proposed to be the most basal clades, and the Trachelipodidae and Porcellionidae are derived clades [14,23; Fig. 1 ]. Although traditional phylogenies are based on morphological characters [ 23 ], the monophyly of Crinocheta and the phylogenetic relationships are supported to some extent by a molecular phylogenetic analysis [ 24 ]. In terrestrial isopods, gas exchange occurs mainly in the exopods of pleopods [ 13 ]. In Crinocheta, basal species inhabiting wet coastal and subterranean environments do not have specialized respiratory structures [ 13 ]. In these species, gas exchange is suggested to occur through the thin ventral epithelium of exopods [ 14 , 22 ]. Considering these findings, the observations in Armadilloniscus in this study allow us to hypothesize that in the ancestral developmental process of the exopods, only the development of the hemolymph sinus and thickening of the dorsal cuticle occurred during postembryonic development (Fig. 10 ). Thus, the respiratory structure of terrestrial isopods was acquired by the addition of novel developmental processes during evolution. For dorsal respiratory fields, the tissue structure of the pleopods differs from the assumed ancestral pleopod at the manca 1 stage, suggesting that lineage-specific changes occurred at manca 1 stage or late in embryonic development. The thin ventral epithelium, which was responsible for gas exchange in the ancestor, protrudes laterally to expand the respiratory surface (Fig. 10 ). On the other hand, in the uncovered lung, it is assumed that the ancestral developmental process is preserved until a certain point in the post-manca stage, but then a new developmental mechanism is acquired that produces folding and depression of the lateral epithelium around the proximal hemolymph sinus (Fig. 10 ). Although it is unclear whether this lateral epithelium originally contributed to gas exchange, the increased size of the epithelium around the sinus probably contributes to the efficiency of respiration. These differences suggest that at least the dorsal respiratory fields of Alloniscus and the uncovered lungs of Nagurus would have different evolutionary origins. Furthermore, considering the developmental processes in covered lungs of Porcellio scaber [ 20 ], it is thought that at least one specific change in the developmental mechanism occurs at early manca 1 stage in covered lungs (Fig. 10 ). It is difficult to specify the position of the epithelium that becomes the lungs because the epithelial invagination occurs during a single developmental stage and the complete lung is established at the next manca stage [ 20 ]. However, given that the Trachelipodidae and the Porcellionidae are closely related [ 24 ] and have similar epithelial modifications, it is possible that covered lungs and uncovered lungs share their origin. These post-embryonic developmental changes in pleopods would be related to efficient gas exchange under the conditions of a fully developed exoskeleton with the cuticle. It has been suggested that the lungs of terrestrial isopods may have evolved with the impermeability of the exoskeleton for water retention [ 25 , 26 ], and a similar phenomenon would have occurred during their ontogeny. In terrestrial amphipods, it has been shown that molecular developmental changes contributed to the thickening of their gills during this expansion into montane environments [ 27 ]. Although amphipods did not evolve lungs, terrestrial isopods should also have some novel molecular developmental mechanisms related to the acquisition of respiratory structures. Analysis of molecular mechanisms and observations of more lineages are needed to elucidate the detailed evolutionary processes of isopod respiratory structures. Conclusions This study shows that the respiratory structures in terrestrial isopods develop mainly during postembryonic development, but the timing and mode of development largely differ among the types of respiratory organs. In particular, the developmental origins of the respiratory structures were different between the two species Nagurus and Alloniscus : in the former, uncovered lungs originated from the epithelium of the lateral surface around the proximal hemolymph sinus of the pleopod, while in the latter, dorsal respiratory fields originated from the ventral epithelium of the pleopod. This would suggest different evolutionary origins of respiratory structures in isopods. Overall, this study provides the basis for evolutionary developmental studies of air-breathing organs in terrestrial isopods and insights into their evolutionary divergence and convergence. Materials and methods Animals Samples of N. okinawaensis were collected from the soil or plantings on the streets of Naha city and around the University of Ryukyu campus, on Okinawa Island, Japan in November 2022. Samples of Al. balssi and Ar. cf. ellipticus were collected from the seashore around the Misaki Marine Biological Station campus in 2021–2023. Species were identified based on previous studies [ 28 – 31 ]. Although the Armadilloniscus species at the site where we collected Ar. cf. ellipticus were previously identified as Ar. japonicus Nunomura, 1984 [ 30 ], it was subsequently suggested that this species was probably the same as Ar. ellipticus [ 32 ]. Therefore, this species was treated as Ar. cf. ellipticus in the present study. Some of the collected isopods were kept in plastic cases filled with moistened fallen leaves to obtain ovigerous females according to a previous study [ 20 ]. Mancae, post-manca juveniles, and adults were observed under a stereomicroscope (SZX16; Olympus, Tokyo, Japan) equipped with a digital camera (DP50; Olympus, Tokyo, Japan) to distinguish their developmental stages. Stages of mancae were defined following a previous study [ 20 ]. Their head width was measured as an indicator of approximate body size according to a previous study [ 33 ] because it is difficult to define the stages of post-manca juveniles based on morphology. For post-manca juveniles and adults, only females were used for observation due to their sexual dimorphisms in pleopods [ 34 ]. Scanning electron microscopy Scanning electron microscopy (SEM) was employed to observe the external morphology of the respiratory structures during development. The samples were prepared according to the methods described in a previous study [ 20 ]. The exopods of the first, second, and third pleopods were observed under a scanning electron microscope (JSM-5510LV; JEOL Ltd., Tokyo, Japan). Histological observations To observe the development processes of the respiratory structures histologically, paraffin sections were made basically according to a previously described method [ 20 ]. The whole bodies were fixed in Bouin's solution for several days and preserved in 70% EtOH. The samples were dehydrated by immersion in increasing concentrations of ethanol, transferred to xylene, and finally embedded in paraffin. Serial sections of the transverse planes were prepared using a microtome (Spencer Lens Co., Buffalo, USA). The thickness of section slices was 8 µm. After deparaffinization, the sections were stained with hematoxylin and eosin. The tissues on the slides were observed under a microscope (BX51; Olympus, Tokyo, Japan) equipped with a digital camera (DP74; Olympus, Tokyo, Japan). Declarations Ethics approval and consent to participate Although all experiments were carried out in Japan, where no ethics approval is required for the maintenance and handling of crustacean species, rearing and all experiments were performed with particular attention to replacement, reduction, and refinement of procedures in order to minimize animal suffering. Consent for publication Not applicable. Competing interests The authors have no competing interests. Funding This work was supported by Grant-in-Aid for JSPS KAKENHI Grant Number 21K18240 to TM, Grant-in-Aid for JSPS KAKENHI Grant Numbers 22KJ1032 to NI from the Ministry of Education, Culture, Sports, and Science, and a grant from the Research Institute of Marine Invertebrates Foundation (RIMI) to NI. Author Contribution NI and TM designed the study. NI performed field sampling and maintenance of the animals and carried out experiments. NI and TM analyzed the data and wrote the manuscript. Both approved the final version of the manuscript. Acknowledgements We thank the lab members for daily discussions. Availability of data and materials All data used and analyzed during the study are available from the corresponding author on reasonable request. References Garwood RJ, Edgecombe, GD. Early terrestrial animals, evolution, and uncertainty. Evol: Educ Outreach. 2011;4:489–501. Selden P. 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Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2024 Read the published version in Developmental Biology Advances → Version 1 posted Reviews received at journal 08 Jun, 2024 Reviews received at journal 05 Jun, 2024 Reviewers agreed at journal 10 May, 2024 Reviewers agreed at journal 10 May, 2024 Reviewers invited by journal 08 May, 2024 Editor assigned by journal 07 Mar, 2024 Submission checks completed at journal 07 Mar, 2024 First submitted to journal 07 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4023002\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":276914887,\"identity\":\"b423c84b-7a32-40f2-92a6-2c76eec6dd70\",\"order_by\":0,\"name\":\"Naoto Inui\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Misaki Marine Biological Station, The University of Tokyo\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Naoto\",\"middleName\":\"\",\"lastName\":\"Inui\",\"suffix\":\"\"},{\"id\":276914888,\"identity\":\"393a5a9c-af9d-4e46-8ebf-56dd48d5dad2\",\"order_by\":1,\"name\":\"Toru Miura\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACZgbGBwwMB+B8AyjNhk8LswFCSwIxWoCSEti04Aa67TxmVTdz7tgz8B9gk2D8YWNscLyB8cMPBr48XFrMDvOY3c7d9iyxQSIBaF1CmpnBmQPMkj0MbMUEtBxOYJBgYJP+k3DYxuBGAoM00LmJDXi0FAO1QBzGANHC/JuQFmagFsYGBrDDDpsBtbARsIWtWBrklzaJxGYLhrQ0Y8kzB9ssewzw+OX84Y2fc7fdsefnP3zwBoONjWHf8ebDN35UHMMZYnDAxsAIcYnCARDD4FgCQS1wIA/RWUOCllEwCkbBKBjmAACzlFDekPV9pwAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Misaki Marine Biological Station, The University of Tokyo\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Toru\",\"middleName\":\"\",\"lastName\":\"Miura\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-03-07 05:34:34\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4023002/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4023002/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s13227-024-00229-z\",\"type\":\"published\",\"date\":\"2024-07-18T00:33:20+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":52403182,\"identity\":\"5214d66b-3a1b-482b-aff7-509e3b9ca43f\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1284496,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMaterial isopod crustaceans (\\u003cstrong\\u003eA\\u003c/strong\\u003e) and schematic diagrams of their respiratory organs\\u003cem\\u003e \\u003c/em\\u003e(\\u003cstrong\\u003eB–D\\u003c/strong\\u003e). \\u003cstrong\\u003eA\\u003c/strong\\u003e\\u003cem\\u003e Nagurus okinawaensis \\u003c/em\\u003ewith uncovered lungs,\\u003cem\\u003e Alloniscus balssi\\u003c/em\\u003ewith dorsal respiratory fields and \\u003cem\\u003eArmadilloniscus\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003ewithout respiratory structures were used in this study. Phylogenetic relationships were based on a previous study [23]. \\u003cstrong\\u003eB \\u003c/strong\\u003eDorsal view of isopods. Dashed square indicates the abdomen. Head width was measured as the indicator of body size. \\u003cstrong\\u003eC\\u003c/strong\\u003e Ventral view of a female abdomen. The first and second pleopods lack endopods. \\u003cstrong\\u003eD\\u003c/strong\\u003e Pleopod structures. The respiratory structures are located on the proximal (lateral) side of exopods of pleopods. Dashed line indicates the location of a transverse plane. Abbreviations: En: endopod; Ex: exopod; Dist: distal; Prox: proximal; Pl: pleopod; Ur: Uropod\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/9b9d4b1ee4157a80e2868c5d.png\"},{\"id\":52403360,\"identity\":\"099eb629-7afa-4783-8420-4920e75c687e\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 08:03:45\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4228504,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdult pleopods of \\u003cem\\u003eN. okinawaensis. \\u003c/em\\u003e\\u003cstrong\\u003eA \\u003c/strong\\u003eDorsal view of exopods of pleopods in SEM images. White arrowheads indicate respiratory structures. \\u003cstrong\\u003eB \\u003c/strong\\u003eEnlarged views of respiratory structures (uncovered lungs).\\u003cstrong\\u003e \\u003c/strong\\u003eOrange arrowheads indicate swollen ridges. \\u003cstrong\\u003eC\\u003c/strong\\u003eTransverse sections of the pleopods. Respiratory structures located in proximal sides. Abbreviations: Res. Str. and Rs: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/c5ad580f94bcd937444eff70.png\"},{\"id\":52403183,\"identity\":\"de0e161c-f13f-44e6-aeb2-30d0c4abffff\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3251628,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMorphological observations of the first (\\u003cstrong\\u003eA\\u003c/strong\\u003e), second (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and third (\\u003cstrong\\u003eC\\u003c/strong\\u003e) pleopods in\\u003cem\\u003e N. okinawaensis\\u003c/em\\u003e during development. Lengths indicate head width of post-manca juveniles.Arrowheads indicate respiratory structures (white) and swollen ridges (orange). Abbreviations: Juv.: post-manca juvenile; Res. Str.: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/423ccfb707c86c3c5bc5fcf9.png\"},{\"id\":52403187,\"identity\":\"01aa53d3-8c78-4ead-a6d9-d5607c924234\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3502988,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHistological observations of the first (\\u003cstrong\\u003eA\\u003c/strong\\u003e), second (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and third (\\u003cstrong\\u003eC\\u003c/strong\\u003e) pleopods in\\u003cem\\u003e N. okinawaensis \\u003c/em\\u003eduring development\\u003cem\\u003e.\\u003c/em\\u003e Arrowheads indicate aggregated cells (white) and proximal hemolymph sinus (orange). Lengths indicate head width of post-manca juveniles. Scale bars indicate 20 μm (Transverse section) and 10 µm (Res. Str.). Abbreviations: Juv.: post-manca juvenile; Res. Str.: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/9b06cecbe0d24b6e783c86ce.png\"},{\"id\":52403362,\"identity\":\"b850dd56-e654-460a-b364-e01d60c5f699\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 08:03:45\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4285217,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdult pleopods of \\u003cem\\u003eAl. balssi. \\u003c/em\\u003e\\u003cstrong\\u003eA\\u003c/strong\\u003e Dorsal view of the exopods of the pleopods in SEM images. White arrowheads indicate respiratory structures. \\u003cstrong\\u003eB\\u003c/strong\\u003eEnlarged views of respiratory structures (dorsal respiratory structures). Arrowheads indicate proximal swollen ridges (orange) and other swollen ridges (black). \\u003cstrong\\u003eC\\u003c/strong\\u003eTransverse sections of pleopods. Respiratory structures located in proximal sides. Arrowheads indicate proximal (orange) and other (black) hemolymph sinuses. Crossed arrows indicate dorsal-ventral and proximal-distal axis. Abbreviations: Hs: hemolymph sinus; Res. Str. and Rs: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/c5078174561e0072028e78ff.png\"},{\"id\":52403361,\"identity\":\"b5a01fe2-f8d6-439b-8b22-4d3d15ef743e\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 08:03:45\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3662393,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMorphological observations of the first (\\u003cstrong\\u003eA\\u003c/strong\\u003e), second (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and third (\\u003cstrong\\u003eC\\u003c/strong\\u003e) pleopods in\\u003cem\\u003e Al. balssi\\u003c/em\\u003e during development. Lengths indicate head width of post-manca juveniles. Arrowheads indicate respiratory structures (white), proximal swollen ridges (orange) and other swollen ridges (black). Abbreviations: Juv.: post-manca juvenile; Res. Str.: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/da64c41c19b1c340a2c500ef.png\"},{\"id\":52403189,\"identity\":\"c52a70d5-b08d-44c6-b622-b71bc88a60b4\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3562907,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHistological observations of the first (\\u003cstrong\\u003eA\\u003c/strong\\u003e), second (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and third (\\u003cstrong\\u003eC\\u003c/strong\\u003e) pleopods in\\u003cem\\u003e Al. balssi\\u003c/em\\u003e during development\\u003cem\\u003e.\\u003c/em\\u003e Arrowheads indicate laterally protruding epithelial tissue (white), proximal hemolymph sinus (orange) and other hemolymph sinuses (black). Lengths indicate head width of post-manca juveniles. Scale bars indicate 50 μm (Transverse section) and 20 µm (Res. Str.). Abbreviations: Juv.: post-manca juvenile; Res. Str.: respiratory structure\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/615e6d1981cac39ad03cdee1.png\"},{\"id\":52403185,\"identity\":\"3396be1a-485b-49fb-8986-fa809cf889de\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":5021452,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdult and manca 1 pleopods of \\u003cem\\u003eAr. \\u003c/em\\u003ecf.\\u003cem\\u003e ellipticus. \\u003c/em\\u003e\\u003cstrong\\u003eA, D\\u003c/strong\\u003e SEM observation. Dorsal view of the exopods of the pleopods in adults (\\u003cstrong\\u003eA\\u003c/strong\\u003e) and manca 1 (\\u003cstrong\\u003eD\\u003c/strong\\u003e). \\u003cstrong\\u003eB, E\\u003c/strong\\u003e Enlarged views of the proximal side in adults (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and manca 1 (\\u003cstrong\\u003eE\\u003c/strong\\u003e). Arrowheads indicate swollen ridges. \\u003cstrong\\u003eC, F\\u003c/strong\\u003e Transverse sections of the pleopods in adults (\\u003cstrong\\u003eC\\u003c/strong\\u003e) and manca 1 (\\u003cstrong\\u003eF\\u003c/strong\\u003e). Arrowheads indicate proximal hemolymph sinus. Crossed arrows indicate dorsal-ventral and proximal-distal axis. Abbreviations: Hs: hemolymph sinus\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/be8056e0ca5071cf0bb2b9fd.png\"},{\"id\":52403190,\"identity\":\"2a763ffe-6d3d-470c-92c2-7c81a3bf5846\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":425609,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSummary of respiratory structure development in focal isopod species. The exopods of the third pleopod are illustrated as representatives. Dorsal views and transverse sections are shown for each stage. \\u003cstrong\\u003eA\\u003c/strong\\u003e Uncovered lungs development in \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e. \\u003cstrong\\u003eB\\u003c/strong\\u003e Dorsal respiratory fields development in \\u003cem\\u003eAl. balssi\\u003c/em\\u003e. \\u003cstrong\\u003eC \\u003c/strong\\u003ePleopodal development in \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e. Abbreviations: Hs: hemolymph sinus; Prox: proximal\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/8f06fd378e42fbecad1b27cd.png\"},{\"id\":52403192,\"identity\":\"19dbce38-aaaf-4790-a316-0b54d0658b35\",\"added_by\":\"auto\",\"created_at\":\"2024-03-11 07:55:45\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":329886,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic diagram of novel developmental modifications in isopod respiratory structures. Pleopods are illustrated as transverse view. Arrows indicate direction of development. Structures and developmental processes of covered lungs in \\u003cem\\u003ePorcellio scaber\\u003c/em\\u003eare based on previous studies [14,20]. Abbreviations: Ex: exopod; En: endopod; plp: pleopods\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/4eaa77703ea93737d4d1d15f.png\"},{\"id\":60629428,\"identity\":\"df5152b6-c63b-4714-9b1b-ee23b7aef378\",\"added_by\":\"auto\",\"created_at\":\"2024-07-19 00:33:43\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":40395590,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4023002/v1/5ce989d8-508c-4fef-9d95-039d0928f484.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Comparisons of developmental processes of air- breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eTerrestrialization is one of the key innovations in the evolutionary history of animals. Terrestrial species are known to have acquired several novel traits that are not found in ancestral aquatic species, such as hard skeletal systems that resist gravity, protective shells or skins that protect against dryness and ultraviolet light, and reproductive systems that do not require water [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. One of the most important changes is in the respiratory system [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Major animal phyla colonizing land, such as vertebrates, arthropods and molluscs, independently acquired air-breathing organs [\\u003cspan additionalcitationids=\\\"CR4 CR5 CR6\\\" citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. However, in many cases, the detailed evolutionary processes of these traits remain unclear because of the extinction of species that had the ancestral or transitional traits [\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Especially in arthropods, which are the most successful land colonizers, many lineages lack aquatic or semiterrestrial species with ancestral traits [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTo study the evolution of respiratory organs, terrestrial isopods (Crustacea, Isopoda, Oniscidea) can be suitable materials. Terrestrial isopods are a unique group in that they have extant species that represent each evolutionary stage of the water-to-land transitions [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Isopod species inhabiting arid terrestrial environments have acquired novel respiratory structures called the pseudotrachea/lung inside their abdominal appendages, pleopods, for air breathing [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe morphology of respiratory structures in isopods are diversified in the most-derived terrestrial isopod lineage, i.e., Crinocheta. The isopod respiratory structures can be classified into three types: dorsal respiratory fields, uncovered lungs, and covered lungs [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Simple dorsal respiratory fields are found in species of relatively basal lineages inhabiting wet environments, while species with complex covered lungs are known in derived lineages inhabiting arid terrestrial environments [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Therefore, it was suggested that the three types of isopod respiratory structures probably share the same origins, which evolved to dorsal respiratory fields, and then to uncovered lungs, and finally to complex, desiccation-resistant covered lungs [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. However, the same type of respiratory organs is seen in several different isopod families [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], so that the evolutionary processes and the homology between organs in different isopods have remained controversial [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eUnderstanding developmental mechanisms is crucial for studying morphological evolution [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. However, despite the importance and diversity of the morphologies of respiratory structures in terrestrial isopods, few developmental studies of these structures have been conducted [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Although postembryonic development of covered lungs in \\u003cem\\u003ePorcellio scaber\\u003c/em\\u003e was described [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], there are only limited descriptions of uncovered lung development with respect to external morphology [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], and nothing is known about development of dorsal respiratory fields.\\u003c/p\\u003e \\u003cp\\u003eIn this study, therefore, to gain insights into the evolutionary trajectories of these respiratory structures, we performed histomorphological studies on the development of these latter two types of respiratory structures. Uncovered lungs in \\u003cem\\u003eNagurus okinawaensis\\u003c/em\\u003e Nunomura, 1992 (Trachelipodidae) and dorsal respiratory fields in \\u003cem\\u003eAlloniscus balssi\\u003c/em\\u003e (Verhoeff, 1928) (Alloniscidae) were observed during postembryonic development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). In addition, to assess possible ancestral states, the pleopods of \\u003cem\\u003eArmadilloniscus\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e (Harger, 1878), which belongs to the basal family Detonidae in Crinocheta, were also observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Based on these observations, together with previous findings, the evolutionary relationships of these respiratory organs are discussed.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eUncovered lungs in\\u003c/b\\u003e \\u003cb\\u003eNagurus okinawaensis\\u003c/b\\u003e\\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAdult structure\\u003c/h2\\u003e \\u003cp\\u003eScanning electron microscopy (SEM) showed that, in \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e, adults had highly wrinkled respiratory structures on exopods of all the pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-B, white arrowheads). All of the respiratory structures were located on the proximal lateral side of each pleopod. On the third to fifth pleopods, these structures were adjacent to swollen ridges (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, yellow arrowheads).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn addition, histological observations on transverse sections of the pleopods were carried out. The dorsal side of the exopods was covered with thicker cuticle compared to the ventral side, but the respiratory structures closer to the dorsal sides were covered with a finely furrowed epithelium and thin cuticle (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Some of these furrows in the structures were deeply invaginated and formed tubular structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, black arrowheads). Inside these structures, hemolymph sinuses were observed and sinuses were partially stained with eosin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). In the first and second pleopods, some parts of the respiratory structures were covered by the dorsal epithelium, whereas in the third to fifth pleopods they were not covered and were continuous with swollen ridges (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). The location of the hemolymph sinuses corresponded to the ridges (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDevelopmental process\\u003c/h2\\u003e \\u003cp\\u003eThe morphological structures of the first, second and third pleopods were observed during postembryonic development, from the first manca stage to the post-manca juvenile stages.\\u003c/p\\u003e \\u003cp\\u003eSEM observations on the dorsal surface of the pleopods showed that no respiratory structures were present in the second and third pleopods during the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Also, no structures were observed in the first pleopod at manca 3 stage, when the first pleopod appeared (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). In the post-manca juvenile stage, a swollen ridge was formed on the proximal lateral side only in the third pleopod (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, yellow arrowheads). After these stages, wrinkled surface structures appeared on the proximal side of all pleopods, followed by the appearance of respiratory structures with surface grooves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, white arrowheads). At the stages when they appeared, these structures consisted of a few grooves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). In later stages, the number of grooves increased and the structures became more complex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn transverse sections, a cell-dense region was observed at the proximal side of the pleopods in the first and second pleopods during the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA-B, white arrowheads). Later, in the post-manca juvenile stages, hemolymph sinuses were observed at the same location, around which the epithelium was partially invaginated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA-B, yellow arrowheads). In contrast, in the third pleopod, the hemolymph sinus was already observed in the manca stages. During development, the surrounding epithelium gradually changed to structures with numerous grooves on the dorsal surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC, yellow arrowheads). The thickness of the cuticle was the same between the dorsal and ventral surfaces during the manca stages. However, during the post-manca juvenile stages, the cuticle was highly thickened on the dorsal surface, except for the respiratory structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eDorsal respiratory fields in\\u003c/b\\u003e \\u003cb\\u003eAlloniscus balssi\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAdult structure\\u003c/h2\\u003e \\u003cp\\u003eSEM observations on pleopods revealed that adults had slightly wrinkled respiratory structures on the exopods of all pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, white arrowheads). All structures were located on the proximal lateral side of the pleopods with swollen ridges in the structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB black arrowheads). In addition, the third to fifth pleopods had distinct swollen ridges at the borders between the structures and the dorsal surface of the pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, yellow arrowheads).\\u003c/p\\u003e \\u003cp\\u003eObservations on transverse sections showed that the dorsal sides of the pleopods were covered by a thick cuticle, while the respiratory structures were covered by a very thin cuticle. Within the structures, three internal hemolymph sinuses were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). All pleopods had similar tissues and the locations of hemolymph sinuses corresponded to the swollen ridges (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, yellow and black arrowheads). The ventral side of the pleopods was composed of columnar epithelial cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDevelopmental processes\\u003c/h2\\u003e \\u003cp\\u003eThe first, second and third pleopods were morphologically observed during the manca stages and the post-manca juvenile stages.\\u003c/p\\u003e \\u003cp\\u003eSEM observation on the dorsal surfaces of pleopods revealed that primordial regions where the respiratory structure is formed were already present in the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, white arrowheads). These regions were separated from the dorsal surface by the lateral sides of the pleopods and became enlarged and wrinkled during development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). As the regions expanded, swollen ridges developed on the lateral side and in the center of the regions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, black arrowheads). In addition, another swollen ridge was observed near the proximal side of the third pleopod in the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC, yellow arrowheads).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eHistological observations showed that the ventral epithelium of the pleopods protruded laterally in the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e, white arrowheads). The protrusions were composed mainly of columnar epithelial cells and corresponded to the primordial respiratory region on the surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). These protruding epithelial tissues expanded with growth (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). In the first pleopods, proximal hemolymph sinuses were observed during the post-manca juvenile stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA, yellow arrowheads). In the second and third pleopods, the proximal hemolymph sinuses were observed during the manca stages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eB-C, yellow arrowheads), and the epithelium around these hemolymph sinuses did not cause major structural changes. As the protruding ventral epithelium expanded and increased in size, additional hemolymph sinuses were observed in the structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e, black arrowheads).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003ePleopods of\\u003c/b\\u003e \\u003cb\\u003eArmadillidium\\u003c/b\\u003e \\u003cb\\u003ecf.\\u003c/b\\u003e \\u003cb\\u003eellipticus\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eFinally, to compare the previous results with the structures in a species lacking special respiratory structures, the pleopods of \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e were examined in an adult and at manca 1 stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). Although no respiratory structures developed on the proximal lateral side of the pleopods, fine scale-like surface structures were observed on the third, fourth and fifth pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB). In addition, swollen ridges were observed on the third to fifth pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB, yellow arrowhead). Observations of histological sections revealed that hemolymph sinuses were located in the proximal sides of all pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC, yellow arrowhead). The cuticle was thicker in the dorsal epithelium of all pleopods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt manca 1 stage, no obvious surface structures were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eD-E), but a cell-dense regions was observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eF) where hemolymph sinuses occur in adults (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eC). No differences in thickness between dorsal and ventral cuticles were observed at this stage.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eComparison of developmental processes between uncovered lungs and dorsal respiratory structures\\u003c/h2\\u003e \\u003cp\\u003eIn previous studies, adult morphologies have been described mainly in species of the genus \\u003cem\\u003eTrachelipus\\u003c/em\\u003e (Trachelipodidae) for uncovered lungs [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e] and in the genus \\u003cem\\u003eOniscus\\u003c/em\\u003e (Oniscidae) for dorsal respiratory fields [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. In this study, the detailed developmental processes were described focusing on uncovered lungs in \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e and on dorsal respiratory fields in \\u003cem\\u003eAl. balssi\\u003c/em\\u003e, in comparison with pleopod structures of the basal lineage \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e, the location of the respiratory structures and the hemolymph sinuses also corresponded to those of \\u003cem\\u003eTrachelipus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Although the proportion of covered area was smaller than in \\u003cem\\u003eTrachelipus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e], the adult respiratory structures in \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e were generally consistent with the features of \\u0026lsquo;uncovered lungs\\u0026rsquo; in \\u003cem\\u003eTrachelipus\\u003c/em\\u003e species. Uncovered lungs of \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e developed mainly by modification of the lateral epithelium around the proximal hemolymph sinus (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA). Histological observations suggested that the wrinkled respiratory surfaces and tubular structures in the groove are formed by epithelial invagination (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe adult respiratory structures in \\u003cem\\u003eAl. balssi\\u003c/em\\u003e revealed in this study agreed with the previous descriptions of dorsal respiratory fields in the genus \\u003cem\\u003eOniscus\\u003c/em\\u003e, except for the number of hemolymph sinuses [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. In this species, the regions that would become the respiratory structure were observed at manca 1 stage, immediately after hatching. These regions were formed by the lateral protrusion of the ventral epithelium, which gradually became surface wrinkles (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003eFurthermore, observations in \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e suggested that in the species lacking respiratory structures, only the proximal hemolymph sinuses and the dorsal cuticle developed during postembryonic development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003eThe formation of a proximal hemolymph sinus and a thickened dorsal cuticle were shared processes in all species (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). In addition, swollen ridges developed on the third through fifth pleopods with the formation of the hemolymph sinus. The previous studies suggested that these swollen structures may play a role in maintaining moisture and fluids around the endopods [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eFor uncovered lungs in \\u003cem\\u003eNagurus\\u003c/em\\u003e, this study showed that the lateral margin/semilunar area, a region homologous to the respiratory structure proposed in previous studies [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], is transformed into the respiratory structure during its development. On the other hand, for the dorsal respiratory fields in \\u003cem\\u003eAlloniscus\\u003c/em\\u003e, the developmental origin of the respiratory structure is not the lateral epithelium of the hemolymph sinus, but a laterally protruding ventral epithelium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB-C). It is known that the hemolymph flow around the respiratory structures largely differs between dorsal respiratory fields in \\u003cem\\u003eOniscus\\u003c/em\\u003e and uncovered lungs in \\u003cem\\u003eTrachelipus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Although the species examined were different, these differences were also supported by the observations in the present study.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEvolutionary origins of respiratory structures in terrestrial isopods\\u003c/h2\\u003e \\u003cp\\u003eAmong Crinocheta lineages with different types of air-breathing organs, the Olibrinidae or Detonidae are proposed to be the most basal clades, and the Trachelipodidae and Porcellionidae are derived clades [14,23; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e]. Although traditional phylogenies are based on morphological characters [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], the monophyly of Crinocheta and the phylogenetic relationships are supported to some extent by a molecular phylogenetic analysis [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn terrestrial isopods, gas exchange occurs mainly in the exopods of pleopods [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. In Crinocheta, basal species inhabiting wet coastal and subterranean environments do not have specialized respiratory structures [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. In these species, gas exchange is suggested to occur through the thin ventral epithelium of exopods [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eConsidering these findings, the observations in \\u003cem\\u003eArmadilloniscus\\u003c/em\\u003e in this study allow us to hypothesize that in the ancestral developmental process of the exopods, only the development of the hemolymph sinus and thickening of the dorsal cuticle occurred during postembryonic development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e). Thus, the respiratory structure of terrestrial isopods was acquired by the addition of novel developmental processes during evolution.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFor dorsal respiratory fields, the tissue structure of the pleopods differs from the assumed ancestral pleopod at the manca 1 stage, suggesting that lineage-specific changes occurred at manca 1 stage or late in embryonic development. The thin ventral epithelium, which was responsible for gas exchange in the ancestor, protrudes laterally to expand the respiratory surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e). On the other hand, in the uncovered lung, it is assumed that the ancestral developmental process is preserved until a certain point in the post-manca stage, but then a new developmental mechanism is acquired that produces folding and depression of the lateral epithelium around the proximal hemolymph sinus (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e). Although it is unclear whether this lateral epithelium originally contributed to gas exchange, the increased size of the epithelium around the sinus probably contributes to the efficiency of respiration. These differences suggest that at least the dorsal respiratory fields of \\u003cem\\u003eAlloniscus\\u003c/em\\u003e and the uncovered lungs of \\u003cem\\u003eNagurus\\u003c/em\\u003e would have different evolutionary origins.\\u003c/p\\u003e \\u003cp\\u003eFurthermore, considering the developmental processes in covered lungs of \\u003cem\\u003ePorcellio scaber\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], it is thought that at least one specific change in the developmental mechanism occurs at early manca 1 stage in covered lungs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e). It is difficult to specify the position of the epithelium that becomes the lungs because the epithelial invagination occurs during a single developmental stage and the complete lung is established at the next manca stage [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. However, given that the Trachelipodidae and the Porcellionidae are closely related [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] and have similar epithelial modifications, it is possible that covered lungs and uncovered lungs share their origin.\\u003c/p\\u003e \\u003cp\\u003eThese post-embryonic developmental changes in pleopods would be related to efficient gas exchange under the conditions of a fully developed exoskeleton with the cuticle. It has been suggested that the lungs of terrestrial isopods may have evolved with the impermeability of the exoskeleton for water retention [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e], and a similar phenomenon would have occurred during their ontogeny.\\u003c/p\\u003e \\u003cp\\u003eIn terrestrial amphipods, it has been shown that molecular developmental changes contributed to the thickening of their gills during this expansion into montane environments [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Although amphipods did not evolve lungs, terrestrial isopods should also have some novel molecular developmental mechanisms related to the acquisition of respiratory structures. Analysis of molecular mechanisms and observations of more lineages are needed to elucidate the detailed evolutionary processes of isopod respiratory structures.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study shows that the respiratory structures in terrestrial isopods develop mainly during postembryonic development, but the timing and mode of development largely differ among the types of respiratory organs. In particular, the developmental origins of the respiratory structures were different between the two species \\u003cem\\u003eNagurus\\u003c/em\\u003e and \\u003cem\\u003eAlloniscus\\u003c/em\\u003e: in the former, uncovered lungs originated from the epithelium of the lateral surface around the proximal hemolymph sinus of the pleopod, while in the latter, dorsal respiratory fields originated from the ventral epithelium of the pleopod. This would suggest different evolutionary origins of respiratory structures in isopods. Overall, this study provides the basis for evolutionary developmental studies of air-breathing organs in terrestrial isopods and insights into their evolutionary divergence and convergence.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003eSamples of \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e were collected from the soil or plantings on the streets of Naha city and around the University of Ryukyu campus, on Okinawa Island, Japan in November 2022. Samples of \\u003cem\\u003eAl. balssi\\u003c/em\\u003e and \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e were collected from the seashore around the Misaki Marine Biological Station campus in 2021\\u0026ndash;2023. Species were identified based on previous studies [\\u003cspan additionalcitationids=\\\"CR29 CR30\\\" citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Although the \\u003cem\\u003eArmadilloniscus\\u003c/em\\u003e species at the site where we collected \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e were previously identified as \\u003cem\\u003eAr. japonicus\\u003c/em\\u003e Nunomura, 1984 [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], it was subsequently suggested that this species was probably the same as \\u003cem\\u003eAr. ellipticus\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Therefore, this species was treated as \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e in the present study. Some of the collected isopods were kept in plastic cases filled with moistened fallen leaves to obtain ovigerous females according to a previous study [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Mancae, post-manca juveniles, and adults were observed under a stereomicroscope (SZX16; Olympus, Tokyo, Japan) equipped with a digital camera (DP50; Olympus, Tokyo, Japan) to distinguish their developmental stages. Stages of mancae were defined following a previous study [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Their head width was measured as an indicator of approximate body size according to a previous study [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e] because it is difficult to define the stages of post-manca juveniles based on morphology. For post-manca juveniles and adults, only females were used for observation due to their sexual dimorphisms in pleopods [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eScanning electron microscopy\\u003c/h2\\u003e \\u003cp\\u003eScanning electron microscopy (SEM) was employed to observe the external morphology of\\u003c/p\\u003e \\u003cp\\u003ethe respiratory structures during development. The samples were prepared according to the methods described in a previous study [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. The exopods of the first, second, and third pleopods were observed under a scanning electron microscope (JSM-5510LV; JEOL Ltd., Tokyo, Japan).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHistological observations\\u003c/h2\\u003e \\u003cp\\u003eTo observe the development processes of the respiratory structures histologically, paraffin sections were made basically according to a previously described method [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. The whole bodies were fixed in Bouin's solution for several days and preserved in 70% EtOH. The samples were dehydrated by immersion in increasing concentrations of ethanol, transferred to xylene, and finally embedded in paraffin. Serial sections of the transverse planes were prepared using a microtome (Spencer Lens Co., Buffalo, USA). The thickness of section slices was 8 \\u0026micro;m. After deparaffinization, the sections were stained with hematoxylin and eosin. The tissues on the slides were observed under a microscope (BX51; Olympus, Tokyo, Japan) equipped with a digital camera (DP74; Olympus, Tokyo, Japan).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e \\u003cp\\u003eAlthough all experiments were carried out in Japan, where no ethics approval is required for the maintenance and handling of crustacean species, rearing and all experiments were performed with particular attention to replacement, reduction, and refinement of procedures in order to minimize animal suffering.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e \\u003cp\\u003eNot applicable.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e \\u003cp\\u003eThe authors have no competing interests.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis work was supported by Grant-in-Aid for JSPS KAKENHI Grant Number 21K18240 to TM, Grant-in-Aid for JSPS KAKENHI Grant Numbers 22KJ1032 to NI from the Ministry of Education, Culture, Sports, and Science, and a grant from the Research Institute of Marine Invertebrates Foundation (RIMI) to NI.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eNI and TM designed the study. NI performed field sampling and maintenance of the animals and carried out experiments. NI and TM analyzed the data and wrote the manuscript. Both approved the final version of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e \\u003cp\\u003eWe thank the lab members for daily discussions.\\u003c/p\\u003e\\u003ch2\\u003eAvailability of data and materials\\u003c/h2\\u003e \\u003cp\\u003eAll data used and analyzed during the study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eGarwood RJ, Edgecombe, GD. Early terrestrial animals, evolution, and uncertainty. Evol: Educ Outreach. 2011;4:489\\u0026ndash;501.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSelden P. Terrestrialisation (Precambrian\\u0026ndash;Devonian). eLS. 2012;2:1\\u0026ndash;6.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHsia CC, Schmitz A, Lambertz M, Perry SF, Maina JN. Evolution of air breathing: oxygen homeostasis and the transitions from water to land and sky. Compr Physiol. 2013;3:849\\u0026ndash;915.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMeyer A, Schloissnig S, Franchini P, Du K, Woltering JM, Irisarri I, Wong WY, Nowoshilow S, Kneitz S, Kawaguchi A, Fabrizius A, Xiong P, Dechaud C, Spaink HP, Volff J, Simakov O, Burmester T, Tanaka EM, Schartl M. Giant lungfish genome elucidates the conquest of land by vertebrates. Nature. 2021;590:284\\u0026ndash;289.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePerry SFS, Wilson RJR, Straus C, Harris MBM, Remmers JEJ. Which came first, the lung or the breath? Comp Biochem Physiol A Mol Integr Physiol. 2001;129:37\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSharma, PP. Chelicerates and the Conquest of Land: A View of Arachnid Origins Through an Evo-Devo Spyglass. Integr. Comp Biol. 2017;57(3):510\\u0026ndash;22.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRodriguez C, Prieto GI, Vega IA, Castro-Vazquez A. Functional and evolutionary perspectives on gill structures of an obligate air-breathing, aquatic snail. PeerJ. 2019;7(1):e7342.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAhlberg PE, Clack JA. Palaeontology: a firm step from water to land. Nature. 2006;440: 747\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eStanden E, Du T, Larsson H. Developmental plasticity and the origin of tetrapods. Nature. 2014;513:54\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003evan Straalen, NM. Evolutionary terrestrialization scenarios for soil invertebrates. Pedobiologia. 2021; 87\\u0026ndash;88:150753.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSfenthourakis S, Hornung E. Isopod distribution and climate change. In: Hornung E, Taiti S, Szlavecz K (Eds) Isopods in a Changing World. ZooKeys. 2018;801:25\\u0026ndash;61.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHornung E. Evolutionary adaptation of oniscidean isopods to terrestrial life: Structure, physiology and behavior. Terr Arthropod Rev. 2011;4:95\\u0026ndash;130.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSfenthourakis S, Myers AA, Taiti S, Lowry, JK. Terrestrial environments. In: M. Thiel, G. Poore, editors. The natural history of the Crustacea: Evolution and Biogeography: volume 8. Oxford: Oxford University Press; 2020. p. 375\\u0026ndash;404.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchmidt C, W\\u0026auml;gele JW. Morphology and evolution of respiratory structures in the pleopod exopodites of terrestrial Isopoda (Crustacea, Isopoda, Oniscidea). Acta Zool. 2001;82:315\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHoese B. Morphologie und Evolution der Lungen bei den terrestrischen Isopoden (Crustacea, Isopoda, Oniscoidea). Zool Jahrb Abt Anat Ontog Tiere. 1982;107(3): 396\\u0026ndash;422.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTaiti S, Paoli P, Ferrara F. Morphology, biogeography, and ecology of the family Armadillidae (Crustacea, Oniscidea). Isr J Zool. 1998;44:291\\u0026ndash;301.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePaoli P, Ferrara F, Taiti S. Morphology and evolution of the respiratory apparatus in the family Eubelidae (Crustacea, Isopoda, Oniscidea). J Morphol. 2002;253:272\\u0026ndash;89.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSilen, L. On the circulatory system of the Isopoda Oniscoidea. Acta Zool. 1954;35:11\\u0026ndash;70.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWagner GP, Lynch VJ. Evolutionary novelties. Curr Biol. 2010;20(2):R48\\u0026ndash;52.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eInui N, Kimbara R, Yamaguchi H, Miura T. Pleopodal lung development in a terrestrial isopod, \\u003cem\\u003ePorcellio scaber\\u003c/em\\u003e (Oniscidea). Arthropod Struct Dev. 2022;71:101210.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVerhoeff KW. Zur Kenntnis der Enteicklung der Trachealsysteme und der Untergattungen von \\u003cem\\u003ePorcellio\\u003c/em\\u003e und \\u003cem\\u003eTracheoniscus\\u003c/em\\u003e. Sber Ges Naturf Freunde Berl. 1917;3:195\\u0026ndash;223.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eUnwin EE. On the structure of the respiratory organs of the terrestrial Isopoda. Pap Proc R Soc Tasman. 1931;1931:37\\u0026ndash;104.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchmidt C. Phylogeny of the terrestrial Isopoda (Oniscidea): A review. Arthropod Syst Phylogeny. 2008;66:191\\u0026ndash;226.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDimitriou AC, Taiti S, Sfenthourakis S. Genetic evidence against monophyly of Oniscidea implies a need to revise scenarios for the origin of terrestrial isopods. Sci Rep. 2019;9(1):18508.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEdney EB, Spencer JO. Cutaneous respiration in woodlice. J Exp Biol. 1955;2:256\\u0026ndash;69.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWright JC, Ting K. Respiratory physiology of the Oniscidea: aerobic capacity and the significance of pleopodal lungs. Comp Biochem Physiol Part A: Mol Integr Physiol. 2006;145:235\\u0026ndash;44.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiu H, Zheng Y, Zhu B, Tong Y, Xin W, Yang H, Jin P, Hu Y, Huang M, Chang W, Ballarin F, Li S, Hou Z. Marine-montane transitions coupled with gill and genetic convergence in extant crustacean. Sci Adv. 2023;9:eadg4011.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNunomura N. Studies on the terrestrial isopod crustaceans in Japan VII. Supplements to the taxonomy-3. Bull Toyama Sci Mus. 1992;15: 1\\u0026ndash;23.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKwon D. Terrestrial isopoda (Crustacea) from Cheju Island, Korea. Korean J Syst Zool. 1995;11:509\\u0026ndash;38.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNunomura N. Terrestrial and Freshwater Isopod Crustaceans in the Coast of the Sagami Sea, Central Japan. Mem Natn Mus Nat Sci. 2006;(42):275\\u0026ndash;83.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBoyko CB, Bruce NL, Hadfield KA, Merrin KL, Ota Y, Poore GCB, Taiti S. World Marine, Freshwater and Terrestrial Isopod Crustaceans database. 2024. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.marinespecies.org/isopoda\\u003c/span\\u003e\\u003cspan address=\\\"http://www.marinespecies.org/isopoda\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e Accessed on 04 Jan 2024.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKarasawa S, Nakada A. Terrestrial isopods (Crustacea: Isopoda: Oniscidea) in Hamamatsu City and the surrounding areas of Shizuoka and Aichi prefectures, Japan. Bull Mus Nat Env Hist Shizuoka. 2020;(13):25\\u0026ndash;37.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJones DT, Hopkin SP. Reduced survival and body size in the terrestrial isopod \\u003cem\\u003ePorcellio scaber\\u003c/em\\u003e from a metal-polluted environment. Environ Pollut. 1998;99: 215\\u0026ndash;23.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAntoł A, Labecka AM, Horv\\u0026aacute;thov\\u0026aacute; T, Zieliński B,Szabla N, Vasko Y, Pecio A, Kozłowski J, Czarnoleski, M. Thermal and oxygen conditions during development cause common rough woodlice (\\u003cem\\u003ePorcellio scaber\\u003c/em\\u003e) to alter the size of their gas-exchange organs. J Therm Biol. 2020);90:102600.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"developmental-biology-advances\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"evod\",\"sideBox\":\"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)\",\"snPcode\":\"13227\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13227/3\",\"title\":\"Developmental Biology Advances\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"appendage, dorsal respiratory fields, hemolymph, homology, pleopodal lung, terrestrialization, uncovered lungs\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4023002/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4023002/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eThe acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be suitable models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., 'uncovered lungs' in \\u003cem\\u003eNagurus okinawaensis\\u003c/em\\u003e (Trachelipodidae), 'dorsal respiratory fields' in \\u003cem\\u003eAlloniscus balssi\\u003c/em\\u003e (Alloniscidae), and pleopods without respiratory structures in \\u003cem\\u003eArmadilloniscus\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e (Detonidae).\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eIn \\u003cem\\u003eN. okinawaensis\\u003c/em\\u003e with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in \\u003cem\\u003eAl. balssi\\u003c/em\\u003e with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in \\u003cem\\u003eAr.\\u003c/em\\u003e cf. \\u003cem\\u003eellipticus\\u003c/em\\u003e, only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eThis study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the timing and mode of development vary among species with different respiratory structures. The positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields, suggesting that these organs derive from different origins despite the similar location of their functional organs. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Comparisons of developmental processes of air- breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-03-11 07:55:40\",\"doi\":\"10.21203/rs.3.rs-4023002/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-08T10:37:04+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-06-05T09:25:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"160358794161891748329369538042259410558\",\"date\":\"2024-05-10T11:33:04+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"193427689318460024615226711846477742774\",\"date\":\"2024-05-10T08:51:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-05-08T11:30:16+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-03-07T06:51:45+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-03-07T06:51:42+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"EvoDevo\",\"date\":\"2024-03-07T05:27:19+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"developmental-biology-advances\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"evod\",\"sideBox\":\"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)\",\"snPcode\":\"13227\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13227/3\",\"title\":\"Developmental Biology Advances\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"facabc0d-9bc7-4530-bf51-3da5a7742371\",\"owner\":[],\"postedDate\":\"March 11th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-07-19T00:33:20+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4023002\",\"link\":\"https://doi.org/10.1186/s13227-024-00229-z\",\"journal\":{\"identity\":\"developmental-biology-advances\",\"isVorOnly\":false,\"title\":\"Developmental Biology Advances\"},\"publishedOn\":\"2024-07-18 00:33:20\",\"publishedOnDateReadable\":\"July 18th, 2024\"},\"versionCreatedAt\":\"2024-03-11 07:55:40\",\"video\":\"\",\"vorDoi\":\"10.1186/s13227-024-00229-z\",\"vorDoiUrl\":\"https://doi.org/10.1186/s13227-024-00229-z\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4023002\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4023002\",\"identity\":\"rs-4023002\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}