Unlocking the Secrets of Regeneration: Histology of Regenerating Arm in Ophiocoma scolopendrina from the Persian Gulf

Unlocking the Secrets of Regeneration: Histology of Regenerating Arm in Ophiocoma scolopendrina from the Persian Gulf | 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 Unlocking the Secrets of Regeneration: Histology of Regenerating Arm in Ophiocoma scolopendrina from the Persian Gulf Nasim Nowruzi, Narges Amrollahi Biuki, Cinzia Ferrario, Michela Sugni This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4641789/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ophiocoma scolopendrina , a prevalent brittle star species in the southernmost intertidal zone of Qeshm Island, serves as an exemplary model for studying echinoderm arm regeneration processes within the Persian Gulf. To elucidate the regenerative mechanisms of these organisms, we conducted a comprehensive investigation of their regenerative structures from a histological perspective. The collected specimens were carefully acclimated to laboratory conditions in aerated seawater aquaria before being transferred to specially treated environments. Adhering to strict ethical protocols, we amputated the arms of the brittle stars and meticulously documented the subsequent regenerative changes at various intervals: 24 hours, 72 hours, and weekly up to six weeks post-amputation. Our findings reveal that Ophiocoma scolopendrina undergoes a triphasic regenerative pathway, encompassing a repair phase, an early regenerative phase, and an advanced regenerative phase. Notably, the temporal progression of these phases differs from that observed in other previously studied species. Initially, the brittle stars effectuate wound closure and healing of the autotomy plane through an epimorphic process, characterized by the migration of epidermal cells and re-epithelialization. Subsequently, the formation of a regenerative blastema within the bud initiates morphogenesis, followed by the differentiation and proliferation of blastemal cells, culminating in the development of the regenerated arm. regeneration Persian Gulf brittle star Echinoderms Epimorphosis Morphallaxis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Regeneration as a post-embryonic developmental process, implies the reconstruction of lost or injured tissues or body parts caused by traumatic or self-induced amputations (Mattson, 1976 ; Goss, 2013 ; Ben Khadra et al., 2018 ). Regeneration can occur at different stages of life (larval and adult) as well as at different levels of biological organization: cells, tissues and organs. Re-growth of lost parts as well as rebuilding the whole body from small parts, all can be considered as regeneration events which can vary a lot among Phyla (Bely and Nyberg, 2010; Rinkevich et AL., 2022). Generally, the extent of regeneration ability among the phylogenetic tree of Metazoan, decreases as much as organisms get more complex (Alvarado, 2000 ). Although, there are some vertebrates like zebrafishes, lizards, mice that can show different range of regenerative abilities (Thouveny and Tassava, 1997 ; Brockes and Kumar, 2002 ; Nakatani et al., 2007 ; Nacu and Tanaka, 2011 ); invertebrates show higher regenerative capabilities than vertebrates. Indeed, some of them are able to completely rebuild their entire body from remaining body fragments or even produce a new individual which is completely the same as the old one as asexual reproduction method as well known, in some starfish like Linkia sp. and Coscinasterias sp. (Thouveny and Tassava, 1997 ; Candia Carnevali, 2006 ; Agata and Inoue, 2012 ). High regenerative ability in echinoderms has increased their chance of survival. Division by fission and the creation of fragments also allows them to rapidly produce colonies adapted to the environmental conditions of the new area. Therefore, the strategic evidences show regeneration has certainly helped these species adaptation as well as increasing their success and survival in marine ecosystems (Ben Khadra et al., 2018 ). All stellate echinoderms have the ability to regenerate their arms, but the timing and the specific processes involved vary among and within classes (Ben Khadra et al., 2018 ). These variations may be related to the particular anatomy, physiology and behavior of each species. Generally, the arms of Crinoidea (Mladenov, 1983 ) and Ophiuroidea, which are thin, delicate and long, regenerate faster than the arms of large Asteroidea. Ophiuroidea have a high regenerative potential, with 80–100% of them showing this ability and more than 70% of their arms regenerating simultaneously (Wilkie, 1978 ; Bourgoin and Guillou, 1994 ; Clements et al., 1994 ; Sköld and Rosenberg, 1996 ; Soong et al., 1997 ; Yokoyama and Amaral, 2010 ). In general, regardless of the class, the regeneration process and its main cellular/histological events can be divided into three main stages: a repair stage, an early regeneration stage and an advanced regeneration stage. The rate of regeneration can considerably vary between the three stellate classes and also within members of the same class. In general, small ophiuroids and crinoids regenerate faster than big epiphytic ophiuroids and asteroids. This might reflect aspects of regeneration mechanisms that are related to size or species (Ben Khadra et al., 2018 ). Also, according to Dupont and Thorndyke (2006), in ophiuroids, depending on the number of segments and missing parts of the arm, the animal will invest more in number or speed of differentiation of regenerated segments. Thus, we chose Ophiocoma scolopendrina as an ophiuroid model for this study; since it’s one of the most abundant brittle stars in the southern intertidal zone of Qeshm island in Iran so it is easy to collect (Fatemi et al., 2010 ). It also has a proper size for histological analyses according to what Ben Khadra and co-workers showed in previous studies and we mentioned above, that the size of ophiuroids affects the regeneration rate; for example, within ophiuroids, small-sized, such as Amphiura filiformis , usually regenerate faster than large epibenthic species, such as Ophioderma longicaudum or Ophioplocus januarii (Biressi et al., 2010 ; Ben Khadra et al., 2018 ). So, we are trying to describe arm regeneration in this species to increase the diversity of brittle star models, from different geographical regions and ecological conditions that can answer this question if the temperature affects the regeneration or not and to prepare information for further studies in the field of comparing the species discussed in this paper with the species that live in other regions with different temperature conditions. In another hand with defining the baseline regenerative process in this species, we can provide the data for more applied research, such as in environmental monitoring, etc. Indeed, the aim of this study is investigating the arm regenerative process after traumatic amputation in O. scolopendrina and comparing the regenerative phases of this species to the previous examined species to give a clearer image of the regeneration process in these brittle stars which lives and adopted to Persian Gulf situation. Materials and methods Animal collection, maintenance and regeneration tests Non-regenerating adult (disc diameter ~ 14 mm) specimens of O. scolopendrina (Fig. 1 .A) were collected on September 26th 2019 at the intertidal zone of South-East coast of Qeshm Island (Fig. 1 .B) (26 ◦ 55 ' 31.23 " N, 56 ◦ 14 ' 18.71 " E). They were immediately transferred to the marine laboratory of Hormozgan University (Bandar-abbas, Iran) where they were left to acclimatize for 12 days and maintained in aerated aquaria filled with sea-water at 25 ◦ C and 39 ppt salinity. Chemical-physical sea water parameters were constantly checked and adjusted if necessary. Animals were fed twice a week with Microvore Microdiet (Brightwell Aquatics), as described in Ferrario et al. ( 2018 ). For the regeneration test, specimens were anaesthetized in 3.5% MgCl 2 (6H 2 O) solution in a 1:1 mix of filtered sea water and distilled water (Blowes et al., 2017 ; Ferrario et al., 2018 ). Traumatic arm amputation was performed using a scalpel. A maximum of two arms per animal were amputated at 1 cm from the disc. Then, animals were left to regenerate in three aquaria with the same chemical-physical sea water parameters as acclimatization aquaria. Three replica groups for each time point were left to regenerate for pre-determined periods, namely 24 and 72 hours (h) and 7, 14, 21, 35 and 42 days post-amputation (p.a.). Then, regenerating arms (stump + regenerate) were collected using a scalpel following the above described anaesthetizing process and treated for the subsequent analyses; but before that, regenerating arm were recorded and photographed under a stereomicroscope and their lengths measured by image j program for calculating the regeneration rate. Light microscopy analysis Regenerating arms were fixed for 48 h at room temperature in Bouin’s fixative, composed of 75 ml of saturated picric acid in distilled water, 25 ml of 40% formaldehyde solution and 5 ml of glacial acetic acid. Following fixation, specimens were decalcified for 5–7 days in 5% Trichloroacetic acid (TCA) solution (Heraeus Kulzer, Technovit 7100 Germany), dehydrated through an increasing ethanol series, immersed in xylene and then embedded in paraffin wax (58–59 ◦ C) using the Tissue processor (DS 2080/H). Serial parasagittal and sagittal sections (5–7 µm in thickness) of regenerated arms were prepared using a MICRODS 4055 microtome and mounted on Albumin coated slides. Following dewaxing in xylene and rehydration, slides were then stained using Milligan trichrome method (Milligan, 1946); this staining differentiates collagenous (blue-green) from non-collagenous (red-violet) tissues (Ben Khadra et al., 2015 ). Slides were examined and photographed under a light microscope equipped with a Nikon camera in the marine laboratory of Hormozgan University. Results Investigating observation of O. scolopendrina arm regeneration by light microscope at 24 h, 72 h, 7 days, 14 days, 21 days, 35 and 42 days After the amputation, have been brought in following paragraphs of this manuscript: Control samples’ regeneration 24 hours p.a. The healing process of the brittle star arm after amputation was observed in sagittal sections of the samples at different time points (Fig. 2 .a). Within 24 hours, a thin layer of epithelial cells covered the wound area, indicating the first sign of healing. This layer was likely formed by a combination of two mechanisms: the expansion of the outer epidermis of the arm and the migration of cells from other tissues. However, this process was not complete yet, as evidenced by the presence of scattered cells in the loose connective tissue below the epithelial layer (Fig. 2 .b). The aboral coelomic cavity and the radial water canal, which are important structures for the brittle star physiology and locomotion, were also closed off at their distal ends. This closure was probably achieved by a combination of cell migration and muscle contraction of the coelomic wall. Adjacent to the radial nerve cord and the radial water canal, large clusters of cells were visible. These cells included undifferentiated coelomocytes, which are involved in immune response, and phagocytes, which are involved in wound cleaning. The water canal cavity also contained free and dispersing coelomocytes and phagocytes. 72 hours p.a. In these sections, the thickness of the epithelial layer that covered the wound part of the arm had increased. However, disruption of this layer and scattered cells in the connective tissue that supported the bottom layer indicated that the process was still incomplete for these animal samples (Fig. 2 .d). The RNC along with the surrounding coelomic cells was also clearly apparent at the taken sections (Fig. 2 .c). 1 week p.a. Sections of this stage (not shown) displayed the thicker epidermis that contained a large number of scattering small cells and phagocytes in a filamentous, dense, mesh-like network of connective tissue. In this developing layer, reconstruction of the coelomic cavity and the hypertrophic radial water canal could also be observed. At these sections, a large number of free cells, coelomic cells, phagocytes, as well as de-differentiating myocytes were visible within the coelomic cavity and the radial water canal. Undifferentiated myocytes were often recognizable among the intervertebral muscles, especially in the unorganized parts around them. 2 weeks p.a. At this stage (not shown), we observed an intense development and elongation of the coelomic cavity and a large group of migrating cells scattered in the connective tissue that was separating from the thick layer of cells that were covered the wound part of the arm at the first stage. At this stage, the accumulation of dedifferentiated myocytes, phagocytes, and coelomocytes can also be observed. On the other hand, podia were also developing. 3 weeks p.a. Signs of regenerated parts and sclerocytes, tissue structures and their pattern of formation and tissue differentiation were visible. Coelomic and neural contents were clearly evident in the regenerated bud of the arm which showed well-developed anatomical and histological features. In general, the regenerated RNC exhibited a pseudo-ganglionic pattern, whereas the fully separated radial water canal exhibited a hypertrophic structure. The large accumulation of migratory cells (mainly coelomocytes), phagocytes, and dedifferentiated myocytes that were often visible in the coelomic cavities, were mainly at the end of the regenerating arm at this stage. The structure of new podia was also visible at this stage. The regenerated miniature arm was developing and increasing in size. 5 weeks p.a. Samples of this stage represent the complete development of the regenerated arm with an advanced level of tissue differentiation. In general, the structure of the serial pseudo-ganglionated structure of the regenerated nerve cord is clearly identified in the regenerated segment. The main coelomic cavity and the other coelom-derived channels, such as the radial water canal, hyponeural sinus, are well developed. In the old stump, the muscles exhibit patterns of rearrangement, and some dedifferentiated myocytes are still present. Undifferentiated coelomocytes, phagocytes, and dedifferentiated myocytes are still scattered in new coelomic canals. From this point on, all structures of miniature arm are formed. 6 weeks p.a. The regenerated section of this stage shows all the general structures of the brittle stars’ arm: the nerve cord with its pseudo-ganglionic structure, the radial water canal with podia, the ossicles and the and muscle tissues and ultimately recognizable skeletal plates with their spines. Regenerate length The lengths (mean ± 0.5 mm) of regenerated arms at the different time-point are reported in Fig. 4 . Overall the regenerate reached approximately 17.65 mm after 42 days. Discussion Despite the differences between the time of regeneration phases of the brittle star studied in this experiment with previous studies (Biressi described 4 stages...in Ben Khadra et al 2018 we described 3 phases also for brittla stars), O. scolopendrina had many similarities in the basic mechanisms of arm regeneration with specimens previously studied. The overall regeneration process, from the amputation to the regeneration of a miniature arm that showed all the structural components of a complete brittle star’s arm, could be divided into three main stages: a repair phase, an early regenerative phase and an advanced regenerative phase, as described in Biressi et al., 2010 . The repair phase This phase included the time between amputation and 48 hours p.a.- It was comparable to the phase observed in other species that Biressi et al. ( 2010 ) studied. The main characteristic of this phase was wound healing and it included: 1. Closure of the coelomic canals and cavities which was the result of the contractions of the coelomic wall and the migration of free cells (Biressi et al., 2010 ). 2. The process of re-epithelialization at the wound area, which was carried out through the epidermal migrating cells, though it wasn’t complete yet. 3. The first signs of rearrangement of tissues that were damaged during the amputation. The presence of loose and net like of connective tissue beneath the external epithelium could be a clear indication that the repair process was not complete yet. The early regenerative phase This phase involved time from 24 hours p.a. to 7 days p.a. The stage began with the strong migration and of cells covering the wound area and eventually resulted in the formation of a sub-epithelial layer containing dense mesenchyme with dedifferentiated cells and phagocytes. These migrating cells, which were usually seen in the coelomic cavities, actually moved rapidly to the ends of the coelomic cavity and RNC segments. The ending structures in this phase were covered by a number of layers made up of migrating cells (Biressi et al., 2010 ). After this time till 14 days p.a. segments grew along the growing end of the RNC and the radial water canal in the injured area. Key events of morphogenesis and differentiation in this phase occurred in the regenerating tip, which is practically associated with the development of pseudo-ganglionic RNC and the differentiation of radial water canal. Indeed, during these regeneration stages, undifferentiated cells together with the undifferentiated myocytes would be involved in different regeneration processes (Alvarado, 2000 ; Biressi et al., 2010 ). The advanced regeneration phase After 14 days post amputation, regenerated arm entered the advanced regenerative phase. In this phase, extensive growth, morphogenesis and differentiation resulted in the formation of a miniature arm. During this phase, the structural patterns of the normal arm gradually became more apparent; its earliest regenerated parts, in particular, acquire distinctive features of the interior and exterior of a fully functional arm, while the tip of it yet appeared to be undifferentiated (Ferrario et al., 2018 ; Biressi et al., 2010 ). Comparison of the regeneration process in O. scolopendrina and species previously studied by other researchers showed similarities to the regeneration different phases and supported the idea that the regeneration in Ophiuroidea, despite to its great diversity, has the same essential characteristics (Biressi et al., 2010 ; Ferrario et al., 2018 ). Conclusion This study showed and followed all three main phases: a repair phase, an early regenerative phase and an advanced regenerative phase, which was different in term of timing with other species studied before (Biressi et al., 2010 , Ben Khadra et al., 2018 ). In the samples obtained from this study, different types of cells were also observed, which according to Bannister and colleagues in 2005, are the first responsible parts for the growth of the regenerated structure. These cells which include coelomocytes, as well as dedifferentiated myocytes from the stump tissues, migrate to the tip area and accumulate there; then with active proliferation and differentiation they formed new cells (Bannister et al., 2005 ; Biressi et al., 2010 ). Declarations Ethic statement Stars sampling steps and animal manipulations were performed according to the ethic certification evaluated by Hormozgan University of Medical science with approval ID: IR.HUMS.REC.1398.181and under observation of Qeshm Department of Environment of Qeshm Free Area organization (QDOE of QFAO). Ophiocoma scolopendrina is not an endangered or protected species. All efforts were made to minimize animal suffering during the whole experimental procedure. Acknowledgment We extend our deepest gratitude to the corresponding author, who has guided this research with utmost precision and dedication. We also thank our esteemed advisors from the University of Milan, Italy, for their expert opinions and valuable guidance, which significantly contributed to the advancement of this scientific work. Our appreciation goes to the laboratory of the Faculty of Science and Technology at the University of Hormozgan for providing a dynamic and well-equipped environment for conducting meticulous experiments. Lastly, we are indebted to Mr. Dakhteh, the Director of Environmental Protection for the Qeshm Free Zone, whose generous support has been instrumental in the success of this project. We should also mention that all authors declare they have no conflicts of interest and we did not receive any budget or funding from other organizations for this research. References Agata, K., & Inoue, T. (2012). Survey of the differences between regenerative and non‐regenerative animals. Development, growth & differentiation , 54 (2), 143-152. Alvarado, A. S. (2000). Regeneration in the metazoans: why does it happen?. Bioessays , 22 (6), 578-590. Bachoo, S. (2002). Experimental Cadmium Contamination of the Echinoid Stomopneustes Variolaris (Echinodermata: Echinoidea): Influence of Dosage and Distribution of the Metal in the Organism (Doctoral dissertation, University of Durban-Westville). Bannister, R., McGonnell, I. M., Graham, A., Thorndyke, M. C., & Beesley, P. W. (2005). Afuni, a novel transforming growth factor-β gene is involved in arm regeneration by the brittle star Amphiura filiformis. Development genes and evolution , 215 (8), 393-401. Ben Khadra, Y., Ferrario, C., Di Benedetto, C., Said, K., Bonasoro, F., Candia Carnevali, M. D., & Sugni, M. (2015). Wound repair during arm regeneration in the red starfish E chinaster sepositus. Wound Repair and Regeneration , 23 (4), 611-622. Ben Khadra, Y., Sugni, M., Ferrario, C., Bonasoro, F., Oliveri, P., Martinez, P., & Carnevali, M. D. C. (2018). Regeneration in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea. In Marine Organisms as Model Systems in Biology and Medicine (pp. 285-320). Springer, Cham. Biressi, A. C. M., Zou, T., Dupont, S., Dahlberg, C., Di Benedetto, C., Bonasoro, F., ... & Carnevali, M. D. C. (2010). Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura filiformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis. Zoomorphology , 129 (1), 1-19. Blowes, L. M., Egertová, M., Liu, Y., Davis, G. R., Terrill, N. J., Gupta, H. S., & Elphick, M. R. (2017). Body wall structure in the starfish Asterias rubens. Journal of anatomy , 231 (3), 325-341. Bourgoin, A., & Guillou, M. (1994). Arm regeneration in two populations of Acrocnida brachiata (Montagu)(Echinodermata: Ophiuroidea) in Douarnenez Bay,(Brittany: France): an ecological significance. Journal of experimental marine biology and ecology , 184 (1), 123-139. Brockes, J. P., & Kumar, A. (2002). Plasticity and reprogramming of differentiated cells in amphibian regeneration. Nature Reviews Molecular Cell Biology , 3 (8), 566. Candia Carnevali, M. D. (2006). Regeneration in Echinoderms: repair, regrowth, cloning. Invertebrate Survival Journal , 3 (1), 64-76. Clements, L. A. J., Bell, S. S., & Kurdziel, J. P. (1994). Abundance and arm loss of the infaunal brittlestar Ophiophragmus filograneus (Echinodermata: Ophiuroidea), with an experimental determination of regeneration rates in natural and planted seagrass beds. Marine Biology , 121 (1), 97-104. Czarkwiani, A., Ferrario, C., Dylus, D. V., Sugni, M., & Oliveri, P. (2016). Skeletal regeneration in the brittle star Amphiura filiformis. Frontiers in zoology , 13 (1), 18. Daviddi, A. (2014). Microscopic anatomy of arm-tip regeneration in the starfish Marthasterias glacialis (Linneaus, 1758) following traumatic amputation. Millan university, Master thesis. Fatemi, S. R., Jamili, S., Valinassab, T., & Kuranlu, N. (2010). Diversity of Ophiuroidea from lengeh portand Qeshm island in the persian gulf. J Fish Aquat Sci , 5 (1), 42-48. Ferrario, C., Khadra, Y. B., Czarkwiani, A., Zakrzewski, A., Martinez, P., Colombo, G., ... & Sugni, M. (2018). Fundamental aspects of arm repair phase in two echinoderm models. Developmental biology , 433 (2), 297-309. Fichet, D., Radenac, G., & Miramand, P. (1998). Experimental studies of impacts of harbour sediments resuspension to marine invertebrates larvae: bioavailability of Cd, Cu, Pb and Zn and toxicity. Marine Pollution Bulletin , 36 (7), 509-518. Goss, R. J. (2013). Principles of regeneration. Elsevier. Lawrence, J. M. (2010). Energetic costs of loss and regeneration of arms in stellate echinoderms. Integrative and comparative biology , 50 (4), 506-514. Mattson, P. (1976). Regeneration. Bobbs-Merrill Company. Mladenov, P. V. (1983). Rate of arm regeneration and potential causes of arm loss in the feather star Florometra serratissima (Echinodermata: Crinoidea). Canadian Journal of Zoology , 61 (12), 2873-2879. Nacu, E., & Tanaka, E. M. (2011). Limb regeneration: a new development?. Annual review of cell and developmental biology , 27 , 409-440. Nakatani, Y., Kawakami, A., & Kudo, A. (2007). Cellular and molecular processes of regeneration, with special emphasis on fish fins. Development, growth & differentiation , 49 (2), 145-154. Phillips, D. J. H., & Rainbow, P. S. (1993). Biomonitoring of trace aquatic contaminants Elsevier Applied Science. New York . Sköld, M., & Rosenberg, R. (1996). Arm regeneration frequency in eight species of Ophiuroidea (Echinodermata) from European sea areas. Journal of Sea Research , 35 (4), 353-362. Soong, K. Y., Shen, Y., Tseng, S. H., & Chen, C. P. (1997). Regeneration and potential functional differentiation of arms in the brittlestar, Ophiocoma scolopendrina (Lamarck)(Echinodermata: Ophiuroidea). Zoological Studies , 36 (2), 90-97. Thouveny, Y., & Tassava, R. A. (1997). Regeneration Through Phylogenesis. Regeneration through phylogenesis in cellular and molecular basis of regeneration, pp 9-43. Wang, H., Zhu, G., Shi, Y., Weng, S., Jin, T., Kong, Q., & Nordberg, G. F. (2003). Influence of environmental cadmium exposure on forearm bone density. Journal of Bone and Mineral Research , 18 (3), 553-560. Wilkie, I. C. (1978). Arm autotomy in brittlestars (Echinodermata: Ophiuroidea). Journal of Zoology , 186 (3), 311-330. Yokoyama, L. Q., & Amaral, A. C. Z. (2010). Ophionereis reticulata (Echinodermata, Ophiuroidea). Iheringia. Série Zoologia , 100 (2), 123-127. Youness, E. R., Mohammed, N. A., & Morsy, F. A. (2012). Cadmium impact and osteoporosis: mechanism of action. Toxicology mechanisms and methods , 22 (7), 560-567. Zyadah, M. A., & Abdel-Baky, T. E. (2000). Toxicity and bioaccumulation of copper, zinc, and cadmium in some aquatic organisms. Bulletin of environmental contamination and toxicology , 64 (5), 740-747. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4641789","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322028132,"identity":"6ca1d23f-daf2-47be-95ae-4af17d6b84eb","order_by":0,"name":"Nasim Nowruzi","email":"","orcid":"","institution":"University of Hormozgan","correspondingAuthor":false,"prefix":"","firstName":"Nasim","middleName":"","lastName":"Nowruzi","suffix":""},{"id":322028133,"identity":"25db63b1-46b0-47dc-88a3-a9c630f5ed13","order_by":1,"name":"Narges Amrollahi Biuki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYLCCBwUMCWzsPaiCPHi1JBgAtfCcIVULg0QOkW7in3b24YMEA7s8Psm3Bx9X5tjYy7ufPcDwo4ZBxrwBuxaJ2+nGBgkGycVs0nnJhme3pTEbnslLYOw5xsAjcwCHNbfT2CQSDJgT26RzzCQbtx1mM2zIMWDgbWDgkcChQx6ipT6xTfIMWAuPYf8bA8a/eLQYQLQcTmyT4AFrkZCXyDFgxmeL4e00ZqBfjie28eQYGzZuSzMwkHhjcFjmmAROLXK30xgffKioTpzffsbwYeM2YIj15xg+fFNjY49LCxanHmBgACLiNQADpIEExaNgFIyCUTAiAABodE/kKsmbFwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1526-6958","institution":"University of Hormozgan","correspondingAuthor":true,"prefix":"","firstName":"Narges","middleName":"Amrollahi","lastName":"Biuki","suffix":""},{"id":322028134,"identity":"3fd3c5cf-de13-4412-8062-cc819bf2ebda","order_by":2,"name":"Cinzia Ferrario","email":"","orcid":"","institution":"University of Milan: Universita degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Cinzia","middleName":"","lastName":"Ferrario","suffix":""},{"id":322028135,"identity":"8189a100-2a62-408a-9dae-b45a6d018b44","order_by":3,"name":"Michela Sugni","email":"","orcid":"","institution":"University of Milan: Universita degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Michela","middleName":"","lastName":"Sugni","suffix":""}],"badges":[],"createdAt":"2024-06-26 09:56:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4641789/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4641789/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61084382,"identity":"44138a4b-90b2-4e44-93ff-dd8aeeb33a67","added_by":"auto","created_at":"2024-07-25 11:29:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":483928,"visible":true,"origin":"","legend":"\u003cp\u003eA: the experimental model \u003cem\u003eO. scolopendrina\u003c/em\u003eand B: Sampling site in Qeshm Island in Persian Gulf (Iran).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/7d31b2a9e3702806582922fc.png"},{"id":61084383,"identity":"dc65095e-718c-414c-9fc9-45960e32b4e9","added_by":"auto","created_at":"2024-07-25 11:29:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1176610,"visible":true,"origin":"","legend":"\u003cp\u003eRegenerated arms of \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e24 h p.a. (a,b) and 72 h p.a. (c,d). Light microscopy. Semi-thin sagittal sections stained with Milligan Trichrome staining method. Arrowhead in both b \u0026amp; d shows the epithelial layer that covered the wound area and arrow indicates migrating cells and coelomocytes. Detail of the thin epithelium (arrowhead) lining the wound: the healing process is still incomplete.\u003cem\u003e As\u003c/em\u003e Aboral arm shield,\u003cem\u003e e\u003c/em\u003e epineural sinus, \u003cem\u003el\u003c/em\u003e ligaments, \u003cem\u003em\u003c/em\u003e intervertebral muscles,\u003cem\u003e n\u003c/em\u003e radial nerve,\u003cem\u003e Os\u003c/em\u003e Oral arm shield, \u003cem\u003erwc\u003c/em\u003e radial water canal.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/5b294ca01c163cf8ca1ccc38.png"},{"id":61084387,"identity":"94787b2d-f56b-4966-b083-709c186ba692","added_by":"auto","created_at":"2024-07-25 11:29:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2744909,"visible":true,"origin":"","legend":"\u003cp\u003eRegenerated arms of \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e. Light microscopy. Semi-thin sections stained with Milligan Trichrome staining method. (a-c): 3 weeks p.a. (d-f) 5 weeks p.a. (g-h) 6 weeks p.a. Accumulation of the cells of the ectoneural component of the radial nerve cord in close association with undifferentiated coelomocytes and phagocytes (arrow in b). The arrowhead in c indicates the accumulation of large numbers of migrating cells at the tip of the regenerated arm. Arrowhead in f indicates the accumulation of cells in regenerated blastema.\u003cem\u003e As\u003c/em\u003e Aboral arm shield,\u003cem\u003ee\u003c/em\u003e epineural sinus, \u003cem\u003el\u003c/em\u003e ligaments, \u003cem\u003em\u003c/em\u003e intervertebral muscles,\u003cem\u003e n\u003c/em\u003e radial nerve,\u003cem\u003e Os\u003c/em\u003e Oral arm shield,\u003cem\u003e p\u003c/em\u003e podia,\u003cem\u003e ra\u003c/em\u003e regenerating arm, \u003cem\u003erwc\u003c/em\u003e radial water canal, \u003cem\u003ev\u003c/em\u003e vertebra.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/63a1ae7b476b1c25edb2631a.png"},{"id":61084385,"identity":"4419292c-ca70-4831-a77c-89121f5600d0","added_by":"auto","created_at":"2024-07-25 11:29:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360142,"visible":true,"origin":"","legend":"\u003cp\u003eThe length of regenerated arm of \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e. Stereomicroscopy.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/be4c0dce286427dc31cd13e7.png"},{"id":61084386,"identity":"a199f0d3-e03f-4b17-89d8-5c500f8bae28","added_by":"auto","created_at":"2024-07-25 11:29:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39765,"visible":true,"origin":"","legend":"\u003cp\u003eThe Diagram displayed how length of regenerated arm of \u003cem\u003eO. scolopendrina\u003c/em\u003e gradually increased during the experiment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/6ed8cf805b40fe36c5cfee78.png"},{"id":61339316,"identity":"223c117a-7952-4d9d-8e1b-64134b7b0e87","added_by":"auto","created_at":"2024-07-29 16:17:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7506098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4641789/v1/c29bf964-71f4-4a34-9604-7de52b2c6df8.pdf"}],"financialInterests":"","formattedTitle":"Unlocking the Secrets of Regeneration: Histology of Regenerating Arm in Ophiocoma scolopendrina from the Persian Gulf","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegeneration as a post-embryonic developmental process, implies the reconstruction of lost or injured tissues or body parts caused by traumatic or self-induced amputations (Mattson, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Goss, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Regeneration can occur at different stages of life (larval and adult) as well as at different levels of biological organization: cells, tissues and organs. Re-growth of lost parts as well as rebuilding the whole body from small parts, all can be considered as regeneration events which can vary a lot among Phyla (Bely and Nyberg, 2010; Rinkevich et AL., 2022). Generally, the extent of regeneration ability among the phylogenetic tree of Metazoan, decreases as much as organisms get more complex (Alvarado, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Although, there are some vertebrates like zebrafishes, lizards, mice that can show different range of regenerative abilities (Thouveny and Tassava, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Brockes and Kumar, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nakatani et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nacu and Tanaka, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e); invertebrates show higher regenerative capabilities than vertebrates. Indeed, some of them are able to completely rebuild their entire body from remaining body fragments or even produce a new individual which is completely the same as the old one as asexual reproduction method as well known, in some starfish like \u003cem\u003eLinkia sp.\u003c/em\u003e and \u003cem\u003eCoscinasterias sp.\u003c/em\u003e (Thouveny and Tassava, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Candia Carnevali, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Agata and Inoue, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHigh regenerative ability in echinoderms has increased their chance of survival. Division by fission and the creation of fragments also allows them to rapidly produce colonies adapted to the environmental conditions of the new area. Therefore, the strategic evidences show regeneration has certainly helped these species adaptation as well as increasing their success and survival in marine ecosystems (Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll stellate echinoderms have the ability to regenerate their arms, but the timing and the specific processes involved vary among and within classes (Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These variations may be related to the particular anatomy, physiology and behavior of each species. Generally, the arms of Crinoidea (Mladenov, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and Ophiuroidea, which are thin, delicate and long, regenerate faster than the arms of large Asteroidea. Ophiuroidea have a high regenerative potential, with 80\u0026ndash;100% of them showing this ability and more than 70% of their arms regenerating simultaneously (Wilkie, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Bourgoin and Guillou, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Clements et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sk\u0026ouml;ld and Rosenberg, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Soong et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Yokoyama and Amaral, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In general, regardless of the class, the regeneration process and its main cellular/histological events can be divided into three main stages: a repair stage, an early regeneration stage and an advanced regeneration stage.\u003c/p\u003e \u003cp\u003eThe rate of regeneration can considerably vary between the three stellate classes and also within members of the same class. In general, small ophiuroids and crinoids regenerate faster than big epiphytic ophiuroids and asteroids. This might reflect aspects of regeneration mechanisms that are related to size or species (Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, according to Dupont and Thorndyke (2006), in ophiuroids, depending on the number of segments and missing parts of the arm, the animal will invest more in number or speed of differentiation of regenerated segments. Thus, we chose \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e as an ophiuroid model for this study; since it\u0026rsquo;s one of the most abundant brittle stars in the southern intertidal zone of Qeshm island in Iran so it is easy to collect (Fatemi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It also has a proper size for histological analyses according to what Ben Khadra and co-workers showed in previous studies and we mentioned above, that the size of ophiuroids affects the regeneration rate; for example, within ophiuroids, small-sized, such as \u003cem\u003eAmphiura filiformis\u003c/em\u003e, usually regenerate faster than large epibenthic species, such as \u003cem\u003eOphioderma longicaudum\u003c/em\u003e or \u003cem\u003eOphioplocus januarii\u003c/em\u003e (Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). So, we are trying to describe arm regeneration in this species to increase the diversity of brittle star models, from different geographical regions and ecological conditions that can answer this question if the temperature affects the regeneration or not and to prepare information for further studies in the field of comparing the species discussed in this paper with the species that live in other regions with different temperature conditions. In another hand with defining the baseline regenerative process in this species, we can provide the data for more applied research, such as in environmental monitoring, etc.\u003c/p\u003e \u003cp\u003eIndeed, the aim of this study is investigating the arm regenerative process after traumatic amputation in \u003cem\u003eO. scolopendrina\u003c/em\u003e and comparing the regenerative phases of this species to the previous examined species to give a clearer image of the regeneration process in these brittle stars which lives and adopted to Persian Gulf situation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal collection, maintenance and regeneration tests\u003c/h2\u003e \u003cp\u003eNon-regenerating adult (disc diameter\u0026thinsp;~\u0026thinsp;14 mm) specimens of \u003cem\u003eO. scolopendrina\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A) were collected on September 26th 2019 at the intertidal zone of South-East coast of Qeshm Island (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B) (26\u003csup\u003e◦\u003c/sup\u003e55\u003csup\u003e'\u003c/sup\u003e31.23\u003csup\u003e\"\u003c/sup\u003eN, 56\u003csup\u003e◦\u003c/sup\u003e14\u003csup\u003e'\u003c/sup\u003e18.71\u003csup\u003e\"\u003c/sup\u003eE). They were immediately transferred to the marine laboratory of Hormozgan University (Bandar-abbas, Iran) where they were left to acclimatize for 12 days and maintained in aerated aquaria filled with sea-water at 25\u003csup\u003e◦\u003c/sup\u003eC and 39 ppt salinity. Chemical-physical sea water parameters were constantly checked and adjusted if necessary. Animals were fed twice a week with Microvore Microdiet (Brightwell Aquatics), as described in Ferrario et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the regeneration test, specimens were anaesthetized in 3.5% MgCl\u003csub\u003e2\u003c/sub\u003e(6H\u003csub\u003e2\u003c/sub\u003eO) solution in a 1:1 mix of filtered sea water and distilled water (Blowes et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ferrario et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Traumatic arm amputation was performed using a scalpel. A maximum of two arms per animal were amputated at 1 cm from the disc. Then, animals were left to regenerate in three aquaria with the same chemical-physical sea water parameters as acclimatization aquaria. Three replica groups for each time point were left to regenerate for pre-determined periods, namely 24 and 72 hours (h) and 7, 14, 21, 35 and 42 days post-amputation (p.a.). Then, regenerating arms (stump\u0026thinsp;+\u0026thinsp;regenerate) were collected using a scalpel following the above described anaesthetizing process and treated for the subsequent analyses; but before that, regenerating arm were recorded and photographed under a stereomicroscope and their lengths measured by image j program for calculating the regeneration rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLight microscopy analysis\u003c/h2\u003e \u003cp\u003eRegenerating arms were fixed for 48 h at room temperature in Bouin\u0026rsquo;s fixative, composed of 75 ml of saturated picric acid in distilled water, 25 ml of 40% formaldehyde solution and 5 ml of glacial acetic acid. Following fixation, specimens were decalcified for 5\u0026ndash;7 days in 5% Trichloroacetic acid (TCA) solution (Heraeus Kulzer, Technovit 7100 Germany), dehydrated through an increasing ethanol series, immersed in xylene and then embedded in paraffin wax (58\u0026ndash;59\u003csup\u003e◦\u003c/sup\u003eC) using the Tissue processor (DS 2080/H). Serial parasagittal and sagittal sections (5\u0026ndash;7 \u0026micro;m in thickness) of regenerated arms were prepared using a MICRODS 4055 microtome and mounted on Albumin coated slides. Following dewaxing in xylene and rehydration, slides were then stained using Milligan trichrome method (Milligan, 1946); this staining differentiates collagenous (blue-green) from non-collagenous (red-violet) tissues (Ben Khadra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Slides were examined and photographed under a light microscope equipped with a Nikon camera in the marine laboratory of Hormozgan University.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eInvestigating observation of \u003cem\u003eO. scolopendrina\u003c/em\u003e arm regeneration by light microscope at 24 h, 72 h, 7 days, 14 days, 21 days, 35 and 42 days After the amputation, have been brought in following paragraphs of this manuscript:\u003c/p\u003e\n\u003ch3\u003eControl samples’ regeneration\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003e24 hours p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe healing process of the brittle star arm after amputation was observed in sagittal sections of the samples at different time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.a). Within 24 hours, a thin layer of epithelial cells covered the wound area, indicating the first sign of healing. This layer was likely formed by a combination of two mechanisms: the expansion of the outer epidermis of the arm and the migration of cells from other tissues. However, this process was not complete yet, as evidenced by the presence of scattered cells in the loose connective tissue below the epithelial layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b). The aboral coelomic cavity and the radial water canal, which are important structures for the brittle star physiology and locomotion, were also closed off at their distal ends. This closure was probably achieved by a combination of cell migration and muscle contraction of the coelomic wall. Adjacent to the radial nerve cord and the radial water canal, large clusters of cells were visible. These cells included undifferentiated coelomocytes, which are involved in immune response, and phagocytes, which are involved in wound cleaning. The water canal cavity also contained free and dispersing coelomocytes and phagocytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003e72 hours p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn these sections, the thickness of the epithelial layer that covered the wound part of the arm had increased. However, disruption of this layer and scattered cells in the connective tissue that supported the bottom layer indicated that the process was still incomplete for these animal samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.d). The RNC along with the surrounding coelomic cells was also clearly apparent at the taken sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.c).\u003c/p\u003e \u003cp\u003e \u003cb\u003e1 week p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSections of this stage (not shown) displayed the thicker epidermis that contained a large number of scattering small cells and phagocytes in a filamentous, dense, mesh-like network of connective tissue. In this developing layer, reconstruction of the coelomic cavity and the hypertrophic radial water canal could also be observed. At these sections, a large number of free cells, coelomic cells, phagocytes, as well as de-differentiating myocytes were visible within the coelomic cavity and the radial water canal. Undifferentiated myocytes were often recognizable among the intervertebral muscles, especially in the unorganized parts around them.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2 weeks p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt this stage (not shown), we observed an intense development and elongation of the coelomic cavity and a large group of migrating cells scattered in the connective tissue that was separating from the thick layer of cells that were covered the wound part of the arm at the first stage. At this stage, the accumulation of dedifferentiated myocytes, phagocytes, and coelomocytes can also be observed. On the other hand, podia were also developing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3 weeks p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSigns of regenerated parts and sclerocytes, tissue structures and their pattern of formation and tissue differentiation were visible. Coelomic and neural contents were clearly evident in the regenerated bud of the arm which showed well-developed anatomical and histological features. In general, the regenerated RNC exhibited a pseudo-ganglionic pattern, whereas the fully separated radial water canal exhibited a hypertrophic structure. The large accumulation of migratory cells (mainly coelomocytes), phagocytes, and dedifferentiated myocytes that were often visible in the coelomic cavities, were mainly at the end of the regenerating arm at this stage. The structure of new podia was also visible at this stage. The regenerated miniature arm was developing and increasing in size.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5 weeks p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSamples of this stage represent the complete development of the regenerated arm with an advanced level of tissue differentiation. In general, the structure of the serial pseudo-ganglionated structure of the regenerated nerve cord is clearly identified in the regenerated segment. The main coelomic cavity and the other coelom-derived channels, such as the radial water canal, hyponeural sinus, are well developed. In the old stump, the muscles exhibit patterns of rearrangement, and some dedifferentiated myocytes are still present. Undifferentiated coelomocytes, phagocytes, and dedifferentiated myocytes are still scattered in new coelomic canals. From this point on, all structures of miniature arm are formed.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6 weeks p.a.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe regenerated section of this stage shows all the general structures of the brittle stars\u0026rsquo; arm: the nerve cord with its pseudo-ganglionic structure, the radial water canal with podia, the ossicles and the and muscle tissues and ultimately recognizable skeletal plates with their spines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRegenerate length\u003c/h2\u003e \u003cp\u003eThe lengths (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm) of regenerated arms at the different time-point are reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Overall the regenerate reached approximately 17.65 mm after 42 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite the differences between the time of regeneration phases of the brittle star studied in this experiment with previous studies (Biressi described 4 stages...in Ben Khadra et al \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e we described 3 phases also for brittla stars), \u003cem\u003eO. scolopendrina\u003c/em\u003e had many similarities in the basic mechanisms of arm regeneration with specimens previously studied. The overall regeneration process, from the amputation to the regeneration of a miniature arm that showed all the structural components of a complete brittle star\u0026rsquo;s arm, could be divided into three main stages: a repair phase, an early regenerative phase and an advanced regenerative phase, as described in Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eThe repair phase\u003c/h3\u003e\n\u003cp\u003eThis phase included the time between amputation and 48 hours p.a.- It was comparable to the phase observed in other species that Biressi et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) studied. The main characteristic of this phase was wound healing and it included: 1. Closure of the coelomic canals and cavities which was the result of the contractions of the coelomic wall and the migration of free cells (Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 2. The process of re-epithelialization at the wound area, which was carried out through the epidermal migrating cells, though it wasn\u0026rsquo;t complete yet. 3. The first signs of rearrangement of tissues that were damaged during the amputation. The presence of loose and net like of connective tissue beneath the external epithelium could be a clear indication that the repair process was not complete yet.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe early regenerative phase\u003c/h2\u003e \u003cp\u003eThis phase involved time from 24 hours p.a. to 7 days p.a. The stage began with the strong migration and of cells covering the wound area and eventually resulted in the formation of a sub-epithelial layer containing dense mesenchyme with dedifferentiated cells and phagocytes. These migrating cells, which were usually seen in the coelomic cavities, actually moved rapidly to the ends of the coelomic cavity and RNC segments. The ending structures in this phase were covered by a number of layers made up of migrating cells (Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). After this time till 14 days p.a. segments grew along the growing end of the RNC and the radial water canal in the injured area. Key events of morphogenesis and differentiation in this phase occurred in the regenerating tip, which is practically associated with the development of pseudo-ganglionic RNC and the differentiation of radial water canal. Indeed, during these regeneration stages, undifferentiated cells together with the undifferentiated myocytes would be involved in different regeneration processes (Alvarado, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe advanced regeneration phase\u003c/h2\u003e \u003cp\u003eAfter 14 days post amputation, regenerated arm entered the advanced regenerative phase. In this phase, extensive growth, morphogenesis and differentiation resulted in the formation of a miniature arm. During this phase, the structural patterns of the normal arm gradually became more apparent; its earliest regenerated parts, in particular, acquire distinctive features of the interior and exterior of a fully functional arm, while the tip of it yet appeared to be undifferentiated (Ferrario et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComparison of the regeneration process in \u003cem\u003eO. scolopendrina\u003c/em\u003e and species previously studied by other researchers showed similarities to the regeneration different phases and supported the idea that the regeneration in Ophiuroidea, despite to its great diversity, has the same essential characteristics (Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ferrario et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed and followed all three main phases: a repair phase, an early regenerative phase and an advanced regenerative phase, which was different in term of timing with other species studied before (Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Ben Khadra et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the samples obtained from this study, different types of cells were also observed, which according to Bannister and colleagues in 2005, are the first responsible parts for the growth of the regenerated structure. These cells which include coelomocytes, as well as dedifferentiated myocytes from the stump tissues, migrate to the tip area and accumulate there; then with active proliferation and differentiation they formed new cells (Bannister et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Biressi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthic statement\u003c/h2\u003e \u003cp\u003eStars sampling steps and animal manipulations were performed according to the ethic certification evaluated by Hormozgan University of Medical science with approval ID: IR.HUMS.REC.1398.181and under observation of Qeshm Department of Environment of Qeshm Free Area organization (QDOE of QFAO). Ophiocoma scolopendrina is not an endangered or protected species. All efforts were made to minimize animal suffering during the whole experimental procedure.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe extend our deepest gratitude to the corresponding author, who has guided this research with utmost precision and dedication. We also thank our esteemed advisors from the University of Milan, Italy, for their expert opinions and valuable guidance, which significantly contributed to the advancement of this scientific work. Our appreciation goes to the laboratory of the Faculty of Science and Technology at the University of Hormozgan for providing a dynamic and well-equipped environment for conducting meticulous experiments. Lastly, we are indebted to Mr. Dakhteh, the Director of Environmental Protection for the Qeshm Free Zone, whose generous support has been instrumental in the success of this project. We should also mention that all authors declare they have no conflicts of interest and we did not receive any budget or funding from other organizations for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgata, K., \u0026amp; Inoue, T. (2012). Survey of the differences between regenerative and non‐regenerative animals. \u003cem\u003eDevelopment, growth \u0026amp; differentiation\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e(2), 143-152.\u003c/li\u003e\n\u003cli\u003eAlvarado, A. S. (2000). Regeneration in the metazoans: why does it happen?. \u003cem\u003eBioessays\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(6), 578-590.\u003c/li\u003e\n\u003cli\u003eBachoo, S. (2002). \u003cem\u003eExperimental Cadmium Contamination of the Echinoid Stomopneustes Variolaris (Echinodermata: Echinoidea): Influence of Dosage and Distribution of the Metal in the Organism\u003c/em\u003e (Doctoral dissertation, University of Durban-Westville).\u003c/li\u003e\n\u003cli\u003eBannister, R., McGonnell, I. M., Graham, A., Thorndyke, M. C., \u0026amp; Beesley, P. W. (2005). Afuni, a novel transforming growth factor-\u0026beta; gene is involved in arm regeneration by the brittle star Amphiura filiformis. \u003cem\u003eDevelopment genes and evolution\u003c/em\u003e, \u003cem\u003e215\u003c/em\u003e(8), 393-401.\u003c/li\u003e\n\u003cli\u003eBen Khadra, Y., Ferrario, C., Di Benedetto, C., Said, K., Bonasoro, F., Candia Carnevali, M. D., \u0026amp; Sugni, M. (2015). Wound repair during arm regeneration in the red starfish E chinaster sepositus. \u003cem\u003eWound Repair and Regeneration\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(4), 611-622.\u003c/li\u003e\n\u003cli\u003eBen Khadra, Y., Sugni, M., Ferrario, C., Bonasoro, F., Oliveri, P., Martinez, P., \u0026amp; Carnevali, M. D. C. (2018). Regeneration in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea. In \u003cem\u003eMarine Organisms as Model Systems in Biology and Medicine\u003c/em\u003e (pp. 285-320). Springer, Cham.\u003c/li\u003e\n\u003cli\u003eBiressi, A. C. M., Zou, T., Dupont, S., Dahlberg, C., Di Benedetto, C., Bonasoro, F., ... \u0026amp; Carnevali, M. D. C. (2010). Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura filiformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis. \u003cem\u003eZoomorphology\u003c/em\u003e, \u003cem\u003e129\u003c/em\u003e(1), 1-19.\u003c/li\u003e\n\u003cli\u003eBlowes, L. M., Egertov\u0026aacute;, M., Liu, Y., Davis, G. R., Terrill, N. J., Gupta, H. S., \u0026amp; Elphick, M. R. (2017). Body wall structure in the starfish Asterias rubens. \u003cem\u003eJournal of anatomy\u003c/em\u003e, \u003cem\u003e231\u003c/em\u003e(3), 325-341.\u003c/li\u003e\n\u003cli\u003eBourgoin, A., \u0026amp; Guillou, M. (1994). Arm regeneration in two populations of Acrocnida brachiata (Montagu)(Echinodermata: Ophiuroidea) in Douarnenez Bay,(Brittany: France): an ecological significance. \u003cem\u003eJournal of experimental marine biology and ecology\u003c/em\u003e, \u003cem\u003e184\u003c/em\u003e(1), 123-139.\u003c/li\u003e\n\u003cli\u003eBrockes, J. P., \u0026amp; Kumar, A. (2002). Plasticity and reprogramming of differentiated cells in amphibian regeneration. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(8), 566.\u003c/li\u003e\n\u003cli\u003eCandia Carnevali, M. D. (2006). Regeneration in Echinoderms: repair, regrowth, cloning. \u003cem\u003eInvertebrate Survival Journal\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(1), 64-76.\u003c/li\u003e\n\u003cli\u003eClements, L. A. J., Bell, S. S., \u0026amp; Kurdziel, J. P. (1994). Abundance and arm loss of the infaunal brittlestar Ophiophragmus filograneus (Echinodermata: Ophiuroidea), with an experimental determination of regeneration rates in natural and planted seagrass beds. \u003cem\u003eMarine Biology\u003c/em\u003e, \u003cem\u003e121\u003c/em\u003e(1), 97-104.\u003c/li\u003e\n\u003cli\u003eCzarkwiani, A., Ferrario, C., Dylus, D. V., Sugni, M., \u0026amp; Oliveri, P. (2016). Skeletal regeneration in the brittle star Amphiura filiformis. \u003cem\u003eFrontiers in zoology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 18.\u003c/li\u003e\n\u003cli\u003eDaviddi, A. (2014). Microscopic anatomy of arm-tip regeneration in the starfish Marthasterias glacialis (Linneaus, 1758) following traumatic amputation. Millan university, Master thesis.\u003c/li\u003e\n\u003cli\u003eFatemi, S. R., Jamili, S., Valinassab, T., \u0026amp; Kuranlu, N. (2010). Diversity of Ophiuroidea from lengeh portand Qeshm island in the persian gulf. \u003cem\u003eJ Fish Aquat Sci\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(1), 42-48.\u003c/li\u003e\n\u003cli\u003eFerrario, C., Khadra, Y. B., Czarkwiani, A., Zakrzewski, A., Martinez, P., Colombo, G., ... \u0026amp; Sugni, M. (2018). Fundamental aspects of arm repair phase in two echinoderm models. \u003cem\u003eDevelopmental biology\u003c/em\u003e, \u003cem\u003e433\u003c/em\u003e(2), 297-309.\u003c/li\u003e\n\u003cli\u003eFichet, D., Radenac, G., \u0026amp; Miramand, P. (1998). Experimental studies of impacts of harbour sediments resuspension to marine invertebrates larvae: bioavailability of Cd, Cu, Pb and Zn and toxicity. \u003cem\u003eMarine Pollution Bulletin\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e(7), 509-518.\u003c/li\u003e\n\u003cli\u003eGoss, R. J. (2013). Principles of regeneration. Elsevier.\u003c/li\u003e\n\u003cli\u003eLawrence, J. M. (2010). Energetic costs of loss and regeneration of arms in stellate echinoderms. \u003cem\u003eIntegrative and comparative biology\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(4), 506-514.\u003c/li\u003e\n\u003cli\u003eMattson, P. (1976). Regeneration. Bobbs-Merrill Company.\u003c/li\u003e\n\u003cli\u003eMladenov, P. V. (1983). Rate of arm regeneration and potential causes of arm loss in the feather star Florometra serratissima (Echinodermata: Crinoidea). \u003cem\u003eCanadian Journal of Zoology\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e(12), 2873-2879.\u003c/li\u003e\n\u003cli\u003eNacu, E., \u0026amp; Tanaka, E. M. (2011). Limb regeneration: a new development?. \u003cem\u003eAnnual review of cell and developmental biology\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e, 409-440.\u003c/li\u003e\n\u003cli\u003eNakatani, Y., Kawakami, A., \u0026amp; Kudo, A. (2007). Cellular and molecular processes of regeneration, with special emphasis on fish fins. \u003cem\u003eDevelopment, growth \u0026amp; differentiation\u003c/em\u003e, \u003cem\u003e49\u003c/em\u003e(2), 145-154.\u003c/li\u003e\n\u003cli\u003ePhillips, D. J. H., \u0026amp; Rainbow, P. S. (1993). Biomonitoring of trace aquatic contaminants Elsevier Applied Science. \u003cem\u003eNew York\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSk\u0026ouml;ld, M., \u0026amp; Rosenberg, R. (1996). Arm regeneration frequency in eight species of Ophiuroidea (Echinodermata) from European sea areas. \u003cem\u003eJournal of Sea Research\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(4), 353-362.\u003c/li\u003e\n\u003cli\u003eSoong, K. Y., Shen, Y., Tseng, S. H., \u0026amp; Chen, C. P. (1997). Regeneration and potential functional differentiation of arms in the brittlestar, Ophiocoma scolopendrina (Lamarck)(Echinodermata: Ophiuroidea). \u003cem\u003eZoological Studies\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e(2), 90-97.\u003c/li\u003e\n\u003cli\u003eThouveny, Y., \u0026amp; Tassava, R. A. (1997). Regeneration Through Phylogenesis. Regeneration through phylogenesis in cellular and molecular basis of regeneration, pp 9-43.\u003c/li\u003e\n\u003cli\u003eWang, H., Zhu, G., Shi, Y., Weng, S., Jin, T., Kong, Q., \u0026amp; Nordberg, G. F. (2003). Influence of environmental cadmium exposure on forearm bone density. \u003cem\u003eJournal of Bone and Mineral Research\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(3), 553-560.\u003c/li\u003e\n\u003cli\u003eWilkie, I. C. (1978). Arm autotomy in brittlestars (Echinodermata: Ophiuroidea). \u003cem\u003eJournal of Zoology\u003c/em\u003e, \u003cem\u003e186\u003c/em\u003e(3), 311-330.\u003c/li\u003e\n\u003cli\u003eYokoyama, L. Q., \u0026amp; Amaral, A. C. Z. (2010). Ophionereis reticulata (Echinodermata, Ophiuroidea). \u003cem\u003eIheringia. \u003c/em\u003e\u003cem\u003eS\u0026eacute;rie Zoologia\u003c/em\u003e, \u003cem\u003e100\u003c/em\u003e(2), 123-127.\u003c/li\u003e\n\u003cli\u003eYouness, E. R., Mohammed, N. A., \u0026amp; Morsy, F. A. (2012). Cadmium impact and osteoporosis: mechanism of action. \u003cem\u003eToxicology mechanisms and methods\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(7), 560-567.\u003c/li\u003e\n\u003cli\u003eZyadah, M. A., \u0026amp; Abdel-Baky, T. E. (2000). Toxicity and bioaccumulation of copper, zinc, and cadmium in some aquatic organisms. \u003cem\u003eBulletin of environmental contamination and toxicology\u003c/em\u003e, \u003cem\u003e64\u003c/em\u003e(5), 740-747.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"regeneration, Persian Gulf, brittle star, Echinoderms, Epimorphosis, Morphallaxis","lastPublishedDoi":"10.21203/rs.3.rs-4641789/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4641789/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e, a prevalent brittle star species in the southernmost intertidal zone of Qeshm Island, serves as an exemplary model for studying echinoderm arm regeneration processes within the Persian Gulf. To elucidate the regenerative mechanisms of these organisms, we conducted a comprehensive investigation of their regenerative structures from a histological perspective. The collected specimens were carefully acclimated to laboratory conditions in aerated seawater aquaria before being transferred to specially treated environments. Adhering to strict ethical protocols, we amputated the arms of the brittle stars and meticulously documented the subsequent regenerative changes at various intervals: 24 hours, 72 hours, and weekly up to six weeks post-amputation. Our findings reveal that \u003cem\u003eOphiocoma scolopendrina\u003c/em\u003e undergoes a triphasic regenerative pathway, encompassing a repair phase, an early regenerative phase, and an advanced regenerative phase. Notably, the temporal progression of these phases differs from that observed in other previously studied species. Initially, the brittle stars effectuate wound closure and healing of the autotomy plane through an epimorphic process, characterized by the migration of epidermal cells and re-epithelialization. Subsequently, the formation of a regenerative blastema within the bud initiates morphogenesis, followed by the differentiation and proliferation of blastemal cells, culminating in the development of the regenerated arm.\u003c/p\u003e","manuscriptTitle":"Unlocking the Secrets of Regeneration: Histology of Regenerating Arm in Ophiocoma scolopendrina from the Persian Gulf","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 11:29:17","doi":"10.21203/rs.3.rs-4641789/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"157a9c1a-4549-4bb0-849f-ac1fe7960161","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-29T16:09:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-25 11:29:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4641789","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4641789","identity":"rs-4641789","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}