Histology and morphometry of the skin of the trident goby Tridentiger brevispinis (Perciformes, Gobiidae)

Histology and morphometry of the skin of the trident goby Tridentiger brevispinis (Perciformes, Gobiidae) | 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 Histology and morphometry of the skin of the trident goby Tridentiger brevispinis (Perciformes, Gobiidae) Hyun-Tae Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1809843/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract The Korean trident goby, Tridentiger brevispinis , lives in adverse habitats that can easily become hypoxic due to low precipitation, regional dry periods, and high amounts of solar radiation. Histological and morphometric studies revealed the goby’s specialized skin (35.4–150.0 µm in thickness), consisting of an epidermis and dermis. The thicker epidermis comprises an outermost surface layer (having taste buds, stratified flattened cells, mucous cells, pigment cells, and stratified polygonal cells), middle layer (having stratified polygonal cells), and stratum germinativum (stratified columnar cells). In particular, the dermis has scales, well-developed vascularization, and a few blood capillaries just above the basement membrane, and a reduced diffusion distance was present in the lateral body. Consequently, adaptations such as thicker epidermis, well-developed vascularization, few blood capillaries, and a reduced diffusion distance may provide cutaneous respiration for survival in poorly oxygenated water during the periodic dry season. cutaneous respiration blood capillary dermal vascularization freshwater goby reduced diffusion distance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Teleosts have a respiratory system that allows for gas exchange of dissolved oxygen absorption and carbon dioxide emission between their body membrane and water (Fernandes, 2016 ). This physiological metabolism is observed in diverse internal organs such as gills (Lefevre et al. 2011 ; Blank and Burggren 2014 ), gastrointestinal surface (Grosell et al. 2010), swim bladder (Fernandes et al. 2012 ), branchial chamber (Sundin et al. 1999 ), labyrinthine organ (Zaccone et al. 2019 ), and skin (Glover et al. 2013 ). Among them, the skin is responsible for 5 to 30% of oxygen absorption in underwater teleosts (Graham 1997 ) that inhabit shallow and stagnant water with lower oxygen levels (Wright 2021 ). In addition, the skin obtains up to 50% percent of supplemental oxygen in amphibious fishes (Graham 2011 ) that are able to move in and out of the water (Ishimatsu 2017 ). For this physiological capability, there are specific skin histological characteristics: 1) intraepidermal blood capillaries developed in various positions of the epidermis, 2) well-developed vascularization along dermal collagen fibers, 3) prominent swollen cells, mucous cells, and club cells, and 4) reduced or absent scales (Kim and Park 2011 ; Glover et al. 2013 ). The trident goby Tridentiger brevispinis prefers to live in slow-flowing streams or reservoirs with a rock, gravel, or pebble bottom, and is distributed along Korean peninsula, Japan, the Kuril Islands, and Sakhalin (Pietsch et al. 2001 ). This goby’s habitat undergoes extreme changes in water level due to dry and rainy seasonal patterns on the Korean peninsula (Kim et al. 2011 ), and may become a periodically slow-flowing water region with low dissolved oxygen content; stagnant water pools are created frequently throughout this aquatic environment. To overcome adverse hypoxic conditions during the dry season, many freshwater fishes have histological adaptations of the skin with blood capillary, epidermal, or dermal modifications (Park 2002 ; Park et al. 2003 a; Park and Kim 2007; Harabawy and Mekkawy 2011 ). While researching fish’s morphology and histology in relation to seasonal changes of the Samcheon-stream, we found obvious blood vessels in T . brevispinis skin. Therefore, the purpose of this study was to describe and analyze the skin structure of T . brevispinis while focusing on cutaneous respiration. Materials And Methods Specimen preparation Eight adult T . brevispinis (85.2 to 11.4 mm in standard length, Fig. 1 ) were caught in Samcheon stream of Jungin-dong, Jeonju-si, Jeollabuk-do (35°46’59”N 127°06’33”E) using a scoop net (10 x 10 mm in nesh) in March 2022. The gobies were immediately anesthetized with 0.05% tricaine methanesulfonate (MS-222; Sigma, St Louis, MO, USA) in the field, and then fixed with 10% formalin solution buffered at pH 7.4 for one day. Experimental procedures obeyed the rules of Jeonbuk National University Institutional Animal Care and Use Committee for animal ethics (2016-12ET-0097). Microscopic investigation Each skin sample (five regions: dorsal snout, operculum, dorsal-caudal region, lateral body, ventral body) of the eight fixed specimens was dissected from the fish’s body, respectively (Fig. 1 ). These fragments were immersed in an ascending series of alcohol (70%, 80%, 90%, 95%, 100%, 100%) for one hour each, cleared in xylene, and then embedded in paraffin-wax (Oxford, paraplast) for 24 hours. The paraffin blocks were five-micrometer serially sectioned using a microtome (Leica 820, Leica Microsystems, Wetzlar, Germany), all paraffin was removed with solvent xylene, dehydrated through descending alcohol series (100%, 100%, 95%, 90%, 80%, 70%, 60%). The skin tissues were stained with hematoxylin and eosin (H&E) and Masson’s trichrome to identify the cell type and clearly identify the epidermis and dermis structures. The image on the stained tissues was obtained by a light microscope (AX10, Carl Zeiss, Germany) and analyzed with Axio vision (LE REL. 4.5, Carl Zeiss, Germany) Statistical analysis SPSS statistical software (statistics version 18.0, IBM, USA) was used for statistical analysis of epidermal thickness (basement membrane to surface) and diffusion distance (blood capillary to surface) between skin regions. Kruskal-Wallis test was applied to mean comparison of epidermal thickness for non-parametric estimation ( p 0.05) determined by Levene’s test. Pearson’s correlation coefficient was measured to identify a correlative interaction between epidermal thickness and diffusion distance. Results Histology The skin of T. brevispinis was classified into two large parts, the epidermis and dermis (Fig. 2 A). The epidermis consisted of the outermost surface layer, middle layer, and stratum germinativum. The dermis contained the stratum laxum and stratum compactum. These five skin regions commonly contain mucous cells (MCs), pigment cells (PCs), stratified columnar cells (SCCs), stratified flattened cells (SFCs), and stratified polygonal cells (SPCs) (Fig. 2 B-D). Taste buds (TB) were only observed in the dorsal snout (Fig. 2 B). The MCs were large oval cell located along the outermost surface layer with a squamous nucleus at its bottom. The cytoplasm showed a faint color or were not stained with H&E (Fig. 2 B). The PCs were small granule-melanophores observed both in between the outermost surface layer and middle layer, and the basement membrane and stratum laxum. PCs demonstrated a deep black color in H&E and Masson’s trichrome staining (Fig. 2 A-D). The SCCs were composed of a single layer of columnar cells along the basement membrane. SCCs showed an oval nucleus with violet color upon staining with H&E and purple color with Masson’s trichrome (Figs. 2 C and D, 3A-D). The SFCs were squamous or cuboidal cells with reduced cytoplasm of pink color stained more deeply than the SPCs in H&E (Fig. 2 B). SPCs had a polygonal shape, unregular cytoplasm, and made up five to ten layers of the middle layer. They had a weak pink cytoplasm on H&E staining. The taste bud was a neuron bundle with a long dendrite extending to the surface. Its nucleus was located at the basal layer of the outermost surface layer (Fig. 2 B). Well-developed dermal vascularization was confirmed in the connective tissue of the three skin regions (operculum, dorsal-caudal region, lateral body) (Figs. 2 C-D, 3 A-D) but little in the dorsal snout (Fig. 2 A) and absent in the ventral body (Fig. 3 E-F). Some blood capillaries wrapped with dermal collagens of the dermis were only observed just below the basement membrane. Occasionally, they protruded slightly into the epidermis of the lateral body (Fig. 3 C and D). Morphometry The epidermal thickness demonstrated regional differences: the dorsal snout showed the highest value (137.3 ± 8.5 µm, 118.9–150.0; mean ± SD, range), followed by the operculum (96.9 ± 5.3 µm, 83.3–109.9), dorsal-caudal region (90.8 ± 7.9 µm, 79.7–105.6), lateral body (88.6 ± 13.8 µm, 65.9–112.1), while the ventral body was the lowest (44.2 ± 5.3 µm, 35.4–52.4). These values showed a highly significant difference in epidermal thickness (Kruskal-Wallis test, χ 2 = 78.944, df = 4, p < 0.001; Fig. 4 A). In addition, the diffusion distance showed regional differences in the minimum distance between blood capillary and the skin surface. The dorsal snout has the highest (189.7 ± 22.4 µm, 141.2–216.9; mean ± SD, range) followed by the dorsal-caudal region (131.9 ± 25.9 µm, 95.7–163.0), operculum (108.3 ± 16.7 µm, 90.7–141.4), and lateral body (88.6 ± 19.0 µm, 51.4–106.7). These values demonstrated a highly significant difference in diffusion distance measurement (one-way ANOVA, df = 3, f = 8.835, p < 0.001; Fig. 4 A). In total, the two parameters (epidermal thickness and diffusion distance) were highly positively correlation in the four skin regions (dorsal snout, operculum, dorsal-caudal region, and lateral body) (Pearson’s correlation coefficient, r = 0.719, p < 0.001; Fig. 4 B). Discussion The fish skin is a body integument that provides diverse barriers and chemical passages as follows: i) the maintenance of water- and ion-osmotic balance (Ghioni et al. 1997 ), ii) a physical barrier to prevent water loss (Sayer 2005 ) and entry of harmful substances (Shephard 1993 ) or potential infective pathogens (Benhamed et al. 2014 ), iii) outer region for color expression (Zarnescu 2017), iv) sensory reception of physical and chemical stimuli (Bleckmann and Zelick 2009 ), and v) cutaneous respiration in the case of some teleosts (Urbina et al. 2014 ). Considering the above reports, the skin of T . brevispinis contained SFCs, SPCs, SCCs, MCs, PCs, and TBs that may enable the maintenance of a stable skin structure (Roberts and Horsley 2014 ), improve tolerance to somewhat turbid water with high amounts of organic materials (Han and An 2013 ; Park and Gwak 2019 ), perceive chemical differences between sour, salty, sweet, and bitter foodstuffs, and adjust to environmental changes in its habitat (Morais 2017 ). Tridentiger brevispinis also shows significant characteristics for cutaneous respiration (Glover et al. 2013 ): 1) a thick epidermis (91.6 ± 31.0 µm, 35.4 − 150.0), 2) well-developed dermal vascularization (in the operculum, dorsal-caudal region, and lateral body), and 3) some blood capillaries with dermal collagen protruding slightly into the epidermis (only in the lateral body). Underwater teleosts or amphibious species have large cells (club cells, mucous cells, swollen cells) that play a major role in absorbing dissolved gas in water and spreading supplemental oxygen to the blood capillary or connective tissue (Park et al. 2001 ; Lauriano et al. 2018 ). Among them, Korean underwater species that inhabit stream regions where water level variation happens frequently (Kim and Park 2002 ) commonly have a thicker epidermis as follows: 53.2 − 111.7 µm in the freshwater goby Rhinogobius brunneus (unpublished) with numerous MCs, 97.5 − 113.5 µm in the Chinese muddy loach Misgurnus mizolepis (Park et al. 2001 ), 87.8 − 137.1 µm in the Korean spined loach Iksookimia koreensis (Park 2002 ) with numerous MCs and club cells, and 59 − 297.0 µm in the Korean eel goby Odontamblyopus lacepedii (Park et al. 2003 b) with abundant MCs and swollen cells. Tridentiger brevispinis has a thicker epidermis as well that is augmented by multi-layered the SPCs and the possession of abundant MCs to promote oxygen diffusion at about 70% of the absorption rate in water (Ultsch and Gross 1979 ). In addition, because a reduced diffusion distance facilitates a higher diffusion velocity of supplementary oxygen, a short length between the blood capillaries and skin surface in many teleosts has been suggested to facilitate cutaneous respiration (Kim and Park 2011 ; Glover et al. 2013 ). So, the reduced diffusion distance of the lateral body in T . brevispinis may be considered the most efficient spot for cutaneous respiration among the five regions studied. In skin vascularization, highly dermal blood vessels are a histological modification for oxygen-carbon dioxide exchange in fish skin (Potter et al. 1995 ; Welsch and Potter 1998 ). In addition, intraepidermal blood capillaries or dermal capillaries near the epidermis allow fish more efficient oxygen absorption than those in the dermis due to the reduced distance between external gas and the blood capillary (Park et al. 2003 a; Park and Kim 2007). So, teleosts living in poorly oxygenated water have been reported to exhibit thicker and wider vascularization and a well-developed dermal papillae of blood capillaries positioned closer to the epidermis. In the lungfish, Neoceratodus forsteri , which is extremely well-adapted to the aerial exposure, abundant blood vessels supplying the papilla and subepidermal capillary network occurs among the fibrous layer of the dermis (Bemis and Northcutt 1992 ). Blood capillaries distributed just below the basement membrane were revealed in M . mizolepis (Park et al. 2001 )d koreensis (Park 2002 ). In addition to such positions of the stratum laxum, the Korean torrent catfish Liobagrus mediadiposalis (Park et al. 2003 a) and the Korean stumpy bullhead Pseudobagrus brevicorpus (Park and Kim 2007) showed blood capillaries in the middle part of the epidermis. All of these examples are best suited to survival in a hypoxic wetland or pool of freshwater prone to drying due to the Korean climate (Kim and Park 2002 ). Tridentiger brevispinis has fine blood capillaries just below the basement membrane protruding into the epidermis and visible only in the lateral body. With this modification, the diffusion distance between blood capillary and surface in T . brevispinis is lowest in the lateral body (88.6 ± 19.0 µm), followed by the operculum (108.3 ± 16.7 µm). This suggests that the skin region of the lateral body of T . brevispinis is the most effective spot for cutaneous respiration. Moreover, well-developed vascularization and blood capillaries near the epidermis in T . brevispinis is a skin modification suitable for obtaining dissolved gas under hypoxic water conditions. Consequently, T . brevispinis exhibits a thicker epidermis, a well-developed vascularization, a few blood capillaries protruding into the epidermis, and reduced diffusion distance in the lateral body, all of which are related to effective cutaneous respiration for survival in a hypoxic habitat during the dry season. Conclusions The Korean trident goby Tridentiger brevispinis showed a thicker epidermis having taste buds, stratified flattened cells, stratified polygonal cells, mucous cells, stratified columnar cells, and pigment cells. Remarkably, the dermis showed well-developed dermal vascularization (in the operculum, dorsal-caudal region, and lateral body) and a few blood capillaries (in lateral body) just above the basement membrane, except for the ventral body (not confirmed). The epidermis thickness was the highest value (137.3 ± 8.5 µm, 118.9–150.0; Mean±SD, Range) and the ventral body was the lowest (44.2 ± 5.3 µm, 35.4–52.4) (Kruskal-Wallis test, χ 2 = 78.944, df = 4, p < 0.001). The diffusion distance was more reduced in the lateral body than other regions (one-way ANOVA, df = 3, f = 8.835, p < 0.001). Epithelial thickness and diffusion distance have a highly significant correlation (Pearson’s correlation coefficient, r = 0.719, p < 0.001). These results demonstrate the goby’s adaptation to cutaneous respiration to overcome hypoxic conditions during periodic dry periods. Abbreviations MC mucous cell PC pigment cell SCC stratified columnar cell SFC stratified flattened cell SPC stratified polygonal cell TB taste bud Declarations Competing interests The author declares no competing interests relevant to the article. Availability of data and materials Not applicable. Funding No funding was received for this work. Author’s contributions Hyun-Tae Kim designed and wrote the manuscript. The author edited and approved the manuscript for publication. Acknowledgements No applicable. References W.E. Bemis, R.G. Northcutt, Skin and blood vessels of the snout of the Australian lungfish, Neoceratodus forsteri , and their significance for interpreting the cosmine of Devonian lungfishes. Acta Zool. 73 , 115–139 (1992) S. Benhamed, F.A. Guardiola, M. Mars, M. Esteban, Pathogen bacteria adhesion to skin mucus of fishes. Vet. Microbiol. 171 , 1–12 (2014) T. Blank, W. Burggren, Hypoxia-induced developmental plasticity of the gills and air‐breathing organ of Trichopodus trichopterus . J. Fish. Biol. 84 , 808–826 (2014) H. Bleckmann, R. Zelick, Lateral line system of fish. Integr. Zool. 4 , 13–25 (2009) N.M. Fernandes, Fish respiration and environment (CRC Press, 2016), p. 408 M.N. Fernandes, A.L. da Cruz, O.T.F. da Costa, S.F. 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Zarnescu, Ultrastructure of the skin melanophores and iridophores in paddlefish, Polyodon spathula . Micron 38 , 81–84 (2007) Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 26 Jul, 2022 First submitted to journal 21 Jul, 2022 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-1809843","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":123954236,"identity":"b1ab807d-fbf3-4f51-80fc-c0e36ac4523f","order_by":0,"name":"Hyun-Tae Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYBACAwY2hgMMFWwgNjORWthAWs6QqoWBsY2BBC3m8m2Jh27O48szON7AbFxBjBbLNrYDh3O3sRUbnDnAnHiGKIcdY28AaUnccCOB+WAD8VrmALXcf0C0FpDDGkC2MDAnEqklLeFwzjG2xJlnEpsNidNy+Jjx55yaY4l9xw8fliRKCxQcA2JGUjQwMNSQpHoUjIJRMApGGAAAxeE2xfs6QbkAAAAASUVORK5CYII=","orcid":"","institution":"Jeonju National University of Education","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hyun-Tae","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2022-06-30 03:55:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1809843/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1809843/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24409375,"identity":"ac1239e2-1af0-419f-bd92-f61dddd6201a","added_by":"auto","created_at":"2022-07-27 16:59:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52597,"visible":true,"origin":"","legend":"\u003cp\u003eThe photograph of \u003cem\u003eTridentiger brevispinis\u003c/em\u003e. The five skin regions are ①, dorsal snout; ②, operculum; ③, dorsal-caudal region; ④, lateral body; ⑤, ventral body, respectively. The bar indicates 5 cm.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1809843/v1/5d0486882d474b0384f1739e.png"},{"id":24409947,"identity":"6eb90a12-60c8-4451-9e72-7040ab78af5b","added_by":"auto","created_at":"2022-07-27 17:04:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":822239,"visible":true,"origin":"","legend":"\u003cp\u003eThe histology of the dorsal snout and operculum skin of \u003cem\u003eTridentiger brevispinis\u003c/em\u003e, stained with hematoxylin and eosin (A, B, C) and Masson’s trichrome (D). A), The skin (dorsal snout) structure classified largely into the epidermal (ED) with outermost surface layer (OS), middle layer (ML) and stratum germinativum (SG) and dermis (DM) with stratum laxum (SL) and stratum compactum (SC); B), the dorsal snout consisting of mucous cells (MC), pigment cells (PC), taste buds (TB), stratified flattened cells (SFC), stratified polygonal cells (SPC); C and D), the operculum showing the stratified columnar cells (SCC) of the ED above the basement membrane (BM), the blood capillaries (yellow asterisk), scale (S) and the PCs in the SL of the DM. The bars indicate 50㎛ in A and 20㎛ in B-D, respectively.\u0026nbsp;\u003c/p\u003e","description":"","filename":"OnlineFig.2revised.png","url":"https://assets-eu.researchsquare.com/files/rs-1809843/v1/9997635a77dba225fcc3b734.png"},{"id":24409377,"identity":"7ca0dea4-39a1-4452-a103-24032c083eb7","added_by":"auto","created_at":"2022-07-27 16:59:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1122047,"visible":true,"origin":"","legend":"\u003cp\u003eThe histology of the dorsal-caudal region (A, B), lateral body (C, D), ventral body (E, F) of \u003cem\u003eTridentiger brevispinis\u003c/em\u003e, stained with hematoxylin and eosin (A, C) and Masson’s trichrome (B, D). A and B), the epidermis (ED) consisting of stratified columnar cells (SCC), pigment cells (PC) distributed along the basement membrane (BM), blood capillaries (yellow asterisk) in the stratum laxum (SL); C and D), the ED consisting of the SCCs, the SPCs, the PCs, and blood capillaries (yellow asterisk) and scales just below the (BM). The scale bars indicate 20 ㎛ in A-D, and 50㎛ in E-F, respectively. \u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"OnlineFig.3revised.png","url":"https://assets-eu.researchsquare.com/files/rs-1809843/v1/837ac5c3ccb242a42f69e3cc.png"},{"id":24409374,"identity":"50cff3cf-a54e-4e97-9a21-1b23438c785c","added_by":"auto","created_at":"2022-07-27 16:59:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16264,"visible":true,"origin":"","legend":"\u003cp\u003eRelative differences (A) in the epidermal thickness and the diffusion distance on the five skin regions; A correlation (B) between two factors, epidermal thickness (x-axis, \u003cem\u003en\u003c/em\u003e = 20) and diffusion distance (y-axis, \u003cem\u003en\u003c/em\u003e = 20). Circle, dorsal snout; triangle, operculum; x, dorsal-caudal region; diamond, lateral body. ET, epithelial thickness; DD, diffusion distance; DT, diffusion distance and epithelial thickness values.\u003c/p\u003e","description":"","filename":"OnlineFig.4revised.png","url":"https://assets-eu.researchsquare.com/files/rs-1809843/v1/9ac3ab96be3eb2bc09db99ee.png"},{"id":24409948,"identity":"9fdfff30-8a07-47ac-b7e6-058a04b59951","added_by":"auto","created_at":"2022-07-27 17:04:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":270315,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1809843/v1/b3ac4f14-b853-4f02-867a-667db945bdf0.pdf"}],"financialInterests":"","formattedTitle":"Histology and morphometry of the skin of the trident goby Tridentiger brevispinis (Perciformes, Gobiidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTeleosts have a respiratory system that allows for gas exchange of dissolved oxygen absorption and carbon dioxide emission between their body membrane and water (Fernandes, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This physiological metabolism is observed in diverse internal organs such as gills (Lefevre et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Blank and Burggren \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), gastrointestinal surface (Grosell et al. 2010), swim bladder (Fernandes et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), branchial chamber (Sundin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), labyrinthine organ (Zaccone et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and skin (Glover et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among them, the skin is responsible for 5 to 30% of oxygen absorption in underwater teleosts (Graham \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) that inhabit shallow and stagnant water with lower oxygen levels (Wright \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, the skin obtains up to 50% percent of supplemental oxygen in amphibious fishes (Graham \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) that are able to move in and out of the water (Ishimatsu \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For this physiological capability, there are specific skin histological characteristics: 1) intraepidermal blood capillaries developed in various positions of the epidermis, 2) well-developed vascularization along dermal collagen fibers, 3) prominent swollen cells, mucous cells, and club cells, and 4) reduced or absent scales (Kim and Park \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Glover et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe trident goby \u003cem\u003eTridentiger brevispinis\u003c/em\u003e prefers to live in slow-flowing streams or reservoirs with a rock, gravel, or pebble bottom, and is distributed along Korean peninsula, Japan, the Kuril Islands, and Sakhalin (Pietsch et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This goby\u0026rsquo;s habitat undergoes extreme changes in water level due to dry and rainy seasonal patterns on the Korean peninsula (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and may become a periodically slow-flowing water region with low dissolved oxygen content; stagnant water pools are created frequently throughout this aquatic environment. To overcome adverse hypoxic conditions during the dry season, many freshwater fishes have histological adaptations of the skin with blood capillary, epidermal, or dermal modifications (Park \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003ea; Park and Kim 2007; Harabawy and Mekkawy \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). While researching fish\u0026rsquo;s morphology and histology in relation to seasonal changes of the Samcheon-stream, we found obvious blood vessels in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e skin. Therefore, the purpose of this study was to describe and analyze the skin structure of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e while focusing on cutaneous respiration.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSpecimen preparation\u003c/h2\u003e \u003cp\u003eEight adult \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e (85.2 to 11.4 mm in standard length, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were caught in Samcheon stream of Jungin-dong, Jeonju-si, Jeollabuk-do (35\u0026deg;46\u0026rsquo;59\u0026rdquo;N 127\u0026deg;06\u0026rsquo;33\u0026rdquo;E) using a scoop net (10 x 10 mm in nesh) in March 2022. The gobies were immediately anesthetized with 0.05% tricaine methanesulfonate (MS-222; Sigma, St Louis, MO, USA) in the field, and then fixed with 10% formalin solution buffered at pH 7.4 for one day. Experimental procedures obeyed the rules of Jeonbuk National University Institutional Animal Care and Use Committee for animal ethics (2016-12ET-0097).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eMicroscopic investigation\u003c/h2\u003e \u003cp\u003eEach skin sample (five regions: dorsal snout, operculum, dorsal-caudal region, lateral body, ventral body) of the eight fixed specimens was dissected from the fish\u0026rsquo;s body, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These fragments were immersed in an ascending series of alcohol (70%, 80%, 90%, 95%, 100%, 100%) for one hour each, cleared in xylene, and then embedded in paraffin-wax (Oxford, paraplast) for 24 hours. The paraffin blocks were five-micrometer serially sectioned using a microtome (Leica 820, Leica Microsystems, Wetzlar, Germany), all paraffin was removed with solvent xylene, dehydrated through descending alcohol series (100%, 100%, 95%, 90%, 80%, 70%, 60%). The skin tissues were stained with hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome to identify the cell type and clearly identify the epidermis and dermis structures. The image on the stained tissues was obtained by a light microscope (AX10, Carl Zeiss, Germany) and analyzed with Axio vision (LE REL. 4.5, Carl Zeiss, Germany)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS statistical software (statistics version 18.0, IBM, USA) was used for statistical analysis of epidermal thickness (basement membrane to surface) and diffusion distance (blood capillary to surface) between skin regions. Kruskal-Wallis test was applied to mean comparison of epidermal thickness for non-parametric estimation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and one-way ANOVA test for parametric estimation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) determined by Levene\u0026rsquo;s test. Pearson\u0026rsquo;s correlation coefficient was measured to identify a correlative interaction between epidermal thickness and diffusion distance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eThe skin of \u003cem\u003eT. brevispinis\u003c/em\u003e was classified into two large parts, the epidermis and dermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The epidermis consisted of the outermost surface layer, middle layer, and stratum germinativum. The dermis contained the stratum laxum and stratum compactum. These five skin regions commonly contain mucous cells (MCs), pigment cells (PCs), stratified columnar cells (SCCs), stratified flattened cells (SFCs), and stratified polygonal cells (SPCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Taste buds (TB) were only observed in the dorsal snout (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe MCs were large oval cell located along the outermost surface layer with a squamous nucleus at its bottom. The cytoplasm showed a faint color or were not stained with H\u0026amp;E (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The PCs were small granule-melanophores observed both in between the outermost surface layer and middle layer, and the basement membrane and stratum laxum. PCs demonstrated a deep black color in H\u0026amp;E and Masson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). The SCCs were composed of a single layer of columnar cells along the basement membrane. SCCs showed an oval nucleus with violet color upon staining with H\u0026amp;E and purple color with Masson\u0026rsquo;s trichrome (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, 3A-D). The SFCs were squamous or cuboidal cells with reduced cytoplasm of pink color stained more deeply than the SPCs in H\u0026amp;E (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). SPCs had a polygonal shape, unregular cytoplasm, and made up five to ten layers of the middle layer. They had a weak pink cytoplasm on H\u0026amp;E staining. The taste bud was a neuron bundle with a long dendrite extending to the surface. Its nucleus was located at the basal layer of the outermost surface layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWell-developed dermal vascularization was confirmed in the connective tissue of the three skin regions (operculum, dorsal-caudal region, lateral body) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D) but little in the dorsal snout (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and absent in the ventral body (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Some blood capillaries wrapped with dermal collagens of the dermis were only observed just below the basement membrane. Occasionally, they protruded slightly into the epidermis of the lateral body (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eMorphometry\u003c/h2\u003e \u003cp\u003eThe epidermal thickness demonstrated regional differences: the dorsal snout showed the highest value (137.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u0026micro;m, 118.9\u0026ndash;150.0; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, range), followed by the operculum (96.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 \u0026micro;m, 83.3\u0026ndash;109.9), dorsal-caudal region (90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9 \u0026micro;m, 79.7\u0026ndash;105.6), lateral body (88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8 \u0026micro;m, 65.9\u0026ndash;112.1), while the ventral body was the lowest (44.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 \u0026micro;m, 35.4\u0026ndash;52.4). These values showed a highly significant difference in epidermal thickness (Kruskal-Wallis test, χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;78.944, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, the diffusion distance showed regional differences in the minimum distance between blood capillary and the skin surface. The dorsal snout has the highest (189.7\u0026thinsp;\u0026plusmn;\u0026thinsp;22.4 \u0026micro;m, 141.2\u0026ndash;216.9; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, range) followed by the dorsal-caudal region (131.9\u0026thinsp;\u0026plusmn;\u0026thinsp;25.9 \u0026micro;m, 95.7\u0026ndash;163.0), operculum (108.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.7 \u0026micro;m, 90.7\u0026ndash;141.4), and lateral body (88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.0 \u0026micro;m, 51.4\u0026ndash;106.7). These values demonstrated a highly significant difference in diffusion distance measurement (one-way ANOVA, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ef\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.835, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In total, the two parameters (epidermal thickness and diffusion distance) were highly positively correlation in the four skin regions (dorsal snout, operculum, dorsal-caudal region, and lateral body) (Pearson\u0026rsquo;s correlation coefficient, \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.719, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe fish skin is a body integument that provides diverse barriers and chemical passages as follows: i) the maintenance of water- and ion-osmotic balance (Ghioni et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), ii) a physical barrier to prevent water loss (Sayer \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and entry of harmful substances (Shephard \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) or potential infective pathogens (Benhamed et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), iii) outer region for color expression (Zarnescu 2017), iv) sensory reception of physical and chemical stimuli (Bleckmann and Zelick \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and v) cutaneous respiration in the case of some teleosts (Urbina et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Considering the above reports, the skin of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e contained SFCs, SPCs, SCCs, MCs, PCs, and TBs that may enable the maintenance of a stable skin structure (Roberts and Horsley \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), improve tolerance to somewhat turbid water with high amounts of organic materials (Han and An \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Park and Gwak \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), perceive chemical differences between sour, salty, sweet, and bitter foodstuffs, and adjust to environmental changes in its habitat (Morais \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eTridentiger brevispinis\u003c/em\u003e also shows significant characteristics for cutaneous respiration (Glover et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e): 1) a thick epidermis (91.6\u0026thinsp;\u0026plusmn;\u0026thinsp;31.0 \u0026micro;m, 35.4\u0026thinsp;\u0026minus;\u0026thinsp;150.0), 2) well-developed dermal vascularization (in the operculum, dorsal-caudal region, and lateral body), and 3) some blood capillaries with dermal collagen protruding slightly into the epidermis (only in the lateral body).\u003c/p\u003e \u003cp\u003eUnderwater teleosts or amphibious species have large cells (club cells, mucous cells, swollen cells) that play a major role in absorbing dissolved gas in water and spreading supplemental oxygen to the blood capillary or connective tissue (Park et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lauriano et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among them, Korean underwater species that inhabit stream regions where water level variation happens frequently (Kim and Park \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) commonly have a thicker epidermis as follows: 53.2\u0026thinsp;\u0026minus;\u0026thinsp;111.7 \u0026micro;m in the freshwater goby \u003cem\u003eRhinogobius brunneus\u003c/em\u003e (unpublished) with numerous MCs, 97.5\u0026thinsp;\u0026minus;\u0026thinsp;113.5 \u0026micro;m in the Chinese muddy loach \u003cem\u003eMisgurnus mizolepis\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), 87.8\u0026thinsp;\u0026minus;\u0026thinsp;137.1 \u0026micro;m in the Korean spined loach \u003cem\u003eIksookimia koreensis\u003c/em\u003e (Park \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) with numerous MCs and club cells, and 59\u0026thinsp;\u0026minus;\u0026thinsp;297.0 \u0026micro;m in the Korean eel goby \u003cem\u003eOdontamblyopus lacepedii\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003eb) with abundant MCs and swollen cells. \u003cem\u003eTridentiger brevispinis\u003c/em\u003e has a thicker epidermis as well that is augmented by multi-layered the SPCs and the possession of abundant MCs to promote oxygen diffusion at about 70% of the absorption rate in water (Ultsch and Gross \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). In addition, because a reduced diffusion distance facilitates a higher diffusion velocity of supplementary oxygen, a short length between the blood capillaries and skin surface in many teleosts has been suggested to facilitate cutaneous respiration (Kim and Park \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Glover et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). So, the reduced diffusion distance of the lateral body in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e may be considered the most efficient spot for cutaneous respiration among the five regions studied.\u003c/p\u003e \u003cp\u003eIn skin vascularization, highly dermal blood vessels are a histological modification for oxygen-carbon dioxide exchange in fish skin (Potter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Welsch and Potter \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In addition, intraepidermal blood capillaries or dermal capillaries near the epidermis allow fish more efficient oxygen absorption than those in the dermis due to the reduced distance between external gas and the blood capillary (Park et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003ea; Park and Kim 2007). So, teleosts living in poorly oxygenated water have been reported to exhibit thicker and wider vascularization and a well-developed dermal papillae of blood capillaries positioned closer to the epidermis. In the lungfish, \u003cem\u003eNeoceratodus forsteri\u003c/em\u003e, which is extremely well-adapted to the aerial exposure, abundant blood vessels supplying the papilla and subepidermal capillary network occurs among the fibrous layer of the dermis (Bemis and Northcutt \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Blood capillaries distributed just below the basement membrane were revealed in \u003cem\u003eM\u003c/em\u003e. \u003cem\u003emizolepis\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e)d \u003cem\u003ekoreensis\u003c/em\u003e (Park \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition to such positions of the stratum laxum, the Korean torrent catfish \u003cem\u003eLiobagrus mediadiposalis\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003ea) and the Korean stumpy bullhead \u003cem\u003ePseudobagrus brevicorpus\u003c/em\u003e (Park and Kim 2007) showed blood capillaries in the middle part of the epidermis. All of these examples are best suited to survival in a hypoxic wetland or pool of freshwater prone to drying due to the Korean climate (Kim and Park \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). \u003cem\u003eTridentiger brevispinis\u003c/em\u003e has fine blood capillaries just below the basement membrane protruding into the epidermis and visible only in the lateral body. With this modification, the diffusion distance between blood capillary and surface in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e is lowest in the lateral body (88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.0 \u0026micro;m), followed by the operculum (108.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.7 \u0026micro;m). This suggests that the skin region of the lateral body of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e is the most effective spot for cutaneous respiration. Moreover, well-developed vascularization and blood capillaries near the epidermis in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e is a skin modification suitable for obtaining dissolved gas under hypoxic water conditions.\u003c/p\u003e \u003cp\u003eConsequently, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrevispinis\u003c/em\u003e exhibits a thicker epidermis, a well-developed vascularization, a few blood capillaries protruding into the epidermis, and reduced diffusion distance in the lateral body, all of which are related to effective cutaneous respiration for survival in a hypoxic habitat during the dry season.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe Korean trident goby \u003cem\u003eTridentiger brevispinis\u003c/em\u003e showed a thicker epidermis having taste buds, stratified flattened cells, stratified polygonal cells, mucous cells, stratified columnar cells, and pigment cells. Remarkably, the dermis showed well-developed dermal vascularization (in the operculum, dorsal-caudal region, and lateral body) and a few blood capillaries (in lateral body) just above the basement membrane, except for the ventral body (not confirmed). The epidermis thickness was the highest value (137.3 \u0026plusmn; 8.5 \u0026micro;m, 118.9\u0026ndash;150.0; Mean\u0026plusmn;SD, Range) and the ventral body was the lowest (44.2 \u0026plusmn; 5.3 \u0026micro;m, 35.4\u0026ndash;52.4) (Kruskal-Wallis test, \u0026chi;\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 78.944, \u003cem\u003edf\u0026nbsp;\u003c/em\u003e= 4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The diffusion distance was more reduced in the lateral body than other regions (one-way ANOVA, \u003cem\u003edf\u003c/em\u003e = 3, \u003cem\u003ef\u003c/em\u003e = 8.835, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Epithelial thickness and diffusion distance have a highly significant correlation (Pearson\u0026rsquo;s correlation coefficient, \u003cem\u003er\u0026nbsp;\u003c/em\u003e= 0.719, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). These results demonstrate the goby\u0026rsquo;s adaptation to cutaneous respiration to overcome hypoxic conditions during periodic dry periods.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emucous cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epigment cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSCC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estratified columnar cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSFC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estratified flattened cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSPC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estratified polygonal cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eTB\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etaste bud\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no competing interests relevant to the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHyun-Tae Kim designed and wrote the manuscript. The author edited and approved the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eW.E. Bemis, R.G. Northcutt, Skin and blood vessels of the snout of the Australian lungfish, \u003cem\u003eNeoceratodus forsteri\u003c/em\u003e, and their significance for interpreting the cosmine of Devonian lungfishes. Acta Zool. \u003cb\u003e73\u003c/b\u003e, 115\u0026ndash;139 (1992)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Benhamed, F.A. Guardiola, M. Mars, M. Esteban, Pathogen bacteria adhesion to skin mucus of fishes. Vet. Microbiol. \u003cb\u003e171\u003c/b\u003e, 1\u0026ndash;12 (2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. Blank, W. 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Micron \u003cb\u003e38\u003c/b\u003e, 81\u0026ndash;84 (2007)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"[email protected]","identity":"applied-microscopy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appm","sideBox":"Learn more about [Applied Microscopy](http://appmicro.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/APPM/default.aspx","title":"Applied Microscopy","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cutaneous respiration, blood capillary, dermal vascularization, freshwater goby, reduced diffusion distance","lastPublishedDoi":"10.21203/rs.3.rs-1809843/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1809843/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Korean trident goby, \u003cem\u003eTridentiger brevispinis\u003c/em\u003e, lives in adverse habitats that can easily become hypoxic due to low precipitation, regional dry periods, and high amounts of solar radiation. Histological and morphometric studies revealed the goby\u0026rsquo;s specialized skin (35.4\u0026ndash;150.0 \u0026micro;m in thickness), consisting of an epidermis and dermis. The thicker epidermis comprises an outermost surface layer (having taste buds, stratified flattened cells, mucous cells, pigment cells, and stratified polygonal cells), middle layer (having stratified polygonal cells), and stratum germinativum (stratified columnar cells). In particular, the dermis has scales, well-developed vascularization, and a few blood capillaries just above the basement membrane, and a reduced diffusion distance was present in the lateral body. Consequently, adaptations such as thicker epidermis, well-developed vascularization, few blood capillaries, and a reduced diffusion distance may provide cutaneous respiration for survival in poorly oxygenated water during the periodic dry season.\u003c/p\u003e","manuscriptTitle":"Histology and morphometry of the skin of the trident goby Tridentiger brevispinis (Perciformes, Gobiidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-27 16:59:51","doi":"10.21203/rs.3.rs-1809843/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2022-07-26T05:13:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Microscopy","date":"2022-07-21T23:55:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-microscopy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appm","sideBox":"Learn more about [Applied Microscopy](http://appmicro.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/APPM/default.aspx","title":"Applied Microscopy","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7232c1d2-6c84-4ea2-a118-85740385178b","owner":[],"postedDate":"July 27th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-07-27T16:59:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-27 16:59:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1809843","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1809843","identity":"rs-1809843","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}