Lymphoid in : Variations on a theme Julien Rességuier, Alf Seljenes Dalum, Louis Du Pasquier, Yaqing Zhang, Erling Olaf Laf Koppang, Pierre Boudinot, Geert Frits Wiegertjes

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Julien Rességuier, Alf Seljenes Dalum, Louis Du Pasquier, Yaqing Zhang, Erling Olaf Laf Koppang, et al.. Lymphoid tissue in teleost gills: Variations on a theme. Biology, MDPI 2020, 9 (6), pp.1-14. ￿10.3390/biology9060127￿. ￿hal-02883166￿

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Article Lymphoid Tissue in Teleost Gills: Variations on a Theme

1, 2, , 3 4 Julien Rességuier †, Alf S. Dalum † ‡, Louis Du Pasquier , Yaqing Zhang , Erling Olaf Koppang 2, Pierre Boudinot 5 and Geert F. Wiegertjes 4,6,*

1 Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway; [email protected] 2 Section of Anatomy, Faculty of Veterinary Science, Norwegian University of Life Sciences, Ullevålsveien 72, 0454 Oslo, Norway; [email protected] (A.S.D.); [email protected] (E.O.K.) 3 Zoology and Evolutionary Biology, University of Basel, 4051 Basel, Switzerland; [email protected] 4 Aquaculture and Group, Department of Animal Sciences, Wageningen University & Research, 6700 AH Wageningen, The Netherlands; [email protected] 5 University of Paris-Saclay, INRAE, UVSQ, VIM, 78350 Jouy-en-Josas, France; [email protected] 6 Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University & Research, 6700 AH Wageningen, The Netherlands * Correspondence: [email protected]; Tel.: +31-317482732 These authors contributed equally to this work. † Present address: Pharmaq Analytiq, Thormøhlens Gate 53D, 5008 Bergen, Norway. ‡  Received: 4 May 2020; Accepted: 12 June 2020; Published: 15 June 2020 

Abstract: In bony fish, the filaments are essential for gas exchanges, but also are vulnerable to infection by water-borne microorganisms. Omnipresent across fish, gill-associated lymphoid tissues (GIALT) regulate interactions with local microbiota and halt infection by pathogens. A special GIALT structure has recently been found in Salmonids, the interbranchial lymphoid tissue (ILT). However, the structural variation of GIALT across bony fish remains largely unknown. Here, we show how this critical zone of interaction evolved across fishes. By labeling a conserved T-cell epitope on tissue sections, we find that several basal groups of possess typical ILT, while modern teleosts have lymphoepithelium of different shape and size at the base of primary gill filaments. Within , neither body size variation between two related species, zebrafish and common , nor morphotype variation, did have a drastic effect on the structure of ILT. Thereby this study is the first to describe the presence of ILT in zebrafish. The ILT variability across fish orders seems to represent different evolutionary solutions to balancing trade-offs between multiple adaptations of and pharyngeal region, and immune responses. Our data point to a wide structural variation in gill immunity between basal groups and modern teleosts.

Keywords: fish; gills; ilt; ; zap70

1. Introduction The pharyngeal pouches and slits are a fundamental structure shared by all deuterostomes. They were primarily involved in filter feeding. Gills are present within pharyngeal pouches of all during embryogenesis, but they fully develop in adult forms of aquatic fishes. This paraphyletic group comprises lampreys and hagfish (Agnathans), cartilaginous fishes () including and rays with a skeleton made of , and bony fishes () with a skeleton made of bone, including the modern bony fishes (Teleostei), [1]. With a vast and complex folded surface, gills are considered crucial for O2 uptake, energy turnover and exchange of ions and CO2.

Biology 2020, 9, 127; doi:10.3390/biology9060127 www.mdpi.com/journal/biology Biology 2020, 9, x FOR PEER REVIEW 2 of 14

(Osteichthyes) with a skeleton made of bone, including the modern bony (Teleostei), [1]. With a vast and complex folded surface, gills are considered crucial for O2 uptake, energy turnover and exchange of ions and CO2. Even if they serve the same general function, gills of sharks and bony fishes, which have been separated for a long time in evolution, are built on totally different anatomical planes [2,3]. The gills of bony are more efficient respiratory organs than those of cartilaginous fish, and their general Biology 2020, 9, 127 2 of 14 organization allowed a reduction of muscular and skeletal elements compared to Chondrichthyes [3– 6]. It stands to reason that the evolution of teleost gills has been driven by respiratory efficiency [7]. Even if they serve the same general function, gills of sharks and bony fishes, which have been All bonyseparated fish gills for are a longenclosed time in in evolution, the pharyngeal are built on pouches totally diff anderent lie anatomical on cartilaginous planes [2,3]. bases The gills which are called gillof arches. bony fish Supported are more e ffibycient these respiratory arches, organsthe gill than respiratory those of cartilaginous surfaces are fish, always and their organized general in two columns organizationof filaments allowed (also called a reduction primary of muscular lamellae) and skeletal that can elements be separated compared by to Chondrichthyes an interbranchial [3–6]. septum extendingIt standsfrom tothe reason gill arch that the (Figure evolution 1). ofThe teleost gas gills exchange has been occurs driven byat respiratorysecondary effi structuresciency [7]. called lamellae All(also bony called fish gillssecondary are enclosed lamellae) in the pharyngealthat extend pouches from each andlie filament. on cartilaginous These lamellae bases which are are hollowed called gill arches. Supported by these arches, the gill respiratory surfaces are always organized in flap-shapedtwo columnsstructures of filaments where an (also intense called primary blood lamellae)flow runs that in-between can be separated supportive by an interbranchial cells called pillar cells. A typicalseptum branchial extending from epithelium the gill arch is (Figuremainly1). composed The of pavement occurs at secondary cells in-between structures calledwhich reside chloride lamellaecells (also (also calledcalled secondary mitochondria-rich lamellae) that extendcells), from mucous each filament. cells and These neuroepithelial lamellae are hollowed cells [3,8]. Filamentsflap-shaped are compartmentalized, structures where an the intense external blood or flow effe runsrent in-between side of a supportive filament cells is differentiated called pillar cells. from the inner or Aafferent typical branchial side and epithelium the interlamellar is mainly composed region of is pavement positioned cells in-betweenin-between. which Water reside flows chloride over the cells (also called mitochondria-rich cells), mucous cells and neuroepithelial cells [3,8]. Filaments are filamentscompartmentalized, from the efferent the aspect external to or the efferent afferent side of aspect, a filament in is the diff erentiatedopposite fromdirection the inner of or the aff erentblood flow, thus maximizingside and the gas interlamellar exchange. region Within is positioned the Teleos in-between.tei, despite Water flowsthis common over the filaments anatomical from the plan, the morphologyefferent of aspectthe gills to thealso aff showserent aspect, large in variations the opposite between direction ofdifferen the bloodt groups flow, thus and maximizing species. gasSalmonids and cyprinidsexchange. have Within well-developed the Teleostei, despiteinterbranchial this common septa, anatomical but in percomorphs plan, the morphology these ofare the incorporated gills into the gillalso showsarches large [9], variationsexemplifying between an diapparentfferent groupsly gradual and species. shortening Salmonids of andthe cyprinidsinterbranchial have septa well-developed interbranchial septa, but in percomorphs these are incorporated into the gill arches [9], across fishexemplifying orders. an apparently gradual shortening of the interbranchial septa across fish orders.

Figure 1. Schematic view of teleost fish gills. (A) Individual gill arches each offering support to two Figure 1.rows Schematic of gill filaments view of in alternateteleost fish positions, gills. forming (A) Individual a sieve-like gill arrangement. arches each Filaments offering are supported support to two rows of bygill interbranchial filaments septain alternate of which thepositions, length varies forming with the a species. sieve-like The septum arrangement. is most reduced Filaments in are supportedmodern by interbranchial teleosts. (B) Schematic septa representationof which the showing length that varies gill filaments with the are species. compartmentalized The septum into is most reduced anin a fferentmodern and anteleosts. efferent side,(B) withSchematic bloodstream representation from the afferent showing (-deprived that gill blood) filaments to the are efferent side (oxygenated blood) and water circulating in the opposite direction. compartmentalized into an afferent and an efferent side, with bloodstream from the afferent (oxygen- deprivedSince blood) the waterto the is efferent an excellent side environment (oxygenated for blood) pathogens and includingwater circulating viruses [10 ]in the the mucosal opposite direction.linings covering fish gills appear vital in providing immune defense mechanisms [11]. Gills have thus a diffuse presence of immune cells. In addition, a specialized lymphoepithelial tissue termed Sincethe the interbranchial water is an lymphoid excellent tissue environment (ILT) was identified for pathogens in 2008 in Atlanticincluding viruses (Salmo [10] salar )[the12 ].mucosal Subsequent studies in Atlantic salmon and rainbow trout (Oncorhynchus mykiss) demonstrated the linings coveringpresence fish of T lymphocytesgills appear embedded vital in providin in a meshworkg immune of reticulated defense epithelial mechanisms cells in [11]. ILT. Only Gills few have thus a diffuseimmunoglobulin presence of immune (Ig)M+ lymphocytes cells. In addition, were detected, a specialized but the structure lymphoepithelial appears rich in cells tissue expressing termed the interbranchialmajor histocompatibilitylymphoid tissue complex (ILT) (MHC)was identified class II+ cells in and2008 IgT in transcripts Atlantic [12 salmon–20]. The (Salmo ILT must salar be ) [12]. Subsequentconsidered studies as in a part Atlantic of the gill-associatedsalmon and lymphoidrainbow tissue trout (GIALT), (Oncorhynchus which is defined mykiss as one) demonstrated of the four the presence of T lymphocytes embedded in a meshwork of reticulated epithelial cells in ILT. Only few immunoglobulin (Ig)M+ lymphocytes were detected, but the structure appears rich in cells expressing major histocompatibility complex (MHC) class II+ cells and IgT transcripts [12–20]. The

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ILT must be considered as a part of the gill-associated lymphoid tissue (GIALT), which is defined as one of the four main mucosal immune compartments found in bony fish [21]. The salmon ILT was recently found positive for the constitutive expression of CCL19, a cytokine normally associated with expressionBiology in2020 lymphoid, 9, 127 organs, suggesting that it could be considered as an immune organ3 of 14in its own right [22]. Here, we characterized histologically the structure of ILT among representative orders of bony main mucosal immune compartments found in bony fish [21]. The salmon ILT was recently found fish (Figurepositive 2), for targeting the constitutive Zap expression70 (Zeta-chain-associated of CCL19, a cytokine normallyprotein associated kinase with70), expressiona phylogenetically in conservedlymphoid protein, organs, to visualize suggesting the that presence it could beof consideredboth T and as NK an immune lymphocytes. in itsWe own compared right [22]. the impact of body sizeHere, on weILT characterized structure, histologically taking advantag the structuree of of a ILT unique among representativeset of samples orders of of African bony barb representingfish (Figure a rare2), “species targeting Zap flock” 70 (Zeta-chain-associated of at least 14 morphotypes protein kinase with 70), areshaped phylogenetically buccal conserved and pharyngeal regions protein,[23,24]. to We visualize also asked the presence whether of both morpholo T and NKgical lymphocytes. adaptation We compared to different the impact feeding of body modes and size on ILT structure, taking advantage of a unique set of samples of African barb representing a rare presumed“species differences flock” of in at leastwater 14 morphotypesflow over the with gill reshaped region buccal would and correlate pharyngeal with regions morphological [23,24]. We also variation in the ILT.asked We whether discuss morphological possible evolutionary adaptation to didrivinfferentg feeding forces modes behind and variation presumed in diff ILTerences structure in water between basal andflow modern over the teleosts. gill region would correlate with morphological variation in the ILT. We discuss possible evolutionary driving forces behind variation in ILT structure between basal and modern teleosts.

Figure 2. Gill-associated lymphoid tissue (GIALT) is omnipresent across fishes. GIALT was not found Figure 2.in Gill-associated the key group of cartilagenouslymphoid tissue fishes but(GIALT) was found is omnipresent in representatives across of many fishes. different GIALT key groupswas not found in the keyacross group ray of finned cartilagenous fishes ( fishes but), was including foun Chondrosteans.d in representatives Names of in many red are different key species key groups across rayanalyzed finned in detailfishes in ( thisActinopterygii work. Taxonomy), including as in [25]. Chondrosteans. Names in red are key species analyzed2. Materials in detail and in Methods this work. Taxonomy as in [25].

2. Materials2.1. Species and Methods Investigated Information about the origin and number of specimens for the different fish species investigated 2.1. Speciesare givenInvestigated in Table S1. The study includes wild-caught species for which only fish with a healthy appearance upon sampling and no obvious histopathological alterations in gill tissue upon histological Information about the origin and number of specimens for the different fish species investigated are given in Table S1. The study includes wild-caught species for which only fish with a healthy appearance upon sampling and no obvious histopathological alterations in gill tissue upon histological evaluation were included in the study. Other fish species were laboratory-raised and also here, only healthy fish were sampled. After euthanasia, gill tissue was transferred to 10% neutral

Biology 2020, 9, 127 4 of 14 evaluation were included in the study. Other fish species were laboratory-raised and also here, only healthy fish were sampled. After euthanasia, gill tissue was transferred to 10% neutral buffered formalin and stored at 4 ◦C before further histological processing, unless indicated otherwise, as also indicated below for confocal imaging. Neither wild-caught nor laboratory-raised fish were subjected to experimental procedures. This study was performed by well-trained staff authorized to perform fish euthanasia. The details about ethical authorizations are provided in Table S1. The study was carried in contact with local ethics committees and followed strictly European legislation.

2.2. Histochemistry Histochemical staining was performed with Hematoxylin/Eosin (H&E) on formalin-fixed material [16]. Briefly, formalin-fixed tissue was paraffin-embedded and processed for histological analysis using standard procedures. Formalin-fixed, paraffin-embedded gill tissue were then sectioned at 4 µm thickness and mounted on poly-L-lysine-coated slides (Superfrost Plus, Thermo Scientific, Braunschweig, Germany). The slides were incubated at 37 ◦C for 24 h, then at 58 ◦C for 24 h, before deparaffinization in xylene. Sections were then rehydrated in graded alcohols to distilled water. Immunohistochemical staining was performed with the anti-Zap-70 rabbit mAb #2705 from Cell Signaling (99F2). The cross-reactivity for the fish Zap-70 protein, a kinase playing a critical role in initiating T-cell responses via the T-cell receptor (TCR) and expressed mainly by T cells and NK cells in human and mice, had been verified for use in common carp [26] and zebrafish [27]. Slides were blocked for 20 min with 0.2% goat normal serum in 5% bovine serum albumin (BSA)/tris-buffered saline (TBS) before incubation with primary antibody of appropriate concentrations diluted in 1% BSA/TBS. Labeled polymer-HRP anti-mouse (Dako EnVison+ System-HRP, Dako, Glostrup, Denmark) was used as the secondary antibody. We examined the omnipresence of GIALT in fishes using the anti-Zap-70 antibody as a universally cross-reactive pan T lymphocyte marker.

2.3. Confocal Imaging Adult zebrafish and medaka (1 year old) were euthanized with an overdose of buffered tricaine, fixed in 4% paraformaldehyde for 24 h at room temperature, cryoprotected in a solution of sucrose 30% for several days, embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Mountain View, CA, USA), flash-frozen in isopentane, and sectioned using a CM1950 cryostat (Leica, Wetzlar, Germany). The resulting 30 µm cryosections were recovered on a Superfrost Plus slide (Thermo Fischer, Waltham, MA, USA). T and NK lymphocytes were labeled using a rabbit anti-Zap70 monoclonal antibody (clone 99F2–Cell Signaling) at 1:200 with overnight incubation at 4 ◦C, followed by a 30 min incubation with a cross-adsorbed goat antirabbit secondary antibody (Jackson, Glendora, CA, USA) conjugated to Alexa647 at 1:250. Cryosections were costained with TRITC conjugated phalloidin (Sigma, St. Louis, MO, USA) at 3 U/mL and DAPI (Sigma) at 5 µg/mL during the incubation with the secondary antibody. Slides were mounted with a prolong-glass mounting medium (Thermo Fischer) and cured at room temperature for 24 h. Acquisitions were made with the Zyla camera of a dragonfly spinning disk confocal microscope (Andor, Belfast, UK), with 40 µm pinholes and either a 20 /0.75-dry × objective or a 60 /1.4-oil-immersion objective. Acquisitions, stitches and deconvolutions were made × using features from the Fusion software. Image analysis was realized using both IMARIS and ImageJ software. Image acquisition and analysis were performed at the NorMIC imaging platform (University of Oslo, Norway).

3. Results

3.1. Variations of Interbranchial Lymphoid Tissue (ILT) across Representative Fish Orders The first report of a defined structure in the GIALT of a teleost fish was the discovery of the ILT in the gills of Atlantic salmon (Figure3A). The ILT of salmon is divided in a proximal portion Biology 2020, 9, 127 5 of 14

(pILT) located at the terminal end of the interbranchial septa (i.e., located at the base of interbranchial clefts) and a less distinct and distal portion (dILT) extending along the primary lamellae, developing toward the tip of filaments along the afferent sides. This structure could be visualized by histochemical staining (Figure3B,D) and appeared to contain a large proportion of lymphocytes. We, therefore, used a monoclonalBiology 2020, 9, x antibody FOR PEER REVIEW against the highly conserved Syk family protein tyrosine kinase Zap-705 of 14 that is expressed by T cells and NK cells, and known to play a critical role in mediating T-cell activation that is expressed by T cells and NK cells, and known to play a critical role in mediating T-cell in response to TCR engagement. The Zap-70 staining allowed us to delineate the ILT of salmon by activation in response to TCR engagement. The Zap-70 staining allowed us to delineate the ILT of enhancing the contrast with surrounding tissues and by confirming that the majority of the lymphoid salmon by enhancing the contrast with surrounding tissues and by confirming that the majority of cells are indeed Zap-70+ and thus likely T lymphocytes (Figure3C,E). the lymphoid cells are indeed Zap-70+ and thus likely T lymphocytes (Figure 3C,E).

FigureFigure 3. 3.Gill-associated Gill-associated lymphoidlymphoid tissue (GIALT) (GIALT) or or interbranchial interbranchial lymphoid lymphoid tissue tissue (ILT) (ILT) of Atlantic of Atlantic salmonsalmon ( Salmo(Salmo salar salar).). All All images images werewere taken taken from from the the second second left left gill gill arch. arch. (A) Macroscopic (A) Macroscopic image image of ofa atransversally transversally dissected dissected gill. gill. The wh Theite white arrow arrow pointspoints to the proximal to the proximal part of the part ILT of (pILT) the ILT as seen (pILT) in as seenimage in image(B,C). (BlackB,C). arrows Black arrowspoint to point the distal to the part distal of partthe ILT of the(dILT) ILT as (dILT) seen asin seenimage in ( imageD,E). The (D ,E). interbranchial septum S forms the bottom of the interbranchial cleft. (B) Hematoxylin/Eosin (H&E) The interbranchial septum S forms the bottom of the interbranchial cleft. (B) Hematoxylin/Eosin staining of the ILT showing intraepithelial lymphoid aggregates. (C) Zap-70 staining of the (H&E) staining of the ILT showing intraepithelial lymphoid aggregates. (C) Zap-70 staining of the corresponding region showing high numbers of Zap-70+ cells (brown). (D) H&E staining of a filament corresponding region showing high numbers of Zap-70+ cells (brown). (D) H&E staining of a filament (sectioned in the coronary plane with regards to the filament cartilage). White arrows point to the (sectioned in the coronary plane with regards to the filament cartilage). White arrows point to the dILT. dILT. (E) Zap-70 staining of the corresponding region showing high numbers of Zap-70+ cells within (E) Zap-70 staining of the corresponding region showing high numbers of Zap-70+ cells within the the epithelium of the afferent aspect of the filament (i.e., dILT). epithelium of the afferent aspect of the filament (i.e., dILT). Given the variation in morphology of the gills across bony fish species and the gradual Given the variation in morphology of the gills across bony fish species and the gradual shortening shortening of the interbranchial septa from “basal” to “advanced” fishes, we used the associated of the interbranchial septa from “basal” to “advanced” fishes, we used the associated interbranchial interbranchial clefts as an anatomical guide through gill morphology to compare the exact location clefts as an anatomical guide through gill morphology to compare the exact location of the ILT across of the ILT across fishes of different orders. Taking advantage of the cross-reactivity of the anti-Zap70 fishesantibody of di fferentacross orders. a wide Taking taxonomic advantage range, of the we cross-reactivity included in of our the anti-Zap70comparative antibody approach across a wideimmunohistochemical taxonomic range, staining we included to confirm in our the comparative presence of lymphocytes approach immunohistochemical in ILT of representative staining fish tospecies, confirm ranging the presence from of lymphocytes to modern teleosts. in ILT of representative fish species, ranging from shark to modernFirst, teleosts. we examined the gills of a representative cartilaginous fish species, the catshark (ScyliorhinusFirst, we examined hesperius the). Here, gills ofthe a interbranchial representative septum cartilaginous connects fish the species, gill arches the catshark to the outer (Scyliorhinus body hesperiuswall, thus). Here, there the are interbranchial no interbranchial septum clefts connects (Figure 4A). the gill We arches could toobserve the outer confined body areas wall, of thus Zap-70+ there are nocells interbranchial in the , clefts which (Figure is located4A). We in couldthe dorsal observe region confined of the gill areas arches, of Zap-70 inside+ thecells interbranchial in the thymus, whichsepta is (Figure located 4B). in The the dorsalantibody region staining of the revealed gill arches, differentiated inside the cortex- interbranchial and medulla-like septa (Figure tissues,4 B).with The antibodya high proportion staining revealed of the medullary-like differentiated cortex-cells be anding Zap-70+ medulla-like (Figure tissues, 4C). The with typical a high cortex- proportion and of themedulla- medullary-like labeling cellspattern being is typical Zap-70 of+ thymus(Figure 4andC). Thehighlights typical the cortex- cross-reactivity and medulla- of the labeling anti-Zap-70 pattern isantibody typical of with thymus the Zap-70 and highlights protein from the cross-reactivityChondrichthyes. ofHowever, the anti-Zap-70 no typical antibody ILT could with be detected the Zap-70 proteinin catshark from Chondrichthyes. by histochemical However, nor by no typicalimmunohistochemical ILT could be detected examination, in catshark despite by histochemical multiple norinvestigations by immunohistochemical using different examination, orientations (transversal, despite multiple sagittal investigations and dorsal). using different orientations (transversal,Second, sagittal among and Actinopterygians, dorsal). we studied the gills of a typical Chondrostean, the Siberian sturgeonSecond, (Acipenser among Actinopterygians,baerii). In this basalwe bony studied fish species, the gills the ofinterbranchial a typical Chondrostean, septa are not connected the Siberian to the outer body wall but have free distal ends allowing for short interbranchial clefts (Figure 4D). sturgeon (Acipenser baerii). In this basal bony fish species, the interbranchial septa are not connected We could detect, by histochemistry, clear and distinct lymphoid aggregates at the base of the clefts to the outer body wall but have free distal ends allowing for short interbranchial clefts (Figure4D). (Figure 4E) that are continuous with the afferent side of the filaments. The presence of these local We could detect, by histochemistry, clear and distinct lymphoid aggregates at the base of the clefts pILT and dILT aggregates was confirmed by the anti-Zap-70 antibody labeling (Figure 4F) and, as (Figure4E) that are continuous with the a fferent side of the filaments. The presence of these local pILT found typical of the ILT from Atlantic salmon, the ILT of sturgeon was always located within the mucosal lining of epithelial cells.

Biology 2020, 9, 127 6 of 14 and dILT aggregates was confirmed by the anti-Zap-70 antibody labeling (Figure4F) and, as found typical of the ILT from Atlantic salmon, the ILT of sturgeon was always located within the mucosal lining of epithelial cells. Biology 2020, 9, x FOR PEER REVIEW 6 of 14

FigureFigure 4. 4.Gill-associated Gill-associated lymphoid lymphoid tissuetissue (GIALT)(GIALT) of Chondrichthyes and and Actinopterygi. Actinopterygi. All All images images werewere taken taken from from the the second second left gillleft archgill arch and orientedand oriented in the in transversal the transver planesal ofplane the gill.of the (A –gill.C) Catshark(A–C) (S.Catshark hesperius ().S. (hesperiusA) Macroscopic). (A) Macroscopic image of image a dissected of a dissected gill. Note gill. that Note the that interbranchial the interbranchial septum septum (S) is continuous(S) is continuous with the with hypodermis the hypodermis of the outer of the body outer surface. body surface. The black The arrowblack arrow points points to the to region the region as seen as seen in images (B,C). (B) Staining of a transversal sectioned gill arch. Note the thymic tissue with in images (B,C). (B) Staining of a transversal sectioned gill arch. Note the thymic tissue with cortex and cortex and medulla within the interbranchial septum. (C) Zap-70 staining of a thymic lobe. Note the medulla within the interbranchial septum. (C) Zap-70 staining of a thymic lobe. Note the high numbers high numbers of Zap-70+ cells (brown) within the medulla (black arrowhead). (D–F) Siberian of Zap-70+ cells (brown) within the medulla (black arrowhead). (D–F) Siberian sturgeon (A. baerii). (D) sturgeon (A. baerii). (D) Macroscopic image of a dissected gill. Note the long but discontinued Macroscopic image of a dissected gill. Note the long but discontinued interbranchial septum (S). The interbranchial septum (S). The black arrow points to the region as seen in images (E,F). (E) H&E black arrow points to the region as seen in images (E,F). (E) H&E staining of the epithelium lining the staining of the epithelium lining the distal part of the interbranchial septum. (F) Zap-70 staining of distal part of the interbranchial septum. (F) Zap-70 staining of the corresponding region shown in (E). the corresponding region shown in (E). Note the high numbers of Zap-70+ cells (brown) within the Note the high numbers of Zap-70+ cells (brown) within the epithelium. (G–I) Bonefish (Albula vulpes). epithelium. (G–I) Bonefish (Albula vulpes). (G) Macroscopic image of a dissected gill. Note the G ( )intermediate Macroscopic length image of ofthe a interbranchial dissected gill. septum Note the (S). intermediate The black arrow length points of the to interbranchialthe region as seen septum in (S).(H The,I). black(H) staining arrow pointsof the toepithelium the region lining as seen the in distal (H,I). part (H) of staining the interbranchial of the epithelium septum. lining (I) Zap-70 the distal partstaining of the interbranchialof the corresponding septum. region (I) Zap-70 shown stainingin (H). Note of the the corresponding high numbers regionof Zap-70+ shown cells in (brown) (H). Note thewithin high numbersthe epithelium. of Zap-70 + cells (brown) within the epithelium.

Third,Third, we we studied studied several several basal basal groups groups ofof teleoststeleosts in which which the the interbranchial interbranchial septa septa and and thus thus also also thethe interbranchial interbranchial clefts, clefts, are are reduced reduced to to anan intermediateintermediate length as as s seeneen in in Atlantic Atlantic salmon. salmon. We We started started withwith Elapomorpha Elapomorpha (eels (eels and and tarpons). tarpons). The The gills gill ofs aof typical a typical Elapomorpha, Elapomorpha, the bonefishthe bonefish (Albula (Albula vulpes ) showedvulpes lymphoid) showed lymphoid aggregates aggregates containing containing a high proportiona high proportion of Zap-70 of Zap-70++ cells both,cells both, at the at basethe base of the interbranchialof the interbranchial cleft (pILT) cleft and(pILT) along and the along aff erentthe afferent side (dILT) side (dILT) of gill of filament gill filament epithelium epithelium (Figure (Figure4G–I), similar4G–I), to similar the ILT to of the Atlantic ILT of Atlantic salmon. salmon. A comparable A comparable structure structure was also was found also found for another for another well-known well- elapomorphanknown elapomorphan species, thespecie Europeans, the European eel (Anguilla eel (Anguilla anguilla). anguilla In addition,). In addition, the groups the ofgroups the cyprinids of the (Ostariophysi)cyprinids (Ostariophysi) also share the also proximal share the and proximal distal structure and distal of ILT,structure as confirmed of ILT, as for confirmed common roachfor common roach (Rutilus rutilus), common carp (Cyprinus carpio) and gudgeon (Gobio gobio; Supplementary Materials (Figure S1)). Details of the ILT of another key cyprinid species, the zebrafish (Danio rerio), will be discussed later in this manuscript.

Biology 2020, 9, 127 7 of 14

(Rutilus rutilus), common carp (Cyprinus carpio) and gudgeon (Gobio gobio; Supplementary Materials (Figure S1)). Details of the ILT of another key cyprinid species, the zebrafish (Danio rerio), will be discussedBiology later 2020, in9, x thisFOR PEER manuscript. REVIEW 7 of 14 Fourth, among , we first focused on a representative spiny-rayed fish (), the EuropeanFourth, perch among (Perca fluviatilisNeoteleostei,). Perch we gillsfirst typicallyfocused showon a very representative short, quasi-absent spiny-rayed interbranchial fish septa and(Acanthopterygii), thus deep interbranchial the European perch clefts ( (FigurePerca fluviatilis5A). This). Perch allows gills for typically a wider show angle very between short, gill filaments,quasi-absent offering interbranchial more space septa at the and base thus of deep the clefts. interbranchial In strong clefts contrast (Figure to 5A). basal This groups allows offor teleosts, a wider angle between gill filaments, offering more space at the base of the clefts. In strong contrast to aggregates of Zap-70+ cells were found only at the base and afferent side of filaments and could not basal groups of teleosts, aggregates of Zap-70+ cells were found only at the base and afferent side of be consideredfilaments ILT-likeand could structures. not be considered Similar ILT-like observations structures. were Similar obtained observations from another were obtained spiny-rayed from fish species;another the Witch spiny-rayed flounder fish (species;Glyptocephalus the Witch cynoglossus flounder (Glyptocephalus). For these spiny-rayed cynoglossus). fishFor these species spiny- without apparentrayed ILT-like fish species structures, without apparent investigation ILT-like of structur gills ines, the investigation sagittal plane of gills (see in the Figure sagittal5D) plane showed (see the presenceFigure of Zap-70 5D) showed+ lymphoid the presence aggregates of Zap-70+ within lymphoid the branchial aggregates epithelium, within the branchial as denoted epithelium, by the presence as of mucousdenoted cells, by the between presence the of proximal mucous cells, part between of the filaments the proximal at their part attachmentsof the filaments to at the their gill arch (Figureattachments5E,F for perch); to the (Figuregill arch5 G–I(Figure for flounder).5E,F for perch); To our (Figure knowledge, 5G–I for suchflounder). lymphoid To ouraggregates knowledge, have such lymphoid aggregates have not been described previously. We consider these aggregates part of not been described previously. We consider these aggregates part of the GIALT. the GIALT.

FigureFigure 5. Absence 5. Absence of interbranchial of interbranchial lymphoid lymphoid tissuetissue (ILT) (ILT) but but the the presence presence of gill-associated of gill-associated lymphoid lymphoid tissue (GIALT) in the interfilamental space in two Neoteleostian species. (A–F) European perch (P. tissue (GIALT) in the interfilamental space in two Neoteleostian species. (A–F) European perch fluviatilis). (A) Macroscopic image of gill in transversal projection. The arrowhead points to the end (P. fluviatilis). (A) Macroscopic image of gill in transversal projection. The arrowhead points to the of the interbranchial cleft as also seen in (B,C). Note the short or missing interbranchial septum. (B,C) end ofstaining the interbranchial of transversal cleft orientation: as also seenno ILT-like in (B, Cst).ructure Note could the short be identified or missing in the interbranchial thin epithelium septum. (B,C) stainingcovering ofthe transversal bottom of the orientation: interbranchial no ILT-likecleft. (D) structure Macroscopic could image be identifiedof gill in a lateral in the projection. thin epithelium covering(E,F the) Sagittal bottom orientation: of the interbranchial Extensive intraepithelial cleft. (D lymphoid) Macroscopic aggregates image between of gill the in proximal a lateral part projection. of (E,F) Sagittalthe filaments orientation: at their attachment Extensive to intraepithelial the gill arch. The lymphoid black arrows aggregates in (D) show between the orientation the proximal in the part of the filamentshistological at their sections attachment (E,F), pointing to the to gillthe transition arch. The between black arrows the gill arch in (D and) show the filament the orientation where the in the histologicalintraepithelial sections lymphoid (E,F), pointing aggregates to theare transitionsituated. (E between) H&E staining; the gill black arch arrowheads and the filament point to wherefree the lamellae, while white arrowheads point to lamellar structures embedded within the intraepithelial intraepithelial lymphoid aggregates are situated. (E) H&E staining; black arrowheads point to free lymphoid aggregate. (F) Immunohistochemical staining using the anti-Zap-70 antibody to indicate lamellae, while white arrowheads point to lamellar structures embedded within the intraepithelial Zap-70+ cells (brown). All images were taken from the second left gill arch. (G–I) Witch flounder (G. lymphoidcynoglossus aggregate.). Sagittal (F) Immunohistochemicalorientation: black arrows show staining orientation using thein the anti-Zap-70 histological antibodysection pointing to indicate Zap-70to+ thecells transition (brown). between All imagesthe gill arch were and taken the filament from thewhere second intrae leftpithelial gill lymphoid arch. (G –aggregatesI) Witch flounderare (G. cynoglossussituated. ).(G–H Sagittal) Extensive orientation: intraepithelial black arrows lymphoid show aggreg orientationates between in the the histological proximal sectionpart of the pointing to thefilament transition at their between attachment the gill to the arch gill and arch. the (I) filamentImmunohistochemical where intraepithelial staining using lymphoid the anti-Zap-70 aggregates are situated.antibody (G to– indicateH) Extensive Zap-70+ intraepithelial cells (brown). All lymphoid images were aggregates taken from between a second the left proximalgill arch. part of the filament at their attachment to the gill arch. (I) Immunohistochemical staining using the anti-Zap-70 3.2. Body Size Variation Does Not Impose Drastic Restructuring of the ILT antibody to indicate Zap-70+ cells (brown). All images were taken from a second left gill arch.

Biology 2020, 9, 127 8 of 14

3.2.Biology Body 2020 Size, 9, x VariationFOR PEER REVIEW Does Not Impose Drastic Restructuring of the ILT 8 of 14

The sizesize of of an an organism organism has has an importantan important impact impact on its physiologyon its physiology and morphology, and morphology, size reduction size oftenreduction leading often to aleading loss of tissueto a loss complexity. of tissue complexi In the immunety. In the system, immune theabsolute system, numberthe absolute of lymphocytes number of haslymphocytes important has consequences important consequences on the diversity on and the structure diversity of and B- andstructure T-cell of repertoires B- and T-cell [28]. repertoires Intuitively, one[28].may, Intuitively, therefore, one expect may, thattherefore, body sizeexpect could that significantly body size could influence significantly the relative influence size and the structure relative ofsize ILT. and structure of ILT. We comparedcompared the ILTILT andand itsits associatedassociated expressionexpression ofof Zap-70Zap-70 describeddescribed for common carp (see Figure S1)S1) withwith that that of of zebrafish zebrafish (D. ( rerioD. rerio), a well-known), a well-known species species widely widely used in used developmental in developmental biology, human-diseasebiology, human-disease modeling modeling and drug and screening. drug screening. Both are Both closely-related are closely-related cyprinid cyprinid species but species an adult but zebrafish,an adult zebrafish, weighing weighing less than oneless gram, than one is thousands gram, is ofthousands times smaller of times than smaller an adult than carp. an Remarkably, adult carp. theRemarkably, gills of zebrafish the gills showed of zebrafish a high showed number a ofhigh Zap-70 number+ cells of locatedZap-70+ at cells the baselocated of theat the interbranchial base of the cleftsinterbranchial (Figure6 )clefts and along(Figure the 6) aandfferent along side the ofafferent the filaments, side of the typical filaments, of ILT typical of common of ILT carp.of common As in carp,carp. scatteredAs in carp, Zap-70 scattered+ lymphoid Zap-70+ cells lymphoid are also found cells onare the also efferent found side on and the interlamellar efferent side region. and Overall,interlamellar the ILTregion. of these Overall, two cyprinidthe ILT of species these oftwo di ffcypriniderent sizes species is elegantly of different structured sizes is in elegantly a highly comparablestructured in manner. a highly comparable manner.

Figure 6. Gill-associated lymphoid lymphoid tissues tissues (GIALT) (GIALT) of zebrafish zebrafish (D. rerio).). Spinning disk confocalconfocal deconvolved imagesimages of of adult adult zebrafish zebrafish gills gills coronal corona sectionsl sections across across and above and theabove interbranchial the interbranchial septum (septumA,B’). ( A(A,B’,B’)). represent (A,B’) represent 10 µm maximum10 μm maximum intensity intensity projection projection (MIP) (MIP) from afrom multifields a multifields stitch stitch (20 × objective)(20× objective) displaying displaying phalloidin phalloidin/DAPI/zap70/DAPI/zap70 labeling labeling for (A for,B) and(A,B DAPI) and/ zap70DAPI/zap70 labeling labeling for (A’ ,B’for). Images(A’,B’). Images were acquired were acquired from 30 fromµm 30 thick μm whole-organismthick whole-organism cryosections, cryosections, while while tissues tissues structures structures are highlightedare highlighted with with phalloidin phalloidin (F-actin, (F-actin, red) red) and and DAPI DAPI (Nuclei, (Nuclei, blue) blue) stainings stainings in order in order to examine to examine the distributionthe distribution of Zap-70 of Zap-70++ cells (white).cells (white). (A,A’ ).(A Zap-70,A’). Zap-70++ cells are cells mainly are mainly concentrated concentrated within thewithin afferent the aspectafferent of aspect the filaments. of the filaments. Note the Note high the number high nu ofmber Zap-70 of Zap-70++ cells within cells within the septum the septum wall, wall, which which then assemblethen assemble into theinto proximal the proximal ILT (pILT) ILT (pILT) at the uppermostat the uppermost layerof layer the interbranchialof the interbranchial septum. septum. (B,B’). Zap-70(B,B’). Zap-70++ cells then cells extend then extend upward upward from thefrom top the of top the of interbranchial the interbranchial septum septum to form to form the distalthe distal ILT (dILT)ILT (dILT) along along the a fftheerent afferent aspect aspect of filaments. of filaments. In addition In addition to the structured to the st lymphoidructured lymphoid tissue observed, tissue Zap-70observed,+ cells Zap-70+ are also cells distributed are also distributed in a diffuse in manner a diffuse among manner the interlamellaramong the interlamellar region and theregion efferent and aspectthe efferent of filaments aspect (ofA’ ,filamentsB’). F, Filament; (A’,B’). La, F, Lamellae;Filament; S,La, Septum; Lamellae; St, Septum S, Septum; top; Sw,St, Septum Septum top; wall; Sw, Af, ASeptumfferent wall; aspect; Af, Il, Afferent interlamellar aspect; region; Il, interlamellar Ef, Efferent re aspect;gion; Ef, Aa, Efferent Afferent aspect; artery; Aa, Vs, Afferent Venous artery; sinus; Vs, Ea, EVenousfferent sinus; artery; Ea, C, Cartilage.Efferent artery; Scale C, bar: Cartilage. 50 µm (A Scale,A’) andbar: 3050 µμmm ((BA,B’,A’).) and 30 μm (B,B’).

We alsoalso comparedcompared thethe expressionexpression of Zap-70Zap-70 we describeddescribed for the spiny-rayed fishesfishes perch and flounderflounder (Figure(Figure5 )5) with with that that of of medaka medaka ( Oryzias (Oryzias latipes latipes), a well-known), a well-known species species widely widely used as used a model as a formodel toxicology for toxicology and developmental and developmental biology. Asbiology. above, As adult above, medaka adult are medaka thousands are ofthousands times smaller of times than smaller than adult perch and flounder. These species are all Percomorphs (Acanthopterygii) with interbranchial septa that have been reduced to a minimum. In the gills of medaka, like the two other species no typical ILT could be identified. Only scattered Zap-70+ lymphoid cells could be observed (Figure 7).

Biology 2020, 9, 127 9 of 14 adult perch and flounder. These species are all Percomorphs (Acanthopterygii) with interbranchial septa that have been reduced to a minimum. In the gills of medaka, like the two other species no typicalBiology 2020 ILT, 9 could, x FOR bePEER identified. REVIEW Only scattered Zap-70+ lymphoid cells could be observed (Figure9 of7 ).14

Figure 7. Gill-associated lymphoid lymphoid tissues tissues (GIALT) (GIALT) of of medaka medaka ( (O.O. latipes ).). SpinningSpinning diskdisk confocalconfocal deconvolved images of adult medaka gills paratransversally sectioned ( A,A’), coronallycoronally sectionedsectioned beneath thethe septumseptum ( B(,BB’,B’),), coronally coronally sectioned sectioned across across the septumthe septum (C,C’ ()C and,C’) coronallyand coronally sectioned sectioned above theabove septum the septum (D). (A )(D is). a ( 10A)µ ism aMIP 10 μ (20m MIPobjective). (20× objective). (A’) is a ( 1A’µ)m is MIP a 1 μ (60m MIPobjective). (60× objective). (B) is a 1 (µB)m is MIP a 1 × × fromμm MIP a 6 fieldsfrom ofa view6 fields stitch of (60view objective)stitch (60× showing objective) two showing successive two gill succ arches.essive (B’ gill) is aarches. reoriented (B’) andis a × zoomedreoriented area and from zoomed (B). ( Carea) is from a 1 µm (B MIP). (C) from is a 1 a μ multifieldsm MIP from stitch a multifields (60 objective). stitch (60× (C’) objective). is a reoriented (C’) × andis a reoriented zoomed area and of zoomed (C). (D )area is a of 1 µ(Cm). MIP(D) is (60 a 1 μobjective).m MIP (60× Images objective). were Images acquired were from acquired 30 µm thickfrom × whole-organism30 μm thick whole-organism cryosections, cryosections, while tissues while structures tissues are structures highlighted are with highlighted phalloidin with (F-actin, phalloidin red) and (F- DAPIactin, (Nuclei,red) and blue)DAPI stainings (Nuclei, inbl orderue) stainings to examine in order the distribution to examine of the Zap-70 distribution+ cells (white). of Zap-70+ (A) Notecells the(white). almost (A) inexistent Note the interbranchialalmost inexistent septum interbranchial (S). Numerous septum Zap-70 (S). Numerous+ cells are Zap-70+ seen scattered cells are among seen gillscattered filaments, among lamellae gill filaments, and septa lamellae without and any septa structural without organization. any structural ( A’organization.) Further magnification (A’) Further illustratingmagnification the illustrating diffuse presence the diffuse of the presence Zap-70+ ofcells the Zap-70+ within the cells very within short the interbranchial very short interbranchial septum and bothseptum the and efferent both the and efferent the afferent and the aspects afferent of theaspect filaments.s of the filaments. The diffuse The distribution diffuse distribution of Zap-70 of+ Zap-cells is70+ consistent cells is consistent all along all the along filaments, the filaments, with cells with present cells present among among the afferent the afferent aspect, aspect, the efferent the efferent aspect andaspect the and interlamellar the interlamellar region (delimitatedregion (delimitated by the yellow by the dotted yellow lines) dotted underneath lines) underneath the septum the ( Bseptum,B’), at the(B,B’ septum), at the (C septum,C’) and (C above,C’) and the above septum the (D septum). Ga, Gill (D arch;). Ga, F, Gill Filament; arch; F, La, Filament; Lamellae; La, S, Lamellae; Septum; St,S, SeptumSeptum; top;St, Septum Af, Afferent top; Af, aspect; Afferent Il, interlamellar aspect; Il, interlamellar region; Ef, E region;fferent aspect;Ef, Efferent Aaa, aspect; Afferent Aaa, arch Afferent artery; Aa,arch A artery;fferent artery;Aa, Afferent Vs, Venous artery; sinus; Vs, Ea, Venous Efferent sinus; artery; Ea, C, Cartilage;Efferent artery; M, skeletal C, Cartilage; muscles; Sm,M, Smoothskeletal muscle;muscles; Pc, Sm, Pillar Smooth cells; muscle; Bl, Blood Pc, vessel Pillar lumen. cells; Bl, Scale Blood bar: vessel 30 µm lumen. (A–C), 20Scaleµm bar: (A’, D30) andμm 10(A–Cµm), (20B’, C’μm). (A’,D) and 10 μm (B’,C’).

3.3. Adaptation of Morphology to Different Feeding Modes Does Not Impose Drastic Restructuring the ILT Adaptive radiation in unstable environments leads to fast morphological change. Classical examples are provided by the beak of Darwin’s finches [29] and by the body shape and armor

Biology 2020, 9, 127 10 of 14

3.3. Adaptation of Jaw Morphology to Different Feeding Modes Does Not Impose Drastic Restructuring the ILT Biology 2020, 9, x FOR PEER REVIEW 10 of 14 Adaptive radiation in unstable environments leads to fast morphological change. Classical examplesphenotype are of stickleback provided by[30]. the The beak structure of Darwin’s of fish jaws finches also [evolves29] and under by the strong body selection: shape and the armorupper phenotypejaw of several ofstickleback species [30]. from The structureLake Victoria of fish and jaws barbels also evolvesfrom Lake under Tana strong showed selection: very fast the upperdivergent jaw adaptive of several changes cichlid upon species food from resource Lake Victoria fluctuations and barbels[31], potentially from Lake affecting Tana showed the cranial very faststructure divergent and gill adaptive arches. changes Barbus uponintermedius food resource is a cyprinid fluctuations fish species [31], potentiallyfrom Lake Tana affecting (Ethiopia) the cranial and structurea hexaploid and taxon gill arches. that formedBarbus a intermediusunique ‘speciesis a cyprinidflock’ consisting fish species of at from least Lake 14 morphotypes. Tana (Ethiopia) Feeding and a hexaploidspecializations taxon introduced that formed rapid a unique reshaping ‘species of bucca flock’l and consisting pharyngeal of at leastregions 14 morphotypes.[23,24] with presumed Feeding specializationsdifferences in water introduced flow over rapid the reshapinggill region ofof buccaldifferent and morphotypes. pharyngeal regionsAccess to [23 long-term,24] with conserved presumed disamplesfferences of inhighly water diverse flow over morphotypes the gill region prompted of different us to morphotypes. examine the diversity Access to in long-term ILT in this conserved unique samplescollection. of Even highly in diversethe gills morphotypes of highly divergent prompted Barbus us tomorphotypes examine the (Figure diversity 8), inalthough ILT in this difficult unique to collection.evaluate in Even detail in because the gills tissues of highly clearly divergent suffered Barbus from morphotypes the long conservation (Figure8), although period, we di ffididcult not to evaluateobserve significant in detail because modifications tissues clearly of ILT suasff foundered from typical the of long closely-related conservation cyprinids period, we such did as not common observe significantcarp and zebrafish. modifications Altogether, of ILT as foundthis dataset typical suggests of closely-related an overall cyprinids conserved such ILT as common structure carp along and zebrafish.cyprinids. Altogether, this dataset suggests an overall conserved ILT structure along cyprinids.

Figure 8. Heads of 66 endemicendemicLabeobarbus Labeobarbusspecies species flock flock in Lakein Lake Tana Tana (column (column (A)) ( andA)) and their their isolated isolated gills (columngills (column (B)) after(B)) after 24-h fixation24-h fixation in formalin in formalin and followed and follow byed 26-year by 26-year storage storage in 70% in alcohol 70% alcohol [24], and [24], a transverseand a transverse view of view lymphoid of lymphoid tissue (ILT) tissue in HE-stained(ILT) in HE-stained gills (Scale gills bar (Scale is 100 barµm, is column 100 μm, (C column)), transverse (C)), viewtransverse of HE-stained view of HE-stained gills (Scale bargills is (Scale 500 µ m,bar columnis 500 μ (Dm,)), column transverse (D)), drawing transverse of gill drawing morphology of gill (columnmorphology (E)). (column The transverse (E)). The drawings transverse of gill drawings morphology of gill are morphology based on scanned are based HE-stained on scanned gills, HE- and lymphocyte-likestained gills, and cell lymphocyte-like accumulations cell are accumulations in blue (column are (E in)). blue (column (E)).

4. Discussion DefinedDefined lymphoid structures associated with fish fish gills have firstfirst been discovered in Atlantic salmon only a few years ago and were named interbranchial lymphoid tissue tissue (ILT). (ILT). In this species, gill filaments are joined about half their length by supportive septa, placing the ILT (i.e., proximal ILT (pILT)) on its distal end, at the base of the interbranchial clefts. This GIALT structure extends to include lymphoid aggregates at the afferent sides of the gill filaments (i.e., distal ILT (dILT)). Given that this structure has been described primarily in Atlantic salmon and rainbow trout, here we

Biology 2020, 9, 127 11 of 14 gill filaments are joined about half their length by supportive septa, placing the ILT (i.e., proximal ILT (pILT)) on its distal end, at the base of the interbranchial clefts. This GIALT structure extends to include lymphoid aggregates at the afferent sides of the gill filaments (i.e., distal ILT (dILT)). Given that this structure has been described primarily in Atlantic salmon and rainbow trout, here we investigated to what extent ILT has evolved between basal fish groups and modern teleosts. Given that both, body size and morphology of the jaw and pharyngeal region can be highly variable across teleosts, potentially modifying the location of immune cells in gills, we also investigated if this would influence the structure of ILT across teleosts. We made use of the fact that Zap-70 is a phylogenetically conserved protein, which helped to visualize the location of T lymphocytes within gills across key orders of bony fish. While GIALT structures are omnipresent across Actinopterygians, our data show that ILT placed at the base of the interbranchial clefts is variable across bony fishes. Its absence in some groups that lack interbranchial septa suggests the presence of a close coevolution of these structures. In this study we used the gradual shortening of the interbranchial septa from “basal” to “advanced” fishes as an anatomical guide through gill morphology to compare the exact location of structured elements of ILT across fishes of different orders. In sharks, the interbranchial septa connect the gill arches to the outer body wall. Hence there are no interbranchial clefts. In shark gills we could not detect a definite ILT in-between the filaments. such as perch and flounder have very short, quasi-absent interbranchial septa and thus have deep interbranchial clefts. It proved difficult to define the lymphoid and largely Zap-70+ aggregates found at the base and afferent side of the filaments of modern teleosts such as perch and flounder, as typical ILT. Medaka does not seem to possess at all the structured lymphoid aggregates found in perch and flounder, which underscores the existence of a large variation in GIALT among fishes, in particular among Percomorpha. In many, although not in all bony fish species we investigated, we did discover the presence of ILT at the base of interbranchial clefts and at the afferent side of filaments. When exposed to turbid water gills are in contact with a tremendous amount of potentially contaminating particles. Food particles and potential pathogens pass through the gills and water flows along GIALT. During respiration the water flow from the efferent (i.e., leading) to the afferent (i.e., trailing) edges of each side to finally meets in the interbranchial clefts, which, therefore, have been suggested as sites of extensive water-mixing [6]. It is easy to imagine that exposure to high numbers of water-borne particles including potential pathogens at these sites could be a driving force for the development and location of mucosal structures such as ILT at the bottom of interbranchial clefts. Even if gills developed initially for ion regulation rather than gas exchange from water [32], it is likely that the base of interbranchial clefts allows for structures such as ILT to be strategically located because of water flow. Indeed, the development of lymphoid tissues in the respiratory pathways of higher vertebrates such as nasopharynx-associated lymphoid tissue, bronchus-associated lymphoid tissue and tonsils appears highly dependent on environmental influences [33–35] and location typically is associated with branching-points of increased interaction between the respiratory medium and the mucosal lining [36]. In gills of fishes with short interbranchial septa, there is less need for upwelling of water along the septal water channels [37], supportive of our observations that ILT placement at the base of the interbranchial clefts is not only variable across bony fishes but could parallel developmental shortening of the interbranchial septa. The environment (temperature, season, salinity, etc.) might have an effect on gill morphology. We never detected alteration of gill integrity such as fusion of lamellae, hyperplasia of epithelia (likely lymphocyte proliferation), tissue damage or change in the cell composition of epithelia, which could suggest the effect of pollutants. Importantly, our analysis of the morphology of ILT in numerous fish species from totally different biotopes (different salinity, different aquatic environment, different regions) argues against these environments having an overruling effect on the presence of ILT. The absence of true ILT in species like flounder and perch was observed from totally different environments, which further supports our conclusions. Finally, even in fish grown in clean facilities (zebrafish, medaka, eel, carp) we did not notice major discrepancies compared to specimens from the wild (e.g., Labeobarbus). Biology 2020, 9, 127 12 of 14

5. Conclusions Our survey across a number of species from diverse taxonomic groups leads to the notion that neither individual size nor anatomy of fish jaws drastically affects the structure of GIALT. Comparison of a number of cyprinids of various sizes (carp, roach, gudgeon, zebrafish) and with various feeding modes and different mouth/head shapes (Barbus species flock) did not reveal drastic differences and we found a comparable fundamental structure of ILT-like structures. Yet, we did notice a major transition that seems to have occurred at the basis of Percomorphs, where the septum virtually disappeared, and gill filaments draw a wide-angle leaving fully accessible the base of the cleft on the gill arch. Our data suggest the “taxonomic rule” determines the ILT structure.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-7737/9/6/127/s1, Table S1. Origin of samples and numbers of individuals; Figure S1: Gill-associated lymphoid tissue (GIALT) or interbranchial lymphoid tissue (ILT) of various teleost species. Author Contributions: Conceptualization, J.R., A.S.D., L.D.P., E.O.K., P.B. and G.F.W.; funding acquisition, E.O.K. and G.F.W.; investigation, J.R., A.S.D., Y.Z., E.O.K. and G.F.W.; methodology, J.R., A.S.D. and Y.Z.; supervision, E.O.K., P.B. and G.F.W.; validation, G.F.W.; visualization, J.R., A.S.D., L.D.P. and Y.Z.; writing—original draft, J.R., A.S.D., L.D.P., E.O.K., P.B. and G.F.W.; writing—review and editing J.R., A.S.D., L.D.P., E.O.K., P.B. and G.F.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Norwegian Research Counsil (FRIMEDBIO-No 144642), and by institutional grants from INRAE, Norwegian University of Life Sciences and WUR. Yaqing Zhang was funded by the China Scholarship Council, National Natural Science Foundation of China. Acknowledgments: We would like to thank Helen Dooley (University of Aberdeen, Scotland) for providing samples of catshark (Scyliorhinus hesperius), Satu Viljamaa-Dirks (Finnish Food Authority, Helsinki, Finland) for providing samples of Siberian sturgeon (Acipenser baerii), Trygve T. Poppe (Pharmaq Analytiq, Bergen, Norway) for providing samples of bonefish (Albula vulpes), and other samples and Leo A.J. Nagelkerke (Wageningen University & Research, The Netherlands) for providing samples of endemic Labeobarbus specimen from Lake Tana, Africa. We would also like to thank O. Bakke (University of Oslo, Norway), H.E. Karlsen (University of Oslo, Drøbak, Norway) and the engineers of the NorMIC imaging platform (University of Oslo, Norway) for their assistance in the project. Conflicts of Interest: The authors declare no conflicts of interest.

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