{"paper_id":"14a43e2f-ca95-4dca-b6a1-cf39d69a1a2e","body_text":"Eye features and retinal photoreceptors of the nocturnal aardvark (Orycteropus afer, 1 \nTubulidentata) 2 \n 3 \nLeo Peichl1,2* (ORCID ID: 0000-0002-8141-0911), Sonja Meimann2, Irina Solovei3 (ORCID 4 \nID: 0000-0002-6813-7279), Irene L. Gügel4 (ORCID ID: 0000-0002-5475-9210), Christina 5 \nGeiger5, Nicole Schauerte5, Karolina Goździewska-Harłajczuk6 (ORCID ID: 0000-0003-6 \n4412-1966), Joanna E. Klećkowska-Nawrot6 (ORCID ID: 0000-0003-1154-8736), Gudrun 7 \nWibbelt7 (ORCID ID: 0000-0002-3456-1303), Silke Haverkamp4 (ORCID ID: 0000-0003-8 \n3084-6544) 9 \n 10 \n1Institute for Clinical Neuroanatomy, Dr. Senckenbergische Anatomie, Goethe University, 11 \nFrankfurt am Main, Germany. 12 \n2Institute of Cellular and Molecular Anatomy, Dr. Senckenbergische Anatomie, Goethe 13 \nUniversity, Frankfurt am Main, Germany. 14 \n3Biozentrum, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany. 15 \n4Department of Computational Neuroethology, Max Planck Institute for Neurobiology of 16 \nBehavior - Caesar, Bonn, Germany. 17 \n5Zoo Frankfurt, Frankfurt am Main, Germany. 18 \n6Department of Biostructure and Animal Physiology, Faculty of Veterinary Medicine, 19 \nWrocław University of Environmental and Life Sciences, Wrocław, Poland. 20 \n7Department of Wildlife Diseases, Leibniz Institute for Zoo and Wildlife Research, Berlin, 21 \nGermany 22 \n 23 \nShort title: Aardvark eye and retinal photoreceptors 24 \n 25 \nKeywords: Mammal, vision, tapetum lucidum, cone photoreceptor, rod photoreceptor, opsin 26 \ncoexpression, thyroid hormones 27 \n 28 \n 29 \n*Corresponding author: 30 \npeichl@em.uni-frankfurt.de 31 \n 32 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n2 \n \n 33 \nAbstract 34 \nThe nocturnal aardvark Orycteropus afer is the only extant species in the mammalian order 35 \nTubulidentata. Previous studies have claimed that it has an all-rod retina. In the retina of one 36 \naardvark, we found rod densities ranging from 124,000/mm² in peripheral retina to 37 \n214,000/mm² in central retina; the retina of another aardvark had 182,000 – 245,000 38 \nrods/mm². This is moderate in comparison to other nocturnal mammals. With opsin 39 \nimmunolabelling we found that the aardvark also has a small population of cone 40 \nphotoreceptors. Cone densities ranged from 300 to 1,300/mm² in one animal, and from 1,100 41 \nto 1,600/mm² in the other animal, with large local variations and no large central-peripheral 42 \ndensity gradient. Overall, cones comprised 0.25-0.9% of the photoreceptors. Both typical 43 \nmammalian cone opsins, longwave-sensitive (L) and shortwave-sensitive (S), were present. 44 \nHowever, there was colocalization of the two opsins in many cones across the retina (35 – 45 \n96% dual pigment cones). Pure L cones and S cones formed smaller populations. This 46 \nprobably results in poor colour discrimination. Thyroid hormones, important regulators of 47 \ncone opsin expression, showed normal blood serum levels. The relatively low rod density and 48 \nhence a relatively thin retina may be related to the fact that the aardvark retina is avascular 49 \nand its oxygen and nutrient supply have to come from the choriocapillaris by diffusion. In 50 \ncontrast to some previous studies, we found that the aardvark eye has a reflective tapetum 51 \nlucidum with features of a choroidal tapetum fibrosum, in front of which the retinal pigment 52 \nepithelium is unpigmented. The discussion considers these findings from a comparative 53 \nperspective. 54 \n 55 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n3 \n \n 56 \nIntroduction 57 \nThe aardvark (Orycteropus afer) is the only extant species of the order Tubulidentata in the 58 \nsupraordinal mammalian clade Afrotheria. It is a medium-sized, pig-like mammal native to 59 \nsub-Saharan Africa (Fig 1). The aardvark is a nocturnal, burrowing species that mostly feeds 60 \non ants and termites (for an overview, see, e.g., [1,2]). The strong legs with sharp claws are 61 \nused to dig out termite and ant nests, as well as abode burrows; the genus name Orycteropus 62 \nmeans ‘burrowing foot.’ The aardvark is considered a ‘living fossil,’ because Orycteropus 63 \nfossils from about 20 million years ago show nearly identical morphological features to those 64 \nof living aardvarks [2]. It is assumed that aardvarks have an acute sense of smell and hearing, 65 \nbut poor eyesight. However, in contrast to the abundant literature available on the eyes and 66 \nretinae of many other mammals, the only substantial study of the aardvark retina known to us 67 \nis that of Victor Franz, published in 1909 [3]. Franz obtained the two eyes of one animal 68 \nhunted at a zoological expedition and examined them in detail macroscopically and 69 \nmicroscopically. The study contains much valuable information, but also some apparent 70 \nerrors, most likely due to the histological methods available at the time. Franz [3] reports a 71 \ncomplete absence of cone photoreceptors in the retina, which appears doubtful in the light of 72 \nmore recent research demonstrating cones in nearly all mammals studied to date (reviews: 73 \n[4,5]). Among the few exceptions without functional cones are some deep-diving whales [6], 74 \nthe subterranean golden moles [7], and Xenarthra [8]. Franz [3] also states that the aardvark 75 \nhas no reflective tapetum lucidum. In contrast, a recent study of the orbital structures and eye 76 \ntunics of young and adult aardvarks reports the presence of a tapetum lucidum [9]. Nocturnal 77 \nphotographs of aardvarks show a strong eyeshine or ‘glow’ (Fig 1). However, this could also 78 \nbe a reflection off the fundus like the ‘red eye effect’ seen, e.g., in human eyes in flash 79 \nphotographs. When we obtained the relatively well-preserved eyes of two aardvark 80 \nindividuals, we studied them with currently available histological approaches to resolve the 81 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n4 \n \nabove discrepancies and to add new observations on aardvark retinal anatomy. The present 82 \npaper reports on general eye features and the photoreceptors. A separate paper shall describe 83 \naardvark retinal bipolar cells, horizontal cells, amacrine cells and ganglion cells.  84 \n 85 \n 86 \nFig 1. Aardvarks at day and night, note the laterally positioned eyes. (A) Aardvark 1, the 87 \npost mortem donor of the studied eye, at daytime. (B, C) Another aardvark, flashlight 88 \nphotographs taken at night. When the head is viewed horizontally, there is a bright whitish 89 \neyeshine indicating a tapetum lucidum; when viewed from above, the weaker eyeshine 90 \nappears orange to red. For details see text. Image sources: (A) Frankfurt Zoo; (B, C) Christina 91 \nGeiger. 92 \n 93 \n 94 \nMethods 95 \nTissue 96 \nThis study has used tissue from aardvarks (Orycteropus afer) that was obtained from zoo 97 \nanimals that had died of natural causes. The aardvark IUCN status is ‘least concern’ and no 98 \nethical approval was required for use of the tissue. The right eye of a nearly 25 years old male 99 \naardvark was obtained when the animal died of old age in the Zoo of Frankfurt am Main, 100 \nGermany. The animal is termed “aardvark 1” here (Fig 1A). It had an age-related cataract and 101 \na mild chronic Uveitis anterior, but the retina appeared macroscopically normal. One day post 102 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n5 \n \nmortem, the eye was enucleated during autopsy, punctured behind the cornea for better 103 \nfixative penetration, and immersion-fixed in 4% formalin for 24 h at 4°C. When punctured, 104 \nthe eye lost some liquefied vitreous and aqueous humor, leading to a collapse of the cornea 105 \nand a slight deformation of the eyeball during fixation. After fixation the external eye 106 \ndimensions were recorded, the eye was cut open behind the cornea, and the posterior eyecup 107 \nwith attached retina was washed in 0.01M phosphate buffered saline (PBS, pH 7.4). The 108 \nretina was carefully dissected from the eyecup after having marked its orientation. It was 109 \ncryoprotected by successive immersion in 10%, 20% and 30% (w/v) sucrose in phosphate 110 \nbuffer (PB, pH 7.4) containing 0.05% sodium azide, and stored frozen at -20°C until further 111 \nprocessing. The eyecup was stored in PBS with 0.05% sodium azide at 4°C.  112 \n 113 \nThe eyes of a nearly 6 years old female aardvark were obtained when the animal died of 114 \nperinatal complications in the Zoological Garden of Wrocław (Poland). The animal is termed 115 \n“aardvark 2” here, it was genetically unrelated to aardvark 1. The eyes were enucleated 116 \nimmediately post mortem and immersion-fixed in 4% buffered formaldehyde solution, they 117 \nhave been used in a previous study of aardvark eye and orbital features [9]. The right eye was 118 \nkept in the fixative for 1 week and then embedded in paraffin for sectioning. The left eye was 119 \npermanently stored in the fixative, and only small pieces of the retina were available for the 120 \npresent study. Probably due to the long fixation time of this eye (ca. 7 years), some of the 121 \nantibodies used here did not work (see below and Results). 122 \n 123 \nFor frozen vertical sections of the retina (i.e., perpendicular to the retinal layers), pieces of 124 \nretina were transferred from 30% sucrose to tissue freezing medium (Leica Biosystems, 125 \nWetzlar, Germany), frozen, sectioned at 16 μm thickness with a cryostat (Leica CM 3050 S, 126 \nWetzlar, Germany), and collected on Superfrost Plus slides (Menzel Gläser, Braunschweig, 127 \nGermany). 128 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n6 \n \n 129 \nFor electron microscopy, small pieces of the sclera and presumed tapetum lucidum were 130 \nstained as previously described [10]. Briefly, the samples were stained in a solution 131 \ncontaining 1% osmium tetroxide, 1.5% potassium ferrocyanide, and 0.15 M cacodylate 132 \nbuffer. The osmium stain was amplified with 1% thiocarbohydrazide and 2% osmium 133 \ntetroxide. The tissue was then stained with 2% aqueous uranyl acetate and lead aspartate. The 134 \ntissue was dehydrated through an 70%–100% ethanol series, transferred to propylene oxide, 135 \ninfiltrated with 50%/50% propylene oxide/epon medium hard formulation (EMbed 812, 136 \nElectron Microscopy Sciences; [11]), and then 100% epon medium hard. The epon medium 137 \nhard infiltrated tissue was transferred into multi-well embedding molds (Electron Microscopy 138 \nSciences) and hardened at 60°C. For scanning electron microscopy (SEM), a few serial 139 \nsections of 50 nm were taken with a Diatome ultra diamond knife and collected on glow 140 \ndischarged silicon wafers and dried on a heating plate at 50 °C until the water was fully 141 \nevaporated. The wafers were mounted with silver paint (Plano) on a sample holder and 142 \nimages were taken with a Supra55 (Leica) SEM. For transmission electron microscopy 143 \n(TEM), 50-nm-thick sections were cut with an Ultra diamond knife and transferred on carbon-144 \ncoated copper grids with a hole size of 35/10 nm (S35/10, Quantifoil, Electron Microscopy 145 \nSciences). Images were recorded with an analytical electron microscope (JEM-2200FS, Jeol) 146 \nat an energy of 200 keV with a CMOS camera (TEM-CAM F416, TVIPS). 147 \n 148 \nFor comparison of the choroid, vertical cryo-sections of the formalin-fixed eye of a captive 149 \nadult African elephant (Loxodonta africana) from a German zoo was used. In agreement with 150 \nCITES regulations the eye was collected during necropsy for pathological investigations 151 \nperformed by the Institute for Zoo and Wildlife Research, Berlin (IZW) after the animal had 152 \nto be euthanized because of severe disease unresponsive to treatment. 153 \n 154 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n7 \n \nImmunohistochemistry 155 \nImmunohistochemistry was performed on vertical sections of the retina, as well as on 156 \nunsectioned retinal pieces from various regions to assess cell populations in flat view. 157 \nImmunolabelling followed standard protocols. Briefly, sections on the slide and free-floating 158 \npieces were preincubated for 1 h in PB with 0.5% Triton X-100 and 10% normal donkey 159 \nserum (NDS). Incubation in the primary antibody/antiserum solution, made up in PB with 3% 160 \nNDS and 0.5% Triton X-100, was overnight at room temperature for sections, and 3 days at 161 \nroom temperature or 4 days at 4°C for unsectioned pieces. Multiple immunofluorescence 162 \nlabelling for simultaneous visualization of several antigens was performed by incubation in a 163 \nmixture of the antisera. Table 1 lists all primary antibodies used. The cone opsin antisera 164 \nJH492, JH455 and sc-14363 have been used in several previous studies to reliably label the 165 \nrespective opsins in a range of mammals [12-16]. 166 \n 167 \nTable 1. Primary antibodies and cell markers used 168 \nAntigen / \nmarker \nImmunogen / \ntarget structure \nAntibody host  \nspecies, catalog #, \nRRID \nDilution Source \nRod opsin N-terminal \nregion of bovine \nrhodopsin1 \nMouse monoclonal, \nName: rho4D2, \nRRID: AB_2315273 \n1:1000 Gift of R. S. Molday, \nUniversity of British \nColumbia Life Sciences \nCentre, Vancouver, \nCanada \nL cone opsin C-terminal 38 \namino acids of \nhuman red cone \nopsin2 \nRabbit polyclonal, \nName: JH 492,  \nRRID: AB_2315259 \n1:2,000 Gift of J. Nathans, Johns \nHopkins University \nSchool of Medicine, \nBaltimore, Maryland, \nUSA \nS cone opsin C-terminal 42 aa \nof human blue \ncone opsin2 \nRabbit polyclonal, \nName: JH 455,  \nRRID: AB_2313807 \n1:5,000 Gift of J. Nathans, Johns \nHopkins University \nSchool of Medicine, \nBaltimore, Maryland, \nUSA \nS cone opsin 20 aa peptide \nmapping near N-\nterminus of \nhuman blue cone \nopsin3 \nGoat polyclonal,  \nCat# sc-14363,  \nRRID: AB_2158332 \n1:500 Santa Cruz Biotechnology \nCtBP2 (C-\nTerminal \nMouse CtBP2, \naa. 361-445 \nMouse monoclonal, \nCat# 612044,  \n1:5000 BD Biosciences \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n8 \n \nBinding \nProtein-2) \nRRID: AB_399431 \nCtBP2 Rat CtBP2, aa \n431-445  \nRabbit polyclonal, \nCat# 193 003, \nRRID: AB_2086768 \n1:5000 Synaptic Systems \nGS \n(Glutamine \nsynthetase) \nMüller glia Mouse monoclonal, \nCat# 610517, \nRRID: AB_397879 \n1:500 BD Bioscience \nGFAP Glial fibrillary \nacidic protein \nMouse monoclonal, \nCat# G3893, \nRRID: AB_477010 \n1:500 Sigma-Aldrich \nH3K4me3 Euchromatin Rabbit polyclonal, \nCat# ab8580, \nRRID: AB_306649 \n1:500 abcam \nH4K20me3 Heterochromatin Mouse monoclonal, \nCMA423 \n1:500 Generated in Hiroshi \nKimura’ lab, Tokyo \nUniversity \nLamins A/C Inner nuclear \nmembrane \nprotein \nMouse serum Undiluted Gift of Harald Herrmann, \nGerman Cancer Research \nCenter \nLBR Inner nuclear \nmembrane \nprotein \nGuinea pig serum 1:50 Generated in Harald \nHerrmann’s lab, German \nCancer Research Center \nPNA-647 \n(Peanut \nagglutinin) \nGeneral cone \nmarker \nCat# L-32460 1:100 Molecular Probes \nNeuN Synthetic peptide \nNeuN \nRabbit monoclonal \n(EPR12763), \nCat# ab177487,  \nRRID: AB_2532109 \n1:100 abcam \nNeuroTrace \n(Ex 530 / \nEm 615) \nFluorescent Nissl \nstain \nCat# N-21482 1:100 Molecular Probes \nIsolectin B4, \nbiotinylated \nBlood vessel \nmarker \nCat# B-1205 1:50 Vector Laboratories \n1Ref. [86], 2Ref. [87], 3Ref. [12]. 169 \n 170 \nBinding sites of the primary antibodies were visualized by indirect immunofluorescence, with 171 \na 1.0-1.5 h incubation of the tissue in the secondary antiserum, or in a mixture of appropriate 172 \nsecondary antisera in the case of several primary antibodies. We used secondary antisera 173 \nconjugated to Alexa 488, Alexa 647, Cy3 and Cy5 in appropriate combinations. Omission of 174 \nthe primary antibodies from the incubation solution resulted in no staining. In addition to 175 \nantisera, we used the fluorescent markers peanut agglutinin (PNA) and NeuroTrace for certain 176 \ncell types (Table 1). After immunolabelling, sections were incubated in a solution of 4,6-177 \ndiamidino-2-phenylindole (DAPI) as a fluorescent nuclear stain to reveal the general retinal 178 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n9 \n \nlayering. Choroidal blood vessels were labeled by biotinylated isolectin B4 (Table 1; 179 \nincubation was 1 h for sections, 2 h for unsectioned pieces) and visualized by a subsequent 180 \n1.0-1.5 h incubation in streptavidin coupled to Alexa 549 or 488. Tissue was coverslipped 181 \nwith an aqueous mounting medium (AquaPoly/Mount, Polysciences Inc., Warrington, PA, 182 \nUSA; or Dako Fluorescence Mounting Medium S3032, Dako North America Inc., 183 \nCarpinteria, CA, USA). 184 \n 185 \nIn retinal pieces from the long-fixed eye of aardvark 2, the S cone opsin antiserum sc14363 186 \ndid not work, hence assessment of the cone opsin pattern was done by sequential double-187 \nlabeling with the two rabbit antisera JH492 against the L cone opsin and JH455 against the S 188 \ncone opsin as follows. One retinal piece was first incubated in a JH492 solution and then in a 189 \ndonkey-anti-rabbit antiserum conjugated to Alexa 488. Then the piece was incubated in a 190 \nJH455 solution and finally in a donkey-anti-rabbit antiserum conjugated to Cy3. This 191 \nsecondary antiserum bound to both primary antisera, hence all cones were labeled by Cy3. 192 \nThe cones also labeled by Alexa 488 were those that contained L cone opsin, and cones only 193 \nlabeled by Cy3 were pure S cones. In a neighboring piece of retina, the order of labeling was 194 \nreversed: First incubation in the JH455 solution and visualization with the donkey-anti-rabbit 195 \nantiserum conjugated to Alexa 488, then incubation in the JH492 solution and visualization 196 \nwith the donkey-anti-rabbit antiserum conjugated to Cy3. Again, all cones were labeled by 197 \nCy3, but here the cones also labeled by Alexa 488 were those that contained S cone opsin, and 198 \nthose only labeled by Cy3 were pure L cones. Combining the data from the two pieces 199 \nprovided the total cone density, the percentages of pure L and S cones, and the proportion of 200 \ncones containing L and S opsin, from which the percentage of dual pigment cones could be 201 \ncalculated for that retinal region.  202 \n 203 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n10 \n \nFor assessment of the retinal vascularization, a retinal piece of 5.5 x 4.0 mm size containing 204 \nthe optic nerve head was stained with a 3,3’-diaminobenzidine (DAB) reaction to selectively 205 \nvisualize the endogenous peroxidase in the vasculature. The retinal piece was washed in 206 \n0.05% Tris buffer (TRIS, pH 7.6), then incubated for 20 min in a solution of 0.05% DAB in 207 \nTRIS, after which hydrogen peroxide was added to the incubation solution at a final 208 \nconcentration of 0.01%, and the incubation was continued for 10 min until the peroxidase 209 \nreaction had fully developed. The reaction was stopped by several washes in TRIS and then 210 \nPB. The retinal piece was flat-mounted on a slide and coverslipped with AquaPoly/Mount. 211 \nFor assessment of the retinal pigment epithelium (RPE) after removal of the retina, pieces of 212 \nthe thin RPE layer from the central and peripheral fundus were gently removed from the 213 \nunderlying tissue, flat-mounted on a slide and coverslipped with AquaPoly/Mount. 214 \n 215 \nImaging and analysis 216 \nThe RPE and the DAB-labelled vasculature of the optic nerve head were analyzed with a 217 \nZeiss Axioplan 2 microscope by differential interference contrast. Micrographs were taken 218 \nwith a CCD camera and the Axiovision LE software (Carl Zeiss Vision, Germany). The 219 \nimmunofluorescence-labelled sections and retinal pieces were analyzed with a laser scanning 220 \nmicroscope (LSM) Olympus FluoView 1000 using the FV 1.7 software (Olympus), or with a 221 \nLeica TCS SP5 or a Leica TCS SP8 confocal microscope. LSM images and z-stack 222 \nprojections were examined with ImageJ (https://imagej.net); cells were counted using the cell 223 \ncounter plugin. Images for illustration were adjusted for brightness and contrast using Adobe 224 \nPhotoshop. Irrespective of the fluorescent dye used to visualize a label, labels are shown in 225 \nthe RGB channels that are most suitable to illustrate label combinations. For the benefit of 226 \nred/green-blind readers, combinations of magenta and green are preferred over red and green. 227 \n 228 \nThyroid hormone 229 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n11 \n \nThyroid hormone (TH), via its receptor TRβ2, is an important regulator of cone spectral 230 \nidentity by repressing S opsin and activating L opsin in developing and adult retina (see 231 \nDiscussion). Because of the L and S opsin co-expression in a large proportion of the aardvark 232 \ncones, we were interested to know whether the serum TH levels in aardvark differ from those 233 \nin other mammals. Frankfurt Zoo, during medical check-ups, had obtained five blood counts 234 \nof aardvark 1 over the last three years of his life, and one blood count of his 21 years old son 235 \n(here termed “aardvark 3”), all including serum TH levels. The blood analysis was done by 236 \nthe commercial veterinary clinical diagnostics laboratory LABOKLIN (Bad Kissingen, 237 \nGermany). 238 \n 239 \nResults 240 \nMost findings reported here came from aardvark 1. The retina of aardvark 2 was used for 241 \ncomparison of the photoreceptor findings. 242 \n 243 \nGeneral eye features 244 \nThe eye of aardvark 1 had an equatorial diameter of ca. 23.0 mm and an axial length of ca. 245 \n21.2 mm. The axial length probably is an underestimate because the cornea had collapsed and 246 \nits original curvature was estimated (Fig 2A). The cornea was elliptical with a naso-temporal 247 \ndiameter of 18.8 mm and a dorso-ventral diameter of 15.5 mm (mean 17.1 mm); the ratio of 248 \nmean corneal diameter to eye equatorial diameter was 0.75, and the ratio of mean corneal 249 \ndiameter to eye axial length was 0.81. The lens diameter was 13.3 mm and the lens thickness 250 \n9.5 mm (Fig 2B). The curvature was stronger at the posterior than at the anterior side of the 251 \nlens (not illustrated). The ratio of lens diameter to eye equatorial diameter was 0.58, the ratio 252 \nof lens thickness to eye axial length was 0.45. The poor pigmentation of the choroid and 253 \nsclera in the present eye confirms the observations by Franz [3]. 254 \n 255 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n12 \n \n 256 \nFig 2. General eye features (aardvark 1). (A) Intact eye, frontal view with attached rectus 257 \nmuscles. The cornea had collapsed when the eye was punctured for better fixative penetration 258 \nto the retina. (B) Anterior part of the opened eye from the vitreous side, showing the lens and 259 \nciliary body. (C) Opened eyecup showing the fundus with the retina in situ. There is a light 260 \nhorizontal band where the retinal pigment epithelium (RPE) is weakly pigmented or 261 \nunpigmented. The transition to the pigmented peripheral RPE is gradual at the dorsal side and 262 \nwith a rather sharp boundary at the ventral side. (D) Eyecup after removal of the retina. Some 263 \nRPE also came off during the preparation, showing an unpigmented, whitish-yellow choroid. 264 \nThe optic disc (OD) is located ventral to the light horizontal band of (C). (E) Optic disc in the 265 \nisolated retina, DAB-reacted for blood vessels. There are only a few capillaries present in the 266 \noptic disc (arrow heads), and no blood vessels exit it to supply the surrounding retina. Around 267 \nthe optic disc there is an accumulation of pigment. (F, G) Light microscopic images of flat-268 \nmounted RPE pieces from peripheral (F) and central fundus (G). In the periphery, all RPE 269 \ncells contain densely packed melanin granules (F); centrally, only very few RPE cells are rich 270 \nin melanin granules, the vast majority of RPE cells contains little or no melanin (G). Eye 271 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n13 \n \ndimensions in (A, C, D) can be determined from the millimeter graph paper in (C). The scale 272 \nbar in (F) applies to (F, G). d, dorsal; n, nasal; t, temporal, v, ventral. 273 \n 274 \nThe fundus in the opened eyecup showed a bright yellowish band extending from the 275 \ntemporal to the nasal periphery. This bright band had the appearance of a tapetum lucidum, its 276 \ndorso-ventral width was larger in the temporal than the nasal fundus. Its relatively sharp 277 \nventral boundary ran along the horizontal midline of the fundus, its dorsal boundary showed a 278 \nmore gradual transition to the pigmented part of the fundus (Fig 2C, D). The ventral half and 279 \nthe dorsal periphery of the fundus were covered by brown-black retinal pigment epithelium 280 \n(RPE). The optic nerve head (optic disc, OD) was located centrally on the temporal-nasal eye 281 \naxis and ventral to the geometric center of the eyecup, about one OD diameter below the 282 \nventral boundary of the bright fundus band (Fig. 2D). When the retina was removed, the 283 \nremaining thin RPE layer consisted of RPE cells (melanocytes) that contained a high density 284 \nof melanin granules in the dark-appearing parts of the fundus (Figs 2D, F, 3A). In the central 285 \nbright-appearing band, the large majority of RPE cells contained very few or no melanin 286 \ngranules, and only some single cells or small cell clusters contained ample melanin (Figs 2G, 287 \n3A). The absence of pigmentation in a horizontal band of the central fundus and the 288 \nassociated tapetum-like reflection explain the eyeshine differences seen at different angles 289 \n(Fig 1B, C). When the eye is seen horizontally, the strong reflectivity of this band produces a 290 \nbright whitish to yellowish eyeshine. When the eye is seen from above, the lower reflectivity 291 \nof the more pigmented ventral fundus produces a fainter orange to red eyeshine that resembles 292 \nthe ‘red eye effect’ seen in flash photographs of human eyes with their pigmented fundus. 293 \n 294 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n14 \n \n 295 \nFig 3. Choroid and tapetum lucidum (aardvark 1). (A) Higher power view of the central 296 \nand ventral midperipheral fundus of the aardvark eye (c.f. Fig. 1). Below the partly removed 297 \nRPE layer, the whitish unpigmented choroid with its red blood vessels is visible. (B) SEM 298 \nmicrograph of subcellular structures from the RPE-facing side of a transverse choroid section, 299 \nshowing collagen fibrils arranged in parallel bundles with different orientations that indicate a 300 \ntapetum lucidum. In the upper image part, the fibrils are longitudinally sectioned; in the 301 \nbottom image part, they are cross-sectioned with round to oval profiles. (C) At higher 302 \nmagnification, the fibrils show the typical cross-striation of native collagen (TEM image). (D) 303 \nIn cross-section, the fibrils show a shell and core of higher electron density (SEM image). (E) 304 \nDifferential interference contrast (DIC) image of a vertical cryo-section of the aardvark 305 \nchoroid (Ch) and tapetum (Tap). The poorly pigmented choroid shows cross-sections of blood 306 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n15 \n \nvessels of various calibers, the tapetum shows a horizontal striation indicating tapetal laminae. 307 \n(F) DIC image of a vertical cryo-section of the choroid and tapetum of an African elephant 308 \nfor comparison. The choroid is strongly pigmented, and the tapetum is thicker and more 309 \nconspicuously striated than in the aardvark. (G) Aardvark vertical cryo-section of the choroid 310 \nand tapetum with blood vessel labelling by isolectin (red). The choroid part contains vessels 311 \nof larger and smaller caliber, the tapetum layer is relatively thin, and the choriocapillaris at the 312 \nborder to the RPE is densely filled with capillaries. (H) African elephant vertical cryo-section 313 \nof the choroid and tapetum with blood vessel labelling by isolectin (red) for comparison. The 314 \nimage shows a vertical choroidal blood vessel supplying the CC capillaries. For the sections 315 \nof (E-H), the choroid has been removed from the sclera, so the sections do not show the full 316 \nthickness of the choroid. (I) Flat view of the aardvark choriocapillaris, labelled by isolectin 317 \n(red) and showing the dense capillary net. The scale bar in (E) applies to (E, F), the scale bar 318 \nin (G) applies to (G-I).  319 \n 320 \nChoroid and tapetum lucidum 321 \nOnce the retina was removed, the thin RPE layer readily detached from the choroid in shreds 322 \n(Figs 2D, 3A). The strongly vascularized choroid was unpigmented and appeared whitish with 323 \na mother-of-pearl-like reflection throughout the fundus; in the fundus regions with pigmented 324 \nRPE, this choroidal reflection was concealed (Fig 3A). Electron microscopy of transverse 325 \nchoroid sections showed that at the RPE-facing side of the choroid, there were fibrillar 326 \nstructures arranged in parallel, with different orientations in neighbouring domains (Fig 3B). 327 \nIn some domains the fibrils were densely packed, in others they were less dense or sparse. 328 \nFibril diameters seen in cross-sections were 120-310 nm, being 150-250 nm in most cases. 329 \nObserved fibril lengths were up to about 7 µm. In TEM images, the fibrils showed the typical 330 \ncross-striation of native collagen, indicating a choroidal tapetum lucidum fibrosum (Fig 3C). 331 \nHowever, in SEM images, cross-sections of the fibrils showed a substructure with a more 332 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n16 \n \nelectron-dense shell and core, which is more characteristic of the rodlets of a tapetum 333 \ncellulosum (Fig 3D). This is addressed in the Discussion. Overall, the presumed tapetum layer 334 \nis thinner and less conspicuously striated than, e.g., in the African elephant, like the aardvark 335 \na member of the Afrotheria with an avascular retina (Fig 3E, F). The boundary of the choroid 336 \nand tapetum to the RPE is formed by the choriocapillaris, a dense capillary net that was 337 \nlabelled by the endothelial cell marker isolectin B4 in vertical sections of both aardvark (Fig 338 \n3G) and African elephant (Fig 3H). The dense mesh of the aardvark choriocapillaris is 339 \nparticularly obvious in flat view (Fig 3I). 340 \n 341 \nGeneral Retina Features 342 \nWe confirm that the aardvark retina is avascular. In the fundus, there were no obvious blood 343 \nvessels emerging from the optic disc or extending across the retina. Staining of blood vessels 344 \nwith DAB in a piece of central retina containing the optic disc revealed a few small capillaries 345 \nwithin the optic disc and confirmed that there were no blood vessels extending outside the 346 \noptic disc and into the retina (Fig 2E). 347 \n 348 \nIn the vertical sections, retinal thickness ranged from ca. 120 µm to ca. 180 µm. This is an 349 \nestimate, given the fact that the sections may not be exactly vertical and that the length of 350 \nphotoreceptor outer segments may not be fully preserved. The layering of the aardvark retina 351 \nconformed to the typical mammalian pattern (Fig 4). The outer nuclear layer (ONL) with the 352 \nphotoreceptor somata was the thickest layer, indicating a dominance of rod photoreceptors, 353 \nwhich is the situation seen in most mammals. The ONL had six to nine soma tiers in more 354 \ncentral retina and five to seven tiers in more peripheral retina. The inner nuclear layer (INL) 355 \nhad three to four soma tiers in central retina and two to three soma tiers in peripheral retina. 356 \nThe ganglion cell layer (GCL) was sparsely populated by somata. The narrower outer 357 \nplexiform layer (OPL) and broader inner plexiform layer (IPL) separated the soma layers. 358 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n17 \n \n 359 \n 360 \nFig 4. General retinal features (aardvark 1). (A) Overview of a vertical retinal section 361 \nlabelled with the fluorescent Nissl stain NeuroTrace, revealing the retinal layering. Cell 362 \nbodies in the outer nuclear layer (ONL) and inner nuclear layer (INL) are stacked in several 363 \ntiers. The photoreceptor outer and inner segments (OS+IS) are also labelled. The ganglion cell 364 \nlayer (GCL) is sparsely populated by cells of various soma sizes. A large soma of a putative 365 \nalpha cell is marked by an arrowhead. (B, C) Double labelling with an antibody against the 366 \nneuronal marker NeuN and with NeuroTrace. NeuN only labels a few presumed amacrine cell 367 \nsomata in the INL and some somata in the GCL (B). The NeuroTrace counterstain shows the 368 \nposition of the NeuN somata in the layers (C). (D, E) Immunolabelling for glutamine 369 \nsynthetase shows the Müller cells forming the retinal glia scaffold (D). Counterstaining with 370 \nDAPI (E) shows that the Müller cells have their somata in the INL and vertically extend their 371 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n18 \n \nprocesses from the inner limiting membrane (ILM) formed by their endfeet to the outer 372 \nlimiting membrane (OLM). The images are maximum intensity projections of confocal image 373 \nstacks. OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bars are 100 µm, scale 374 \nbar in (C) applies to (B-E). 375 \n 376 \nMüller cells, the scaffolding radial macroglia of the retina, were specifically labelled by an 377 \nantibody against glutamine synthetase (Fig 4D, E). They were present at a high density and 378 \nhad the mammalian-typical morphology. Their somata were located in the INL. Their inward 379 \nprocesses traversed the IPL and terminated in the Müller cell endfeet that ended at the inner 380 \nlimiting membrane (ILM), separating the retina from the vitreous. In some of the cells these 381 \nprocesses bifurcated and formed two endfeet. Their outward processes encircled the 382 \nphotoreceptor somata in the ONL and ended at the outer limiting membrane (OLM) that lies 383 \nbetween the ONL and the photoreceptor inner segments. An antibody against glial fibrillary 384 \nacidic protein (GFAP) did not label any structures (not illustrated). Hence, there are no 385 \nastrocytes in the aardvark retina, as mammalian astrocytes specifically express GFAP [17]. 386 \nThis is in line with the absence of retinal blood vessels (see Discussion). Furthermore, the 387 \nabsence of GFAP label in the Müller cells indicates that the studied retina was healthy. The 388 \nMüller cells of healthy retinae have very low or no GFAP expression, but show reactive 389 \ngliosis with dramatically upregulated GFAP expression to practically all forms of retinal 390 \nstress, i.e. to various retinal diseases and injuries (reviews: [18,19]. 391 \n 392 \nRod photoreceptors 393 \nIn the retina of aardvark 1, rod photoreceptors were identified by labelling with an antibody to 394 \nrod opsin (Fig 5A, B), and by their characteristic nuclear morphology (Fig 5C, D). Their outer 395 \nsegments showed the most intense rod opsin label and formed a densely packed layer at the 396 \nouter retinal surface (Fig 5A). The used antibody rho4D2 also, but less intensely, labelled the 397 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n19 \n \nother parts of the rod cells, particularly the soma cytoplasm in the ONL and the axonal ending 398 \nin the OPL (Fig 5B), which is common in mammals. The large majority of the photoreceptor 399 \nsomata in the ONL showed rho4D2 label and hence are rod somata. This fits the very low 400 \naardvark cone densities (see below).  401 \n 402 \n 403 \nFig 5. Rod photoreceptors (aardvark 1). (A, B) Vertical retinal section, rod opsin label 404 \n(green). (A) The rod outer segments are strongly labelled, DAPI counterstaining (blue) shows 405 \nthe retinal nuclear layers. (B) Overexposure of the same field shows less strong opsin label in 406 \nthe rod somata in the outer nuclear layer (ONL) and the rod axonal spherules in the outer 407 \nplexiform layer (OPL). Clearly, the vast majority of ONL somata belong to rods. (C-G) DAPI 408 \nnuclear staining of various retinal neurons to reveal their heterochromatin arrangement. (C) 409 \nOverview of a DAPI stained section, nuclei in the ONL (top) are more intensely stained than 410 \nthose in the INL (middle) and GCL (bottom), confirming the appearance seen in (A). (D) The 411 \nrod nuclei show a “semi-inverted” nuclear architecture with most of the heterochromatin 412 \nclustered in the nuclear centre, often in two aggregates, but with some extensions towards the 413 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n20 \n \nnuclear periphery. (E) In contrast, the cone nuclei (arrowhead) have several smaller 414 \nheterochromatin clusters localized towards the nuclear periphery (conventional nuclear 415 \narchitecture). The same conventional heterochromatin arrangement is seen in cells of the INL 416 \n(F) and in retinal ganglion cells (G). (H) In the rods, euchromatin (immunolabelled by anti-417 \nH3K4me3, green) is located mostly in the nuclear periphery and in the gaps between the 418 \nheterochromatin clusters (DAPI, red). (I) In the rod nuclei, heterochromatin (immunolabelled 419 \nby anti-H4K20me3, green) colocalizes with the DAPI staining (red), the merge of the labels 420 \nappears yellow. (J-L) Immunolabelling for lamin A/C (red) and LBR (green), counterstained 421 \nwith DAPI (blue). The aardvark retina shows a presence of lamin A/C in the neuronal nuclei 422 \nin all layers (J, K). As a positive control for labeling with the anti-LBR antibody, the nucleus 423 \nof a microglial cell (arrowhead) expressing LBR but not lamin A/C is shown in (K). LBR 424 \nlabel is only present in microglial cells, not in any neurons. (L) A rod nucleus (left) and a 425 \ncone nucleus (right, arrowhead) with labelled lamin A/C. For layer abbreviations, see Fig 3. 426 \nScale bar in (B) applies to (A, B). 427 \n 428 \nIn the vast majority of eukaryotic cells, the euchromatin is located in the centre of the nucleus 429 \nand the heterochromatin in the nuclear periphery. In contrast, the rod nuclei of nocturnal 430 \nmammals have a unique inverted chromatin architecture with heterochromatin aggregated in 431 \nthe nuclear centre and euchromatin arranged at the nuclear periphery. This unusual chromatin 432 \narrangement evolved as an adaptation to night vision because it reduces light scattering in the 433 \nthick retinae of nocturnal species, enhancing their ability to detect low intensity light [20-22]. 434 \nIn the nocturnal aardvark, the heterochromatin in the rods was clustered in two large central 435 \ngranules, thus the nuclei seem to be inverted (Fig 5D, H, I). At the same time, the internal 436 \nheterochromatin exhibited several protrusions towards the nuclear envelope (Fig 5D), making 437 \nthese nuclei semi-inverted. All other retinal cells, including the cones, had the conventional 438 \nnuclear architecture with heterochromatin attached to the nuclear periphery or nucleolus (Fig 439 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n21 \n \n5E-G). The nuclear envelope protein lamin A/C was present in all retinal nuclei including the 440 \nrod nuclei, whereas the lamin B receptor (LBR) of the nuclear envelope was only present in 441 \nretinal microglial cells, not in any retinal neurons (Fig 5J-L). The functional implications of 442 \nthe nuclear inversion and the role of nuclear envelope proteins in this process are addressed in 443 \nthe Discussion. 444 \n 445 \nIn the retina of aardvark 1, photoreceptor densities (and hence rod densities) were estimated 446 \nfrom counts of ONL nuclei in DAPI-stained vertical sections at 16 positions from the visual 447 \nstreak region, midperipheral and peripheral retina. The observed range was about 124,000 – 448 \n214,000 photoreceptors (rods)/mm², with densities decreasing from central to peripheral 449 \nretina. In the retina of aardvark 2, photoreceptor densities were estimated from counts of ONL 450 \nsomata at seven positions in vertical paraffin sections from central and midperipheral retina. 451 \nHere the range was about 182,000 – 245,000 photoreceptors (rods)/mm². The ONL had five to 452 \nnine tiers of photoreceptor somata like the ONL of aardvark 1. It is possible that aardvark 2 453 \nhad higher rod densities than aardvark 1, but the larger shrinkage of paraffin-embedded tissue 454 \nmay also have artefactually increased the cell density.  455 \n 456 \nCone photoreceptors 457 \nThe cone photoreceptors were identified by labelling with antisera to the longwave-sensitive 458 \n(L) cone opsin and the shortwave-sensitive (S) cone opsin in retinal sections (Fig 6A, B) and 459 \nin flat-mounted retinal pieces from various retinal regions (Fig 6C, S1 Fig, S2 Fig). The two S 460 \nopsin antisera sc14363 (directed against an N-terminus epitope) and JH455 (directed against a 461 \nC-terminus epitope) showed complete colocalization of labelling (not illustrated). This is 462 \nevidence that a full-length, functional S opsin is present. Interestingly, a very large proportion 463 \nof aardvark cones throughout the retina showed co-expression of the L and S opsin.  464 \n 465 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n22 \n \n 466 \nFig 6. Cone photoreceptors (aardvark 1). (A, B) Vertical retinal sections double-467 \nimmunolabelled for S cone opsin (A1, B1, red) and L cone opsin (A2, B2, green). In the 468 \nmerged images, DAPI counterstaining in blue shows the retinal nuclear layers (A3, B3). Most 469 \ncones co-express S and L opsin, arrowheads point to pure S cones. In many cones of the 470 \nregion shown in (A), both the S opsin and the L opsin label extend throughout the cone from 471 \nthe OS to the cone pedicle in the OPL; in the region shown in (B), the L opsin label is 472 \nrestricted to the OS. (C) Double immunolabelled cones in a flat-mounted piece from ventral 473 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n23 \n \nmidperipheral retina. S opsin label (C1, magenta) and L opsin label (C2, green) are 474 \ncolocalized in most cones (C3). Three of the pure S cones are indicated by arrowheads, pure L 475 \ncones are not present in this field. The aureole seen around most cones is an artefact; as the 476 \ncones were photographed from the vitreous side of the retina, there is light scatter at many 477 \ncellular structures. (D) Vertical section double-immunolabelled for S opsin (D1, in red, 478 \nshowing the S cone pedicles in the OPL) and the synaptic ribbon marker CtBP2 (D2, in 479 \ngreen). Most of the small CtBP2 structures in the OPL are ribbons of rod spherules; the merge 480 \n(D3) shows that cone pedicles do not have the ribbon/CtBP2 clusters seen in other mammals. 481 \nAs expected, many somata in the INL are also CtBP2-labelled. (E) Flat-mounted retinal piece 482 \ndouble-labelled for S opsin (E1, magenta) and CtBP2 (E2, green). The focus is on the cone 483 \npedicles in the OPL. The merge (E3) confirms the absence of cone-typical ribbon/CtBP2 484 \nclusters at the pedicle. (A-E) are maximum intensity projections of confocal image stacks. 485 \nThe stack in (C) starts at the level of the intensely labelled cone outer segments and ends at 486 \nthe cone soma level. In this region, faint S opsin label extended throughout the cone, whereas 487 \nL opsin label was restricted to the outer segment in most of the cones. The stack in (E) is of 3 488 \nfocal images spaced 0.5 µm apart. For layer abbreviations, see Fig. 3. Scale bar in (B3) is 100 489 \nµm and applies to (A, B); scale bar in (C3) is 50 µm; scale bars in (D3) and (E3) are 20 µm. 490 \n 491 \nIn sample fields across the retina of aardvark 1, between 35% and 96% were such dual 492 \npigment cones. A substantial proportion of the cones were pure S cones without L opsin 493 \nexpression (some marked in Fig 6A-C). In dorsal retina, between 2% and 37% of the cones 494 \nwere pure S cones, in ventral retina, this percentage was between 22% and 65%. Hence, it 495 \nappears that the proportion of pure S cones is markedly higher in ventral than dorsal retina, 496 \nbut the large variation of percentages between sampling fields also indicates a high local 497 \nvariability. Only a small proportion of the cones were pure L cones without S opsin 498 \nexpression. In many counting fields containing between 100 and 300 cones, pure L cones 499 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n24 \n \nwere absent; in other fields, pure L cones comprised 0.3% to 7% of the cones with no obvious 500 \nregional trend.  501 \n 502 \nIn the retina of aardvark 2, the proportions were somewhat different. In several counting 503 \nfields from a sample region in far peripheral retina, 67-80% of the cones were co-expressing 504 \nL and S opsin, 15-21% were pure L cones, and 6-12% were pure S cones. In another region 505 \nfrom an unknown but probably also relatively peripheral location, 86-95% of the cones were 506 \nco-expressing L and S opsin, 2-6% were pure L cones, and 3-8% were pure S cones (S1 Fig). 507 \n 508 \nThese percentages have to be considered with some reservation. First, the relative intensity of 509 \nthe L and S opsin label varied considerably between cones, in some cones one of the labels 510 \nwas barely above background. This was particularly obvious in aardvark 2 (S1 Fig) but can 511 \nalso be seen in aardvark 1 (Fig 6C). Hence, assessment of opsin co-expression has a 512 \nsubjective component. Second, in many retinal regions, the L opsin label was restricted to the 513 \ncone outer segments (Fig 6B), whereas the S opsin label typically extended throughout the 514 \ncone including the soma, axon, and cone pedicle (Fig 6A, B, D, E). The cone opsin labelling 515 \nrevealed some degree of post mortem outer segment damage, some outer segments were 516 \nelongated, others were just small stumps (see Fig 6C). Hence, S opsin-labelled cones could be 517 \nidentified rather reliably by focusing through the outer retina to their soma level, whereas L 518 \nopsin expression may have been missed in cases of outer segment loss. Nevertheless, there 519 \nwere many sampling fields where the cone outer segments were partially preserved, so the 520 \nabove proportions of pure S and L cones are at least semi-quantitatively reliable. 521 \n 522 \nThe synaptic ribbon marker CtBP2 mainly revealed the single synaptic ribbons of rod 523 \nsynaptic endings (rod spherules) in the OPL (Fig 6D, E). These synaptic ribbons often showed 524 \nthe horseshoe shape that is typical for mammalian rod ribbons (Fig 6E2). In contrast to the 525 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n25 \n \nsituation in other mammals, CtBP2 did not show the typical clustering of ribbons in the cone 526 \npedicles; there were only a few or no CtBP2 puncta at the pedicles of S cones. This is 527 \naddressed in the Discussion.  528 \n 529 \nThe general cone marker peanut agglutinin (PNA) labelled the S cone pedicles in the aardvark 530 \nretina (S2 Fig). This was evident by the overlapping label of the pedicles by the S opsin 531 \nantiserum and PNA. The lateral displacement of the pedicles against the cone outer segments 532 \n(S2C Fig) is mostly due to slight tissue shearing during the mounting of the retinal pieces and 533 \ncould be followed by tracing the stained S cone axons through the ONL in the image stacks. 534 \nThe PNA label of cone pedicles varied in intensity and size; in a few S cone pedicles it was 535 \nabsent or too faint to be detected. Surprisingly, the L cone pedicles did not show PNA 536 \nlabelling. This was checked in several L cones found in appropriately stained retinal pieces. 537 \nTwo pure L cones are present in the field shown in S2 Fig. It remains open whether PNA does 538 \nnot label the L cone pedicles at all, or whether the label is below the detection threshold of our 539 \nstaining. 540 \n 541 \nFor aardvark 1, cone densities were assessed in more than 70 sample fields in flat-mounted 542 \npieces coming from various positions across central and peripheral retina. The sampled tissue 543 \nincluded a large piece of temporal retina that contained the presumed area centralis. Total 544 \ncone densities, i.e., of S cones, L cones and dual pigment cones, showed no strong central-545 \nperipheral gradient. Highest densities of up to 1,100 – 1,300 cones/mm² were present in a 546 \nregion about 4 mm temporal to the optic disc. Density minima of about 300 cones/mm² were 547 \npresent in a few fields in dorsal peripheral retina, but many other fields in dorsal, ventral, and 548 \nnasal periphery had densities of 400 to 1,100 cones/mm². In pieces from midperipheral retina, 549 \ndensities ranged from 600 to 1,000 cones/mm². In the region of the bright (unpigmented) 550 \nhorizontal fundus band described above (see Fig 2C), the cone density did not increase, fields 551 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n26 \n \nfrom a temporal and a nasal region of the band had 600 – 900 cones/mm². This suggests that 552 \nthe band does not represent a retinal specialization (visual streak) at the level of the cone 553 \npopulation. With rod densities of up to 214,000/mm² in central and down to 124,000/mm² in 554 \nperipheral retina, the above cone densities correspond to cone percentages of 0.25 – 0.85% 555 \namong the photoreceptors, with a rough average of approximately 0.5% cones over most of 556 \nthe retina. For aardvark 2, total cone densities could only be determined in the two small 557 \nperipheral pieces of retina described above. They ranged from 1,100 to 1,600 cones/mm². 558 \nThese densities are somewhat higher than those of aardvark 1. With the higher rod densities 559 \nof 182,000 – 245,000/mm², this again amounts to cone percentages of 0.5 – 0.9% of the 560 \nphotoreceptors. 561 \n 562 \nThyroid hormone levels 563 \nGiven the high incidence of cone opsin co-expression in the aardvark and the important role 564 \nof thyroid hormone (TH) in regulating cone spectral identity, we looked at the serum TH 565 \nlevels available for aardvark 1 (five measurements within 2.5 years) and his adult son 566 \naardvark 3 (one measurement) in comparison to published serum TH levels in some 567 \nrepresentative mammals (Table 2). To our knowledge, these are the first published serum TH 568 \nlevels for aardvarks. The serum values of total thyroxine (tT4), total triiodothyronine (tT3), 569 \nfree T4 (fT4) and the biologically active form free T3 (fT3) are very similar for aardvark 1 570 \nand his son, and are also similar to or higher than the respective values in other mammals. 571 \nThis suggests that aardvarks are not hypothyroid and that the S opsin dominance is not related 572 \nto a lack of TH (see Discussion).  573 \n 574 \n 575 \nTable 2. Thyroid hormone serum levels in different mammals 576 \n(Different units in the publications have been converted to unify the table) 577 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n27 \n \nSpecies / Animal tT4 \n(µg/dl) \ntT3 \n(ng/dl) \nfT4 \n(ng/dl) \nfT3 \n(pg/ml) \nTSH \n(ng/ml) \nAardvark 11 12 – >15 226.3 – 390.4 1.54 – 1.93 2.28 – 4.23 <0.03 \nAardvark 31 >15 304.2 2.25 3.32 nd \n      \nAsian elephant2 11.12 126.72 0.87 1.39 0.97 \nAfrican elephant2 10.76 123.27 0.93 1.41 0.56 \nManatee3 4.5 – 8.3 140 – 160 1.3 – 1.6 nd nd \nCow4 3.9 – 8.8 70 – 250 1.3 – 2.4 2.3 – 7.8 nd \nDog5,6 1.5 – 1.6  \n(1.53 – 2.25) \n76 – 84 \n(nd) \n~1.75 \n(0.98 – 1.57) \n~1.15 \n(nd) \nnd \n(0.07 – 0.26) \nHuman7 5 – 12 80 – 220 0.7 – 1.9l 2.3 – 4.1 0.006 – 0.06 \n 578 \ntT4 = total thyroxine T4, tT3 = total triiodothyronine T3, fT4 = free T4, fT3 = free T3; TSH = 579 \nThyroid-stimulating hormone, thyrotropin; nd = not determined. 580 \n1Frankfurt Zoo animals (see Methods); 2Ref. [88]; 3Ref. [89]; 4Ref. [90]; 5Ref. [91]; 6Ref. [92]; 7UCLA 581 \nHealth Website (https://www.uclahealth.org/medical-services/surgery/endocrine-surgery/conditions-582 \ntreated/thyroid/normal-thyroid-hormone-levels), fT3 level from Cleveland Clinic 583 \n(https://my.clevelandclinic.org/health/diagnostics/22425-triiodothyronine-t3) 584 \n 585 \n 586 \nDiscussion 587 \nThe aardvark’s designation as a ‘living fossil’ [2] suggests that its eye and retina also may 588 \nshow prototypic, primordial mammalian features. On the other hand, the aardvark ancestor 589 \ncould already have been specialized on feeding on ants and termites, which speaks against a 590 \n‘basic general’ mammal. Hence, we were interested to study the aardvark’s eye and retina 591 \nwhen the opportunity arose. Early mammals are assumed to have gone through a ‘nocturnal 592 \nbottleneck’ with associated adaptations to low-light vision (see, e.g., [23-27]). Most extant 593 \nnocturnal mammals possess eye and retina features reflecting such an evolutionary adaptation: 594 \nThe eye optics with a proportionately large lens and cornea serves increased light capture. The 595 \nretina has a dominance of the more light-sensitive rod photoreceptors and only a small 596 \nminority of cone photoreceptors (review: [5]). The features of the aardvark eye and retina fit 597 \nthis ‘nocturnal’ category. 598 \n 599 \nUntil 2022, the only detailed study available on the aardvark eye and retina was by Victor 600 \nFranz, dating back to 1909 [3]. It claimed a pure rod retina without cones, but concedes that 601 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n28 \n \nthe tissue conservation may not have been sufficient to prove the absence of cones. The 602 \nabsence of cones has since been repeated in various printed summaries and websites on 603 \naardvark biology and vision ([28] page 326, [29] page 579, [1,30,31]; website examples: 604 \n[32,33]). Franz [3] also claimed the absence of a tapetum lucidum, but conceded in a later 605 \nhandbook chapter that a tapetum lucidum fibrosum may be present ([34] page 1214). 606 \nRecently, Paszta and colleagues published a study on general features of the aardvark eye and 607 \nextraocular structures that included a brief description of the retina [9]. This paper similarly 608 \nclaimed an absence of cones, but reported a tapetum lucidum. Walls [23] also listed the 609 \naardvark as having ‘eye shine’ and a trace of a tapetum fibrosum (his Table VII, p. 241). The 610 \npresent study has assessed these claims. 611 \n 612 \nThe external dimensions of the eye of aardvark 1 are similar to those reported previously 613 \n[3,9]. The corneal size and lens are large compared to total eye size, the ratios of corneal 614 \ndiameter to eye equatorial diameter (0.75), of corneal diameter to eye axial length (0.81), of 615 \nlens diameter to eye equatorial diameter (0.58), and of lens thickness to eye axial length 616 \n(0.45) are within the range seen across nocturnal mammals [25,35]. However, these 617 \nparameters overlap between nocturnal, cathemeral/crepuscular, and diurnal mammals, so their 618 \ndiagnostic value is limited (for discussion see, e.g., [25], but also [36]). 619 \n 620 \nTapetum lucidum 621 \nSome nocturnal, crepuscular, and arrhythmic mammals have a reflective choroidal tapetum 622 \nlucidum behind the retina to increase the amount of light absorbed by the photoreceptors. To 623 \nobservers the tapetal reflection appears as ‘eye shine.’ Morphologically, the two most 624 \ncommon tapetum types are a ‘tapetum cellulosum’ where the reflecting structures termed 625 \nrodlets are located intracellularly (found in Carnivora), and a ‘tapetum fibrosum’ where the 626 \nreflecting structures termed fibrils are located extracellularly (found in Artiodactyla, Cetacea 627 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n29 \n \nand Perissodactyla) (reviews: [37-39]). Our own observation (Fig 1) as well as images and a 628 \nvideo in the internet (https://www.youtube.com/watch?v=apNr2HSrx6s), last accessed on 629 \nOctober 17, 2024; aardvark shown from minute 2:11) indicate that the aardvark may have a 630 \ntapetum lucidum. Hence, it is surprising that Franz [3], Matas et al. [40] and Freeman et al. 631 \n[41] describe the absence of a tapetum in the aardvark eye. In contrast, Walls [23] and Paszta 632 \net al. [9] describe the presence of a tapetum, which they consider to be a choroidal tapetum 633 \nfibrosum. 634 \n 635 \nOur more detailed histological observations confirm a choroidal tapetum. The ultrastructural 636 \nappearance of the tapetal fibrils with their cross-striation (Fig 3C) indicates that the fibrils 637 \nconsist of collagen, which is typical for a tapetum fibrosum [38,42]. On the other hand, in 638 \ncross-section, the aardvark tapetal fibrils show a substructure with concentric zones of 639 \ndifferent electron-density (Fig 3D). This is reminiscent of the substructure of the zinc-640 \ncontaining rodlets of the tapetum cellulosum in ferret and dog [43,44]. As our tissue fixation 641 \nwas not optimized for electron microscopy, the substructure of the aardvark tapetal fibrils 642 \nmay be an artefact. Unfortunately, our material did not allow us to determine whether the 643 \ndomains with their changing fibril orientations are contained intracellularly (suggesting a 644 \ntapetum cellulosum) or whether they are extracellular (suggesting a tapetum fibrosum). 645 \nWeighing all evidence, we think that the aardvark tapetum lucidum is of the fibrosum type. It 646 \nwould certainly seem unlikely that the aardvark has a mix of both tapetum types. In mammals 647 \nwith a tapetum lucidum (whether of the cellulosum or fibrosum type), the RPE cells in front 648 \nof the tapetum are unpigmented or only minimally pigmented, such that the tapetum can in 649 \nfact function as a reflector (reviews: [23] page 232; [38]). This is also the case in the aardvark. 650 \n 651 \nAcross mammals, the optically relevant properties of tapetum fibrosum fibrils and tapetum 652 \ncellulosum rodlets are similar. Both have diameters of about 100-200 nm and are nearly 653 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n30 \n \nhexagonally arranged with a spacing of about one fibril/rodlet diameter, suitable for 654 \nconstructive interference (e.g., sheep: [42]; bovine: [45]; cat: [46,47]; overviews: [38,39,48]). 655 \nThe aardvark fibrils have comparable dimensions, but their spacing is more disordered. In 656 \nmany domains they are loosely spaced and do not show a tight hexagonal arrangement. Also, 657 \nthe aardvark tapetum layer is relatively thin and the tapetum lamination is not as conspicuous 658 \nas in other species. Overall, the aardvark appears to have a rudimentary version of a tapetum 659 \nlucidum. Nevertheless, it obviously shows the typical ‘eye shine.’ 660 \n 661 \nAvascular retina 662 \nThe aardvark retina is avascular, as observed by Franz [3] and Matas et al. [40], and 663 \nconfirmed here. This also explains why we did not see any GFAP-labelled astrocytes. Retinal 664 \nastrocytes are neuroglia cells restricted to the optic nerve fibre layer and associated with blood 665 \nvessels as well as with retinal ganglion cell axons. In species with limited retinal 666 \nvascularization, they only occur in the vascularized regions, and in avascular retinae they are 667 \ncompletely absent (review: [17]). Avascular retinae have been found in several mammals 668 \nfrom different orders [23,49-52]. There is no convincing common explanation for why the 669 \nretinae of some species are avascular whereas those of most other species are vascularized to 670 \nvarious extents. Damsgaard and Country [52] conclude from their large data survey that the 671 \nancestral mammal had an avascular retina, and that retinal vascularization was dynamically 672 \ngained and lost throughout subsequent mammalian evolution depending on species-specific 673 \nvisual needs for retinal processing capacity, neuron numbers and hence retinal thickness. 674 \nMammalian avascular retinae are generally thinner than vascularized ones, commonly below 675 \n150 µm (reviews: [51,52]). The reason is that they have to be supplied with oxygen by 676 \ndiffusion from the choroidal capillary network, and the maximum oxygen diffusion distance 677 \nhas been modeled to be about 143 µm [53], although this value has been questioned by some 678 \nauthors [54]. At 120-180 µm, the aardvark retina is slightly thicker than other avascular 679 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n31 \n \nretinae, but it is still thinner than many vascular nocturnal retinae, particularly having a 680 \nthinner ONL and lower photoreceptor density (see below).  681 \n 682 \nChase [51] also stated that mammals with avascular retinae lack a tapetum lucidum and 683 \nassumed that this is because a tapetum would add to the tissue thickness that has to be reached 684 \nby choroidal oxygen. The aardvark tapetum lucidum is not in line with that assumption. The 685 \nelephants, the horse and the zebra are further examples of species with a tapetum lucidum and 686 \nan avascular retina [49,50]. In fact, the choriocapillaris, the actual release site of oxygen and 687 \nnutrients to the retina, is located in front of the choroidal tapetum, directly facing the RPE and 688 \nretina. Figure 3E-H shows this for the aardvark and elephant. The aardvark choriocapillaris 689 \nforms a very dense capillary mesh (Fig 3I) that obviously suffices to adequately supply the 690 \nretina. We conclude that Chase’s assumption of an incompatibility of a (choroidal) tapetum 691 \nlucidum with an avascular retina is not tenable. 692 \n 693 \nRods 694 \nTypically for a nocturnal mammal, the vast majority of the aardvark photoreceptors are rods; 695 \nthe proportion of only around 0.5% cones is low even among nocturnal species (review: [5]). 696 \nHowever, the estimated rod densities of 124,000 – 214,000/mm² for aardvark 1 and of 697 \n182,000 – 245,000/mm² for aardvark 2 are rather low in comparison to those of many other 698 \nnocturnal mammals, which may range from 200,000 rods/mm² to more than 700,000 699 \nrods/mm² [5]. These low rod densities and the correspondingly thinner ONL most likely are 700 \ncorrelated with the avascularity of the aardvark retina. The nocturnal Microchiroptera also 701 \nhave avascular retinae (review: [51]), and rod densities in the greater horseshoe bat average 702 \nabout 370,000 rods/mm², complemented by about 2.5% cones [55]. The avascular retina of 703 \nthe nocturnal to crepuscular rabbit has between 300,000 rods/mm² in central and 130,00 704 \nrods/mm² in peripheral retina, complemented by about 4 – 6% cones [56]. Hence, even among 705 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n32 \n \nnocturnal mammals with avascular retinae, the aardvark rod density is in the lower range, 706 \nsuggesting a low adaptive pressure for good nocturnal vision. 707 \n 708 \nAlso typical for nocturnal mammals is an inverted architecture of the rod nuclei [20]. Franz 709 \n[3] noted the central clustering of heterochromatin in the aardvark rods and described it as 710 \n‘nuclei with two opposing chromatin bodies that looked like being in mitosis.’ We here give a 711 \ndetailed description of these rod nuclei (Fig 4). As analyzed by Solovei and colleagues 712 \n[20,22], the central position of the inactive heterochromatin and peripheral position of the 713 \nactive euchromatin are assumed to be strongly disadvantageous for nuclear functions, but they 714 \nhave an optical advantage. The densely packed heterochromatin core is highly refractive and 715 \nshows the physical properties of a light-focusing lens. Hence, the rod nuclei in the ONL form 716 \ncolumns of microlenses that act as ‘light guides.’ This strongly reduces light scattering in the 717 \nONL, which is particularly important for nocturnal mammals with their thicker ONL and the 718 \nneed to capture a large proportion of the few photons available at night. Obviously, inverted 719 \nrod nuclei are an evolutionary adaptation to improve nocturnal vision. The rods of diurnal 720 \nmammals have a conventional nuclear architecture, because they do not have to maximize 721 \nphoton capture. 722 \n 723 \nThe aardvark has semi-inverted rod nuclei. Most likely, these are less effective focusing 724 \nlenses than fully inverted rod nuclei. Both the relatively low rod density compared to other 725 \nnocturnal mammals and the semi-inverted rod nuclei support the assumption that for the 726 \naardvark with its reliance on smell and hearing, there was no strong evolutionary pressure for 727 \nan optimally adapted nocturnal retina, and that its night vision sensitivity is lower than that of 728 \nmany other nocturnal mammals. 729 \n 730 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n33 \n \nIn conventional nuclei, heterochromatin is tethered to the nuclear envelope by either lamin 731 \nA/C or the lamin B receptor (LBR); the inverted rod nuclei of nocturnal mammals are 732 \ncharacterized by the absence of both tethers [57]. The semi-inverted architecture of the 733 \naardvark rod nuclei might be explained by expression of one of these proteins. We tested this 734 \nwith antibodies to LBR and to lamin A/C. Whereas no aardvark retinal neurons showed LBR 735 \nimmunoreactivity, all of them showed lamin A/C immunoreactivity. The lamin A/C antibody 736 \nclearly marked the rod’s nuclear periphery, although admittedly weaker compared to the 737 \nnuclei of the INL and GCL. This is similar to the weak LBR expression in the semi-inverted 738 \nrod nuclei of, e.g., goat and cow [57].  739 \n 740 \nCones 741 \nFrom today’s perspective, the claim that the aardvark retina completely lacks cones [3,9] was 742 \nbased on histological approaches that are not suitable to detect sparse populations of cones. 743 \nThe most reliable approach to identify cones is by immunolabelling them with antibodies to 744 \ncone opsins, which we have done in the present study. The immunolabelling showed the 745 \npresence of L and S opsin in a low-density population of aardvark cones. 746 \n 747 \nThe two common mammalian cone opsins are the L opsin (also termed LWS opsin, peak 748 \nsensitivity λmax in the green to yellow part of the spectrum) and the S opsin type 1 (SWS1 749 \nopsin, λmax in the blue, violet or UV part of the spectrum [58]. The opsin antisera used here 750 \nrecognize all spectral variants of the respective opsins. Partial gene sequencing has identified 751 \nL and S opsin genes in the aardvark, and judging from the tuning-relevant amino acids, the 752 \naardvark S opsin has been suggested to be UV-sensitive, and the L opsin to have its λmax at 753 \n527-533 nm (Supplementary Table S5 in [7]). UV tuning is the ancestral tuning of the 754 \nvertebrate SWS1 opsin and has been maintained in a number of nocturnal mammals (reviews: 755 \n[59-61]). Our opsin immunolabelling gives evidence that the L and S opsins are indeed 756 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n34 \n \nexpressed in aardvark cones, and provides information about the population density and 757 \ntopographical distribution of the cones.  758 \n 759 \nA less normal feature of the aardvark cones is, that many of them co-express the two opsins 760 \nacross the retina and are ‘dual pigment’ cones. An artefactual double-labelling due to cross-761 \nreactivity between the two opsin antisera can be excluded, because the tissue also shows pure 762 \nL and S cones. Many nocturnal mammals have the two opsins in separate cone populations 763 \nand thus the basis for dichromatic color vision. This is the canonical mammalian condition of 764 \nbeing spectrally selective (reviews: [4,5,62]). Dual pigment cones were reported in the ventral 765 \nretinae of the house mouse, guinea pig and rabbit [63,64]. Since then, opsin co-expression in 766 \nall cones across the retina has been found in the pouched mouse Saccostomus campestris and 767 \nthe Siberian dwarf hamster Phodopus sungorus [65]. The molecular mechanisms responsible 768 \nfor the co-expression of both opsins in some cones and its suppression in other cones is only 769 \npartly understood. During rat and gerbil retinal development, all cones first express S opsin, 770 \nand prospective L cones then successively switch to L opsin expression with an intermediate 771 \nphase of opsin co-expression; this suggests that S opsin expression may be the default 772 \npathway when an L opsin-activating mechanism is absent or suppressed [66]. One such factor 773 \nis thyroid hormone (see below).  774 \n 775 \nIn the retina of aardvark 1, the majority of single pigment cones are pure S cones (regionally 776 \nvarying from 2% to 65% of the cones); pure L cones are a sparse population of about 0.3 – 777 \n7%. In the peripheral retina of aardvark 2, there are somewhat more pure L cones (2 – 21%) 778 \nthan pure S cones (3 – 12%). If the aardvark has colour-processing (cone-opponent) retinal 779 \nganglion cells, most of their input will be from dual pigment cones. What they may be able to 780 \nuse for colour vision are the spectrally different sensitivities of pure L and S cones vs. dual 781 \npigment cones. The rods may also contribute to colour vision in mesopic light conditions 782 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n35 \n \n([58] and references therein). However, given the very low cone density and the dominance of 783 \ndual pigment cones, it is likely that the aardvark at best has feeble colour vision [62]. The low 784 \ncone density also makes good photopic visual acuity (cone-based spatial resolution) unlikely.  785 \n 786 \nOur opsin immunolabelling showed qualitatively different mixtures of the two opsins in 787 \ndifferent cones, but did not allow to quantitatively access the absolute amounts of the two 788 \nopsins per cone. Hence, we cannot comment on the presumed dominant spectral sensitivity of 789 \nthese dual-pigment cones. However, the much higher fraction of S opsin-containing cones in 790 \ncomparison to most other mammals suggests that the aardvark retina has a relatively high 791 \ncone-based sensitivity in the shortwave (blue to UV) range. This is the case, e.g., in 792 \nMicrochiroptera with a similar S opsin dominance [67]. It may be an evolutionary adaptation 793 \nto the spectral composition of twilight, which contains higher proportions of short 794 \nwavelengths than full daylight (see, e.g., [68,69]). Twilight is the most likely situation that 795 \nnocturnal animals may encounter during their active phases, and cones contribute to vision at 796 \nthis mesopic light level. On the other hand, many nocturnal mammals facing the same twilight 797 \nconditions, e.g., rats [70], flying foxes [71], colugos [14], nocturnal lemurs [72], and Canidae 798 \n[73], have low proportions of S cones (reviews: [4,5,60]). Moreover, a substantial number of 799 \nnocturnal mammals are completely lacking S cones (review: [74]). That makes the hypothesis 800 \nof a special shortwave adaptation to twilight less plausible. 801 \n 802 \nAs assessed in the retina of aardvark 1, cone density changes across the retina are small, 803 \nranging from a shallow maximum of up to 1,300 cones/mm² in a region of temporal retina to 804 \nminima of 300 cones/mm² at some peripheral locations, but up to 1,100 cones/mm² in other 805 \nperipheral regions, representing 0.25–0.85% of the photoreceptors. The horizontal band of 806 \nreduced RPE pigmentation seen in the eye’s fundus has no higher cone density than the 807 \nsurrounding midperiphery. Thus, the cone topography does not suggest specialized regions of 808 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n36 \n \nparticularly high cone-based visual performance like a prominent area centralis or visual 809 \nstreak. Such specialisations are present in many other mammals (reviews: [75-77]), but often 810 \nmost prominent in the retinal ganglion cells. The cone density in the small parts of the retina 811 \nof aardvark 2 that could be studied is slightly higher, but due to the higher rod densities there, 812 \nthe cones also represent only 0.5–0.9% of the photoreceptors. Together with the different 813 \nproportions of pure L cones, pure S cones and dual pigment cones in the two individuals, it 814 \nappears that there is some interindividual variability in the detailed characteristics of the cone 815 \npopulation, even though the basic properties of a low cone-to-rod ratio and a dominance of 816 \ndual pigment cones are preserved. Alternatively, these differences between the old male 817 \naardvark 1 and the younger female aardvark 2 could be sex-related or age-related. A larger 818 \nsample of aardvark retinae would be needed to study these aspects. 819 \n 820 \nA further unusual feature of the aardvark cones is that the cone pedicles do not show the 821 \ntypical cluster of CtBP2-positive ribbons. In other mammals, the cone pedicles have a large 822 \nnumber of presynaptic sites with ribbons (reviews: [78,79]). The tissue quality did not allow 823 \nan ultrastructural analysis, so we could not determine whether the aardvark cones have only 824 \nfew ribbon synapses per pedicle, or whether there are many that do not label for CtBP2. A 825 \nlow ribbon density may impair the signal transmission performance of the aardvark cones. 826 \n 827 \nThyroid hormone (TH) 828 \nIn mouse early postnatal development, TH, through its receptor TRβ2, is a crucial regulator of 829 \ncone spectral identity by repressing S opsin and activating L opsin [80-82]. Even in adult 830 \nmouse and rat, pharmacological suppression of serum TH reversibly activates S opsin and 831 \nrepresses L opsin in all cones [83]. It may be that dual pigment cones are the consequence of a 832 \n(genetic) defect of the switch-off mechanism for S opsin expression during developmental L 833 \nopsin activation [84]. Hence, we were interested to know whether the aardvark has unusually 834 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n37 \n \nlow TH levels that might correlate with the high proportion of dual pigment cones. The data 835 \npresented in Table 2 show that this is not the case, the aardvark has serum TH levels that are 836 \nsimilar to or higher than those of other mammals, particularly those of the elephants and the 837 \nmanatee that belong to the same clade Afrotheria as the aardvark. The other listed species 838 \ncover a broad range of mammals, and all of them have a normal cone complement with a 839 \nmajority of pure L cones and a ca. 10% minority of pure S cones (with the exception of the 840 \nmanatee, for which no cone population data exist). This suggests that aardvarks are not 841 \nhypothyroid and that the S opsin dominance is not related to a lack of TH. Also, elevated 842 \nserum levels of thyroid-stimulating hormone (TSH) would be a first sign of hypothyroidism, 843 \nbut they appear low in aardvark 1 (Table 2). However, there could be other deficits in the 844 \nchain of thyroid hormone action that we were unable to study here. For example, one crucial 845 \ncomponent is the nuclear T3 receptor TRβ2; in TRβ2 knockout mice, all cones express S 846 \nopsin and none express M opsin [80]. Another component is the monocarboxylate transporter 847 \n8 (MCT8), a plasma membrane transporter allowing TH access to the cones; in MCT8 848 \nknockout mice, cone opsin expression resembles that in hypothyroid or TRβ2 knockout mice 849 \n[85].  850 \n 851 \nConclusions 852 \nThe aardvark eye and retina have the typical features seen in nocturnal mammals: light-853 \nsensitive optics with a large lens and a large cornea, a reflective tapetum lucidum, and a rod 854 \ndominance with a very low cone density. Three unusual features are, (i) that the retina is 855 \navascular with the corollary of being thinner and hence having a lower rod density than many 856 \nother nocturnal mammals, (ii) that the rod nuclei are only semi-inverted and not fully inverted 857 \nas in other nocturnal mammals, (iii) that there is opsin co-expression in a large proportion of 858 \nthe cones. Hence, nocturnal visual sensitivity probably is lower than in many other nocturnal 859 \nmammals, and cone-based visual acuity and colour vision certainly are poor.  860 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n38 \n \n 861 \nRegarding the designation of the aardvark as a living fossil [2] and the question whether this 862 \nentails a primordial, prototypical mammalian retina, one can state that the massive presence of 863 \ndual pigment cones argues against a primordial character of the aardvark retina. The most 864 \ncommon pattern in non-mammalian vertebrate retinae, as in mammalian retinae, are single 865 \nopsin cones. The most parsimonious assumption is that the last synapsid ancestor of the 866 \nmammals, and hence also the first mammal, had single opsin cones. Therefore, dual pigment 867 \ncones are a derived feature, and the aardvark retina is not a primordial mammalian retina as 868 \nfar as the cones are concerned. 869 \n 870 \n 871 \nAcknowledgments 872 \nWe thank Robert Molday and Jeremy Nathans for kindly providing antibodies. The technical 873 \nassistance of Alena Konoplew, Elke Laedtke and Carola Tröger is gratefully acknowledged. 874 \nWe also thank Radosław Ratajszczak, Wojciech Paszta and Krzysztof Zagórski (Wrocław 875 \nZoo) for their help in collecting research material and for providing information on the animal 876 \nfrom which the eyes were taken. 877 \n 878 \n 879 \nReferences 880 \n1. Shoshani J, Goldman CA, Thewissen JGM. Orycteropus afer. Mammalian Species. 881 \n1988; 300:1–8. 882 \n2. Shoshani J. Tubulidentata (Aardvarks). In: Encyclopedia of Life Sciences. 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Breed-specific reference intervals for assessing thyroid function in seven dog 1139 \nbreeds. J Vet Diagn Invest. 2015; 27:716–727. doi: 10.1177/1040638715606953. 1140 \n 1141 \n 1142 \n 1143 \nSupporting information 1144 \nS1 Fig. Cone photoreceptors. Sequentially double immunolabelled cones in two 1145 \nneighbouring flat-mounted pieces from an unknown location in midperipheral to peripheral 1146 \nretina (aardvark 2). The piece of field 1 was first incubated with the rabbit L opsin antiserum 1147 \nJH492, visualized with an Alexa488-coupled secondary antiserum (green). Then it was 1148 \nincubated with the rabbit S opsin antiserum JH455, visualized with a Cy3-coupled secondary 1149 \nantiserum (magenta). The merge shows that all cones are labelled by Cy3, because this 1150 \nsecondary antiserum bound to JH455 as well as JH492. The pure S cones are exclusively 1151 \nlabelled by Cy3 (arrowheads), all Alexa488-labelled cones contain the L opsin. The 1152 \nneighbouring piece of field 2 was first incubated with the rabbit S opsin antiserum JH455, 1153 \nvisualized with the Cy3-coupled secondary antiserum (magenta). Then it was incubated with 1154 \nthe rabbit L opsin antiserum JH492, visualized with the Alexa488-coupled secondary 1155 \nantiserum (green). The merge shows that all cones are labelled by Alexa488, because this 1156 \nsecondary antiserum bound to JH492 as well as JH455. The pure L cones are exclusively 1157 \nlabelled by Alexa488 (arrowheads), all Cy3-labelled cones contain the S opsin. The relative 1158 \namount of L and S opsin (i.e., labelling intensity) differs between cones. For details of the 1159 \nprocedure see Methods. The images are maximum intensity projections of confocal image 1160 \nstacks. Scale bar is 50 µm and applies to all images. 1161 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint \n\n50 \n \n 1162 \nS2 Fig. Cone photoreceptors and their synaptic pedicles in the outer plexiform layer. 1163 \nFlat-mounted piece of retina (aardvark 1), triple immunolabelled for S opsin, L opsin and 1164 \nPNA. (A) Focus on the outer segments of the opsin-immunolabelled cones; most cones 1165 \nexpress both opsins (A1, S opsin; A2, L opsin; A3, merge). The field contains two pure L 1166 \ncones. (B) Focus on the cone pedicles in the outer plexiform layer; the S opsin-labelled 1167 \npedicles (B1) are also labelled by PNA (B2), as shown in the merge (B3). (C) Schematic 1168 \nillustration of the cones identified in A (S cones, magenta circles; L cones, green circles; dual 1169 \npigment cones, bipartite circles) and the PNA-labelled pedicles identified in B (grey squares). 1170 \nThe pairing between outer segments and pedicles (connecting lines) was checked by 1171 \nfollowing the S opsin-labelled cone axons through the image stacks. The pedicles of the two 1172 \npure L cones show no PNA label. For two pedicles at the edges of the image, the 1173 \ncorresponding outer segments lie outside the frame. Due to faint labelling of some cones, not 1174 \nall cones shown in (C) can be seen in (A) and (B). Scale bar in (C) is 50 µm and applies to all 1175 \nimages. 1176 \n 1177 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.09.622767doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}