Living in darkness: Exploring adaptation of Proteus anguinus in 3 dimensions by X-ray imaging

GigaScience, 2022, 11,1–8 DOI: 10.1093/gigascience/giac030 DATA NOTE Li ing in da kness: Explo ing adap a ion o P o eus anguinus in 3 dimensions by X- ay imaging Ma ké a Tesaˇ o á 1, Lucia Mancini 2, Edga do Mau i3, G ego Aljanˇ ciˇ c4, Magdalena N˘ ap˘ a u¸s-Aljanˇ ciˇ c4,5,RokKos anjšek 6, Lilijana Bizjak Mali 6,TomášZikmund 1, Ma ké a Kaucká 7, Fede ica Papi3,JanaGoyens 8, Anass Bouchni a 9,10, And eas Hellande 9, Igo Adameyko 11,12 and Joze Kaise 1,* 1Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyˇ no a 123, B no, 61200, Czech Republic 2Ele a-Sinc o one T ies e S.C.p.A., S.S. 14 - km 163,5 in A ea Science Pa k, Baso izza, T ies e, 34149, I aly 3Speleo i a ium E win Pichl, Ad ia ic Speleology Socie y, Via Guido Reni, 2/C, T ies e, 34123, I aly 4Ins i u e Tula Ca e Labo a o y, Oldhamska 8a, K anj, 4000, Slo enia 5Resea ch Cen e o he Slo enian Academy o Sciences and A s: Ka s Resea ch Ins i u e, Ti o g 2, Pos ojna, 6230, Slo enia 6Uni e si y o Ljubljana, Bio echnical Facul y, Depa men o Biology, Veˇ cna po 111, Ljubljana, 1000, Slo enia 7Max Planck Ins i u e o E olu iona y Biology, Augus -Thienemann-S . 2, Plon, 24306, Ge many 8Labo a o y o Func ional Mo phology, Uni e si y o An we p, Uni e si ei splein 1, Wil ijk, 2610, Belgium 9Depa men o In o ma ion Technology, Uppsala Uni e si y, Box 337, Uppsala, 755 01, Sweden 10Depa men o In eg a i e Biology, Uni e si y o Texas a Aus in, Aus in, 78712, Texas, USA 11Medical Uni e si y o Vienna, Cen e o B ain Resea ch, Depa men o Neu oimmunology, Spi algasse 4, 1090 Vienna, Aus ia 12Ka olinska Ins i u e , Depa men o Physiology and Pha macology, Solna agen 9, 17165 Solna, Sweden ∗Co espondence add ess: Joze Kaise , Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu ky ˇ no a 123, B no, 61200, Czech Republic. E-mail: [email p o ec ed].cz Abs ac Backg ound: Ligh less ca es can ha bou a wide ange o li ing o ganisms. Ca e animals ha e e ol ed a se o mo phological, physi- ological, and beha iou al adap a ions known as oglomo phisms, enabling hei su i al in he pe pe ual da kness, na ow empe - a u e and humidi y anges, and nu ien sca ci y o he sub e anean en i onmen . In his s udy, we ocused on adap a ions o skull shape and senso y sys ems in he blind ca e salamande ,P o eus anguinus,also known as olm o simply p o eus— he la ges ca e e a- pod and he only Eu opean amphibian li ing exclusi ely in sub e anean en i onmen s. This ex ao dina y amphibian compensa es o he loss o sigh by enhanced non- isual senso y sys ems including mechano ecep o s, elec o ecep o s, and chemo ecep o s. We compa ed de elopmen al s ages o P. anguinus wi h Ambys oma mexicanum, also known as axolo l, o make an exempla y compa ison be ween ca e- and su ace-dwelling paedomo phic salamande s. Findings: We used con as -enhanced X- ay compu ed mic o omog aphy o he 3D segmen a ion o he so issues in he head o P. anguinus and A. mexicanum. Senso y o gans we e isualized o elucida e how he animal is adap ed o li ing in comple e da kness. X- ay mic oCT da ase s we e p o ided along wi h 3D models o la al, ju enile, and adul specimens, showing he ca ilage o he chond oc anium and he posi ion, shape, and size o he b ain, eyes, and ol ac o y epi helium. Conclusions: P. anguinus s ill keeps some o i s sec e s. Ou high- esolu ion X- ay mic oCT scans oge he wi h 3D models o he ana omical s uc u es in he head may help o elucida e he na u e and o igin o he mechanisms behind i s adap a ions o he sub e anean en i onmen , which led o a se ies o oglomo phisms. Keywo ds: P o eus anguinus,Ambys oma mexicanum, olm, axolo l, X- ay mic oCT, mic o omog aphy, salamande , ca e animal, sub e - anean adap a ions Da a Desc ip ion Con ex P o eus anguinus, also known as he olm o simply p o eus, has a ac ed he a en ion o scien is s and animal ade s o cen- u ies. P. anguinus is an apex p eda o o he ka s unde g ound wa e s. I s p esence indica es he s abili y o ood chains in he sub e anean ecosys em. I s geog aphic dis ibu ion is limi ed o he Dina ic Ka s ; i anges om he Gul o T ies e in I aly, h ough he sou he n hal o Slo enia,coas al mainland o C oa ia and pa s o Bosnia and He cego ina, as a as adjacen pa s o Mon eneg o. Wi h i s ex emely agmen ed and limi ed habi a , P. anguinus is pa icula ly ulne able o pollu ion o g oundwa e . Some popula ions ha e been locally des oyed o endange ed by pollu ion o habi a des uc ion [1,2]. P. anguinus is an impo an objec o esea ch om a leas 2 pe spec i es. Fi s , P. anguinus played a his o ical ole du ing he o ma ion o mode n science om he 17 h o 19 h cen u y, puzzling he minds o mos p omi- nen ea ly na u alis s, om Val aso o Linnaeus, Scopoli, Cu ie , and Humbold , and om Lama ck o Da win and Goe he. Second, P. anguinus has po en ial o answe he ques ions o he science o oday and he u u e (e.g., egene a ion, ca e- ela ed adap a- ions, eno mous genome, and conse a ion o sub e anean bio- di e si y) [3]. P. anguinus was i s men ioned by Janez Vajka d Val aso in 1689. I s scien i ic name P o eus anguinus was gi en by Josephus Nicolaus Lau en i in 1768, and hus P. anguinus became he i s Recei ed: Augus 27, 2021. Re ised: Janua y 6, 2022. Accep ed: Feb ua y 27, 2022 C The Au ho (s) 2022. Published by Ox o d Uni e si y P ess GigaScience. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h ps://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 2|GigaScience, 2022, Vol. 11, No. 1 axonomically desc ibed ca e animal in he wo ld [4]. In he pas 2 cen u ies, mo e de ailed esea ch has been pe o med on P. an- guinus. Two o he cen al igu es o he ea ly P. anguinus esea ch we e Žiga Zois, a na u alis om Ljubljana, who i s desc ibed i s beha iou and conduc ed he ea lies physiological and ecologi- cal obse a ions. Then, Ka l on Sch eibe s, a Viennese zoologis , was he i s o explo e he ana omy o P. anguinus [5]. This mys- e ious animal, which e ains la al ea u es a he adul s age, s a ed o in e es he scien i ic communi y in he ea ly 19 h cen- u y, wi h a ocus on i s sec e i e mode o ep oduc ion. In 1859, P. anguinus se ed as an example o blind ca e animals in he a- mous monog aph On he O igin o Species, whe e Cha les Da win a ibu ed he educ ion o eyes wholly o hei disuse in da kness [6]. P. anguinus also became a model species in classical s udies o compa a i e ana omy o he la e 19 h cen u y. In he 20 h cen u y, mo e sys ema ic esea ch was enabled by o e coming he inac- cessibili y o P. anguinus’s sub e anean habi a in cap i i y o ca e labo a o ies wo ldwide, including F ance, Slo enia, and Ge many; he s udies ocused on ca e- ela ed physiology,ecology,beha iou , and molecula phylogeny [3]. Fo una ely, he a en ion o P. an- guinus and i s habi a has g adually ecei ed an impo an cul- u al and conse a ion a i ude (1951 p o ec ed species in Slo e- nia, 1971 Ramsa con en ion, 1979 Be n con en ion, and 1992 EU Habi a Di ec i e). The yea 2019 ep esen ed ano he miles one in he esea ch o P. anguinus. The public p esen a ion o he P. anguinus genome sequencing p ojec [7] aimed o deciphe he P. anguinus gene ic in o ma ion coded in i s genome, which is ∼15 imes la ge han he human genome. Obliga e ca e-dwelle s o en esul in a se o speci ic mo - phological, physiological, and beha iou al ai s (i.e., oglomo - phism). Compa ed wi h hei epigean ela i es, ca e-dwelling an- imals may ha e phylogene ically e ained olde senso y p ope - ies, imp o ed hem, o acqui ed new ones, enabling hei su - i al in da k habi a s [8].P. anguinus e ol ed a ange o adap a ions such as he loss o pigmen a ion, slow me abolism,and capabili y o su i ing ex eme s a a ion. Mo eo e , he loss o eyesigh is compensa ed by o he specialized senses ha enable na iga ion in comple e da kness. The mode o ca e li e and o he biological peculia i ies o P. anguinus and o he oglobion s e oke he po- en ial ole o unde wa e audio-, mechano-, elec o-, and mag- ne o ecep ion [9,10]. Specimens o P. anguinus a e aluable s udy ma e ial because hey a e p o ec ed and ulne able. Ou knowledge o P. anguinus la al and ju enile s ages is s ill sca ce. Fo hese easons, we aimed o use P. anguinus specimens om exis ing collec ions o a oid collec ing hem om na u e. These animals died by na u- al causes and hei bodies we e p ese ed. We accessed he col- lec ion, applied non-des uc i e s aining and a non-des uc i e imaging me hod (X- ay compu ed mic o omog aphy [mic oCT]), and hen e u ned he specimens back o he collec ion. In his way, we ob ained high-quali y and high- esolu ion 3D da a wi h- ou needing o kill any animal. X- ay mic oCT has become a powe ul me hod in de elopmen- al biology o explo ing mo phological changes in 3 dimensions. Geome ic mo phome ics based on X- ay mic oCT has al eady been used p e iously o an explo a o y analysis o he mo phol- ogy o he c anial os eology in he whi e and black subspecies o P. anguinus [11,12]. Howe e , wi hou con as -enhanced ech- niques, less dense issues such as senso y o gans would be un- ecognizable o inaccu a ely cap u ed owing o insu icien con- as o de ailed 3D analysis. In ou s udy, we use s aining o so issues by phospho ungs ic acid (PTA) o iodine o isualize so issues in he head o P. anguinus. Fo he i s ime, we look a ol- ume ic in e nal s uc u es by using a non-des uc i e imaging echnique and we show he senso y o gan wi h a high spa ial and con as esolu ion. P. anguinus has been an impo an objec o s udy in he his o y o in e na ional na u e esea ch, in iguing scien is s. Thanks o s udies in he pas 300 yea s [5], he mys e ies o his ca e amphib- ian a e slowly being un a elled. Howe e , P. anguinus s ill lea es gaps in ou knowledge abou i s ecology, e olu ion, and physiol- ogy. Ou high- esolu ion mic oCT scans, oge he wi h 3D models o ana omical s uc u es in he head, could help o elucida e he na u e and o igin o he mechanisms behind i s adap a ions o he sub e anean ligh less en i onmen , which led o he acquisi ion o he oglomo phic ea u es. Me hods Sample p epa a ion App o al o he cap u e, handling, main enance, and b eeding in cap i i y o he animals used in he s udy was g an ed by he Minis y o he En i onmen and Spa ial Planning o he Re- public o Slo enia, Slo enian Agency o he En i onmen (Pe - mi s Nos. 35701-36/01, 35601-95/2009-4, and 35601-132/2014-4), by he I alian Republic, F iuli Venezia Giulia Region (Pe mi Nos. 4105/6MU4/95/04/12),and by he I alian Republic, Minis y o Eco- logical T ansi ion (Pe mi s Nos. 3006/015590-93, 39/04). No ani- mals we e killed o his s udy. Ou expe imen al plan in ol ed scanning P. anguinus and he axolo l Ambys oma mexicanum sam- ples a di e en s ages o s udy a ious pa s o he skull and sen- so y o gans. All 5 P. anguinus specimens (NCBI: xid221568) we e s o ed in 75% e hanol.Specimens o A. mexicanum (NCBI: xid8296) had been ea ed a he Speleo i a ium E win Pichl (I aly) since 2004 and in- cluded a 5-yea -old adul emale (died in 2009) wi h a o al leng h o 194 mm, and a la a o 6 days old (died in 2012) wi h a o- al leng h o 11 mm. Specimens o P. anguinus included 1 la a, 3 weeks old (died in 2007), leng h 23 mm, cap i e b eeding o ig- ina ing om he Pos ojna-Planina Ca e Sys em, om he collec- ion o he Tula Ca e Labo a o y (Slo enia); 1 ju enile, 35 mm long, deceased a he collec ion si e in sp ing nea Me ko i´ c (C oa- ia), om he collec ion o he Depa men o Biology, Bio echnical Facul y, Uni e si y o Ljubljana (Slo enia); 1 adul , sex unknown, leng h 276 mm, collec ed in Pos ojna-Planina Ca e Sys em (Slo e- nia,1989),died in cap i i y o he Speleo i a ium in 1999, om he collec ion o he Speleo i a ium E win Pichl (I aly). We modi ied a con as -enhancing p o ocol ini ially de eloped by B ian Me sche [13] ha has been success ully applied on sala- mande issues be o e [14]. La al samples o bo h P. anguinus and A. mexicanum, and ju enile P. anguinus we e s ained wi h 1% PTA in 90% me hanol o 7 weeks. The solu ion was exchanged wi h a esh one once a week. The adul P. anguinus and A. mex- icanum specimens we e s ained wi h 2% iodine (ins ead o PTA) in 90% me hanol o 6 weeks o ensu e ha he con as ing agen would pene a e he en i e sample because iodine pene a es be - e han PTA. Subsequen ly, he samples we e g adually ehy- d a ed in e hanol se ies (90%, 80%, 70%, and 50%), 1 day o each concen a ion (i.e., 4 days o ehyd a ion). The samples we e hen s abled in polyamide ubes illed wi h 1% low-mel ing aga ose gel o p e en sample mo emen du ing CT scan. Image acquisi ion The head egion o he samples was scanned by using a labo a- o y X- ay mic oCT sys em GE Phoenix | ome|x L 240 (Wayga e Technologies / Bake Hughes Digi al Solu ions GmbH, Wuns o , Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 Explo ing adap a ion o P o eus anguinus |3 Ge many). The sys em was equipped wi h a 180 kV/15 W maxi- mum powe nano ocus X- ay ube and a high-con as la -panel de ec o DXR250 wi h 2,048 ×2,048 pixels esolu ion and (200 × 200) μm2pixel size. A o al o 2,000 p ojec ions o e a o al scan angle o 360◦we e acqui ed wi h an exposu e ime o 900 ms pe p ojec ion. Each p ojec ion was cap u ed 3 imes, and an a e age o he signal was used o imp o e he signal- o-noise a io. The ac- cele a ion ol age o he X- ay ube was se o 60 kV and he ube cu en o 200 μA o la al and ju enile P. anguinus;80kVand 250 μA we e used, espec i ely, o ol age and cu en o adul samples. The X- ay beams o lowe ene gies we e il e ed wi h a 0.2-mm- hick aluminium pla e o la al and wi h a 1-mm- hick aluminium pla e o adul samples. The oxel sizes o he econ- s uc ed da a we e as ollows: 3.5 μm o ju enile P. anguinus, 5.8 μm o la al P. anguinus,25μm o adul P. anguinus, 2.8 μm o la al A. mexicanum, and 27.5 μm o adul A. mexicanum. Tomog aphic da a p ocessing The omog aphic econs uc ion was pe o med using GE phoenix da os|x 2.0 so wa e (Wayga e Technologies / Bake Hughes Dig- i al Solu ions GmbH, Wuns o , Ge many) (Phoenix Da os|x 2.0, RRID:SCR_017996). A segmen a ion p ocedu e was hen applied o econs uc ed slices. The A izo 7.1 (The mo Fishe Scien i ic, Wal ham, MA, USA) (A izo 3D So wa e, RRID:SCR_014431) im- age p ocessing so wa e was used o semi-au oma ic segmen- a ion [15–17] o s uc u es in he head. To educe he load o he 3D segmen a ion olume, e e y hi d slice was manually seg- men ed, and he es was calcula ed by linea in e pola ion be- ween manually segmen ed slices [17]. We con e ed he semi- manually segmen ed models in o polygonal meshes andimpo ed his in VG S udio MAX 2.2 so wa e (Volume G aphics GmbH, Heidelbe g, Ge many) (VG S udio MAX, RRID:SCR_017997) o 3D isualiza ions. Da a alida ion and quali y con ol By con as -enhanced X- ay mic oCT scan, we we e able o i- sualize he in e nal s uc u es o he P. anguinus head. Figu e 1 shows he manually segmen ed ca ilaginous chond oc anium,as well as he posi ion and he shape o he b ain, he emnan eyes, and he ol ac o y epi helium.A conside able po ion o he c anial skele on in he adul P. anguinus specimen emains ca ilaginous. A alida ion o he semi-au oma ic segmen a ion p ocedu e is p esen ed in Fig. 2. The 3D models we e c ea ed by an ope a o based on he g ey-scale alue con as and he shape o he s uc- u es. The de ailed p ocedu e is desc ibed in he Me hods sec ion and ollowed ou p e ious s udy [17]. In Fig. 3, we compa e he mic oCT da ase s and segmen a ions o he in e nal head s uc u es o he la al and adul specimens o bo h P. anguinus and A. mexicanum.The head o he oglobio ic P. anguinus is na owe and mo e elonga ed in compa ison wi h he epigean A. mexicanum, which li es in open su ace bodies o wa e bu no unde wa e ca es. Ou 3D segmen a ions show emnan eyes a la al and ju enile s age P. anguinus, bu no emnan o eyes was no iced in he adul specimen. The p og essi e degen- e a ion o he eye in he de elopmen o P. anguinus may lead o he appa en disappea ance o he eye in he adul animal [18]. By con as , 3D models o A. mexicanum clea ly show he eyes, o- ge he wi h an op ic ne e ha leads o he b ain, in bo h he la - al and adul s ages. Howe e , he absence o emnan eyes and op ic ne es in ou da a could be caused by low con as o hese s uc u es in mic oCT da a because he segmen a ion was done manually by he ope a o and based on hei g ey-scale alues and hei shape. Reuse po en ial Museum- ype documen a ion o a e and endange ed species The p esen ed da ase s gi e insigh no only in o he de elopmen- al biology o P. anguinus. Toge he wi h 3D da ase s o A. mexi- canum, hey a e impo an s udy ma e ials o he conse a ion e - o s o p ese e hese endange ed amphibians. Acco ding o he global assessmen o he In e na ional Union o Conse a ion o Na u e (IUCN), 43% o amphibian species a e in decline while 32% a e h ea ened wi h ex inc ion [19]; P. anguinus is cu en ly classi- ied as ulne able. The X- ay mic oCT me hod enables ana omi- cal s udies wi hou damaging he mo phology o he specimens and is he e o e excep ionally app op ia e o s udying endan- ge ed species wi h a limi ed amoun o a ailable specimens. Semi- au oma ically segmen ed images and he ex ac ed 3D models could also be aken as an inpu in o a machine lea ning algo- i hm. The ield o image p ocessing is becoming domina ed by deep lea ning algo i hms and con olu ional neu al ne wo ks [20]. C ea ing an online da abase could also be bene icial o s uden s udies and dis ance lea ning. Especially, las yea showed he impo ance o easy access o online s udy ma e ials because o Co id-19 es ic ions. Pe spec i es: Cellula esolu ion Using mic oCT scan wi h a con en ional X- ay sou ce, we ob- ained da a o excellen quali y ha depic single cells in he ca i- laginous elemen s (Fig. 4). Despi e he ac ha he cells can be i- sually de ec ed, hei au oma ic segmen a ion and quan i ica ion is challenging. The po en ial o X- ay mic oCT imaging wi h syn- ch o on sou ces o he s udy o 3D cell dis ibu ion was demon- s a ed in ou p e ious s udy on salamande limbs [12], and he po en ial o biomedical applica ions was shown be o e [21,22]. The da a wi h cellula esolu ion can be used as he inpu o he s udy o pola iza ion o cells in he ex acellula ma ix in sala- mande limbs o o ma hema ical modelling o join o ma ion [14]. Resea ch ou look The mo phology o he c anium ca ies impo an in o ma ion e- la ed o mechanics in ol ed in eeding, as well as compe i i e, e- p oduc i e, and an i-p eda o y beha iou . E en small di e ences in c anial skele on may ha e impo an biomechanical and eco- logical implica ions [23]. The mos de ailed desc ip ions o he P. anguinus skull a e hose o Doli o-Dob o olsky [24,25], I ano i´ c e al. [11], Papi e al. [12], and Bizjak Mali and Ske [26]. Mic oCT con as -enhanced da a included o e all 3D in o ma ion and a u he segmen a ion. Wo king on he p o ided da a can be aken as an inpu o a ious ype o analyses (Fig. 5). The isualiza- ion o muscles o he uppe and lowe jaw could be used o bi ing analyses ha may shed ligh on he p eda o y abili ies o P. anguinus. In gene al, ou da ase s will help o in es iga e how e olu ion- a y changes in he shape o he head and i s in eg a ed muscu- loskele al appa a us, ne ous sys em, and senso y o gans would help o oglomo phic adap a ion. Because P. anguinus is a ca e- dwelling animal, he c anio acial design unde wen a numbe o ca e- ela ed e olu iona y adap a ions, acqui ing p o ac ed and longi udinally elonga ed skele al elemen s, in compa ison wi h he epigean A. mexicanum, as e ealed by compa a i e mic oCT Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 4|GigaScience, 2022, Vol. 11, No. 1 Figu e 1: 3D econs uc ions o P. anguinus head based on X- ay mic oCT da a. La a ( op), ju enile (middle), and adul (bo om) P. anguinus. Images in he i s column show semi- anspa en 3D ende ings o he head wi h skin in do sal iew. Do sal, la e al, and on al iews o he segmen ed and colo -coded in e nal so s uc u es a e shown in he second o he ou h columns. Figu e 2: Accu acy alida ion o semi-au oma ic segmen a ions o X- ay mic oCT da a in ju enile P. anguinus. Raw CT image (le ) and he co esponding segmen ed image (middle) h ough he ans e se plane (g een) o he head o a ju enile P. anguinus ( igh ). scanning analysis and in-dep h 3D analysis. Indeed, when com- pa ed o P. anguinus,A. mexicanum exhibi s a wide skull wi h a massi e jaw. Elonga ed skull, body, and limbs a e common ea- u es o oglomo phism o o ganisms inhabi ing sub e anean habi a s [27,28]. Only 14 ca e-obliga e species a e known (all ple hodon id and p o eid salamande s), bu his numbe is likely o be an unde es ima e because o c yp ic species [29]. In his ega d, he esul s o ou s udy p o ide insigh in o e olu ion- a y ends in adap i e mo phological ai s as a esul o con e - gen e olu ion ac oss phyla. E en wi hou e olu iona y compa i- son ac oss phyla, ou esul s enable u he s udies on e olu ion o he skele al pa s in he adap i e landscapes indi idually o closely ela ed species. No only e olu iona y ques ions bu also de elopmen al pa - e ns can be ackled wi h hese da a. Fo ins ance, o in es i- ga e whe he he elonga ed skull shape de elops e enly wi h he g ow h o he animal, we in es iga ed he la al P. anguinus. The la a o P. anguinus, al hough small, e ealed i s minia u ized p o ac ed chond oc ania wi h simila spa ial p opo ions o he adul o m. Thus, he elonga ed body and s e ched c anium may indica e possible bene i s a any phase o de elopmen . Such com- pa a i e de elopmen al and g ow h- ela ed s udies open up new oppo uni ies o look in o he dynamics o skele al shape de el- opmen in ela ion o he speci ics o he en i onmen . Howe e , when i comes o he adap a ions in senso y o gans, he 3D analysis o he head e ealed majo di e ences in isual Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 Explo ing adap a ion o P o eus anguinus |5 Figu e 3: Compa ison o he head and in e nal so - issue ana omy o he ca e-dwelling P. anguinus wi h A. mexicanum in la al and adul specimens. Images in he i s ow show 3D ende ings o he head wi h skin in do sal iew. The second ow shows colo -coded segmen ed b ain, ca ilage, bones, and eyes wi h op ic ne e and ol ac o y epi helium. The hi d ow shows hese s uc u es wi hou bone and ca ilage o be e cla i y. Figu e 4: Images a nea cellula -le el esolu ion showing he ca ilaginous elemen s in ju enile P. anguinus ob ained by mic oCT. The whi e do s ep esen cell nuclei. 3D de ail o he ca ilaginous i s basib anchial elemen o he hyob anchial appa a us in en al iew (yellow; op ow) wi h3 o hogonal CT slices along he on al, sagi al, and ans e se planes (second ow). and ol ac o y sys ems o P. anguinus and A. mexicanum.Fi s ,P. an- guinus is blind, a ypical oglomo phic ai ha is explained by he oglobio ic way o li e. The eye de elopmen in P. anguinus la a begins as in o he amphibians. The eg ession o he almos no mally o med eyes s a s soon a e ha ching and g adually leads o a conside able educ ion o he eyes in adul P. anguinus [18], while su ace-dwelling salamande s de elop ully unc ional eyes. The mo phological compa isons o he P. anguinus de elop- men al s ages wi h hei g adual educ ion o eyes may imp o e ou knowledge on he mechanisms o eye degene a ion in P. angui- Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 6|GigaScience, 2022, Vol. 11, No. 1 Figu e 5: Po en ial use o con as -enhanced X- ay mic oCT da a. Segmen a ion o c anio acial muscles and ea laby in h in A. mexicanum and P. anguinus. nus and possibly o he simila blind ca e-dwelle s. Mo eo e , he eyeballs de elop in a complex conjunc ion wi h muscula appa- a us [30] and op ical ne e. He e, in P. anguinus, i is possible o ques ion how he induc ion and degene a ion o auxilia y issues is achie ed du ing he deg ada ion o he p e-shaped eyeballs. The animals wi h eye eg ession a e gene ally known o inhabi ca es; he e o e ision is nea ly useless o hem. The blind Mex- ican ca e ish (As yanax mexicanus) is one o he popula model sys ems o s udy he loss o ision and de elopmen al a es o he isual sys em [31]. Howe e , blind ish and o he model o - Downloaded om h ps://academic.oup.com/gigascience/a icle/doi/10.1093/gigascience/giac030/6562166 by VUT B no use on 06 May 2022 Explo ing adap a ion o P o eus anguinus |7 ganisms, including P. anguinus, s ill ha e a g ea po en ial o com- pa ison wi h o he eyeless e eb a es [32]. The e o e, ou esul s and da ase s may enable new insigh s in o e olu iona y ends in he eye de elopmen and degene a ion s a egies in ca e-dwelle s ac oss e eb a e clades. Ol ac ion plays an impo an ole in he li e o salamande s [8]. The analysis o 3D- ende ed ol ac o y o gans e ealed s ik- ing di e ences be ween he su ace-dwelling A. mexicanum and he ca e-dwelling P. anguinus. Elonga ed and ube-shaped ol ac- o y ca i ies in P. anguinus likely eme ge as ano he adap a ion o he ca e en i onmen ,whe e enhanced ol ac ion capabili ies pose an ad an age in he absence o isual signals [33]. Compa ing o A. mexicanum, he elonga ed ol ac o y ca i ies o P. anguinus migh enable a highe dynamic ange o sensi i i y, owing o a mo e e i- cien longi udinal di usion o signals upon en y ia he nos ils. In line wi h his, he ol ac o y ne es o P. anguinus a e also con- side ably elonga ed, which is explained by a a he long os al pa o he skull in P. anguinus. The e o e, s udies o e olu iona y di e gence o he ol ac o y sys em and i s sensi i i y and gene al design could bene i om he deposi ed da ase s. Finally, salamande s ep esen a well-es ablished model sys- em o he esea ch o egene a ion,and he undamen al p inci- ples o mul i- issue egene a ion ha e al eady been e ealed [34]. Regene a ion o P. anguinus has been desc ibed p e iously, ye he p o ided da a o e an impo an insigh in o he e olu iona y di - e ences in egene a ion among salamande s wi h undamen ally di e en li es yles. Da a A ailabili y The da ase s unde lying his a icle a e a ailable in he GigaScience Da abase eposi o y [35]. We p o ide econs uc ed slices as DI- COM image s acks and segmen ed s uc u es in STL o ma o he head egion o 3 specimens (1 la a, 1 ju enile, and 1 adul ) o P. anguinus and 2 specimens (1 la a and 1 adul ) o A. mexicanum. Fo segmen ed s uc u es, we also p o ide segmen ed masks as DICOM image s acks—1 s ack o each s uc u e. The olde s a e s uc u ed so ha each olde ep esen s 1 sample con aining a olde wi h DICOM s ack, a olde wi h STL iles, and a olde wi h segmen ed masks. The DICOM image s acks can be opened in any image iewe suppo ing his o ma ; we ecommend ImageJ o iewing he da a [36]. To explo e da ase s in 3 dimensions, spe- cialized ee iewe s a e a ailable—D ish i [37], D agonFly (Objec Resea ch Sys ems [ORS], Inc., Mon eal, QC, Canada), o o he s. Fo analysis and u he segmen a ion, we ecommend he use o ITK-SNAP [38] o comme cial so wa e, e.g., A izo o VG S udio MAX. A de ailed desc ip ion and a manual o segmen a ion o bi- ological da a can be ound in ou p e ious wo ks [17,39]. The STL iles can be also explo ed in 3D mesh iewe s: popula ee open- sou ce so wa e, e.g., MeshLab [40] o Blende [41], as well as in he ske ch ab eposi o y [42]. Abb e ia ions DICOM: Digi al Imaging and Communica ions in Medicine; mi- c oCT: X- ay compu ed mic o omog aphy; PTA: phospho ungs ic acid; STL: S anda d T iangle Language. Compe ing In e es s The au ho s decla e ha hey ha e no compe ing in e es s. Funding We acknowledge CzechNanoLab Resea ch In as uc u e sup- po ed by MEYS CR (LM2018110). M.T. acknowledges g an CEITEC VUT-J-21-7145, he B no Ci y Municipali y as a B no Ph.D. Tal- en Schola ship Holde and Ma ina Roeselo a Memo ial Fellow- ship. J.G. was unded he Fonds We enschappelijk Onde zoek– Vlaande en by a pos doc o al ellowship (G an No. 12R5118N). Au ho s’ Con ibu ions M.T.: W i ing—o iginal d a , Visualiza ion; L.M.: Concep ualiza- ion, Me hodology; E.M.: In es iga ion, W i ing—o iginal d a ; G.A.: In es iga ion, W i ing—o iginal d a ; M.N.A.: In es iga ion, W i ing—o iginal d a ; R.K.: Valida ion, W i ing— e iew & edi - ing; L.B-M.: Valida ion; T.Z.: P ojec adminis a ion; M.K.: Me hod- ology; W i ing— e iew & edi ing; F.P.: Me hodology; J.G.: Da a cu a ion, W i ing— e iew & edi ing; A.B.: Da a cu a ion; A.H.: P ojec adminis a ion; I.A.: Concep ualiza ion, P ojec adminis- a ion; J.K.: Funding acquisi ion, Supe ision. 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