A qualitative and quantitative stereological study of the microscopic morphology of the nervous ganglia of the bivalve peppery furrow shell (Scrobicularia plana)

A QUALITATIVE AND QUANTITATIVE STEREOLOGICAL STUDY OF THE MICROSCOPIC MORPHOLOGY OF THE NERVOUS GANGLIA OF THE BIVALVE PEPPERY FURROW SHELL (SCROBICULARIA PLANA) SUKANLAYA TANTIWISAWARUJI TESE DE DOUTORAMENTO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM CIÊNCIAS BIOMÉDICAS D 2015 SUKANLAYA TANTIWISAWARUJI A quali a i e and quan i a i e s e eological s udy o he mic oscopic mo phology o he ne ous ganglia o he bi al e peppe y u ow shell (Sc obicula ia plana) Tese de candida u a ao g au de Dou o em Ciências Biomédicas subme ida ao Ins i u o de Ciências Biomédicas Abel Salaza da Uni e sidade do Po o. O ien ado – Edua do Jo ge Sousa da Rocha Ca ego ia – P o esso Ca ed á ico A iliação – Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o, Po ugal Coo ien ado : U haiwan Ko i adhi Ca ego ia – P o esso Associado A iliação – Depa men o Zoology, Facul y o Science, Kase sa Uni e si y, Bangkok, Thailand Coo ien ado : Ma ia João Tomé da Rocha Ca ego ia – P o esso Auxilia A iliação – Ins i u o de Ciências Biomédicas Abel Salaza , Uni e sidade do Po o, Po ugal - I - OBJECTIVES The mic oscopic mo phology o bi al es is s ill poo ly s udied, pa icula ly in quan i a i e e ms, and ha o peppe y u ow shell (Sc obicula ia plana) is no excep ion. Wi h his scena io in mind, he gene al aim o he Thesis was o en ich he knowledge o he bi al es’ cen al ne ous sys em, e iewing he s a e o a and un eiling new quali a i e and quan i a i e cy ological and his ological da a in S. plana, namely using echnical app oaches ha we e ei he ne e o seldom used in he neu oscience o bi al es. Based on hese b oad objec i es, he speci ic aims we e: To be e unde s and he mic oscopic ana omy o he cen al ne ous sys em, by conduc ing an unp eceden ed h ee-dimensional (3D) compu e assis ed econs uc ion o S. plana ganglia, while es ima ing o he i s ime hei o al olumes and su ace a eas in he 3D models, and he ela i e olumes o co ex and medulla. Wi hin his scope, we wan ed o s a s udying whe he sexual di e ences exis ega ding ganglion size and i s in e nal composi ion, in iew o he key oles o he neu osec e o y neu ons in go e ning gonadal ma u a ion, pa icula ly in emales. To examine he gene al his ology and cy ology o he neu al cells in S. plana, egis e ing i s main aspec s and looking a e any s ill undesc ibed ea u es, while ying a i s iden i ica ion o po en ially neu osec e o y neu ons — iz. hose pu a i ely p oducing se o onin and dopamine — compa a i ely ac oss he ce eb al, pedal and isce al ganglia, and conside ing he gonadic sex. To s a s udying, wi h design-based s e eological me hods, heo ized s uc u al di e ences be ween he a ious ne ous ganglia ypes, speci ically connec ed wi h in insic ea u es o hose elemen s, like hei di e en body loca ions and dis inc unc ions, and in iew o he gende and gonadal ma u a ion s aging (compa ing adul s wi h ma u ing s. spen gonads). The expec able neu al cell a ge s would be hose iden i ied by he p e ious his ological and ul as uc u al s udy. To wo k u he on he possible e ec s o he animal’s gende in he s uc u e o he cen al ne ous sys em, and, in pa allel, o s a explo ing he hypo hesis ha i adul bi al es, including S. plana, con inue o g ow du ing i s li espan, hen i s ne ous ganglia may con inue o de elop wi h age — a leas un il senescence, i i happens — and e en ually gains in cellula i y and o he mo pho unc ional changes plausible occu . Implemen ing design-based s e eological s a egies, one aim was hus o sea ch o changes in ganglion size and in numbe o dis inc neu al elemen s. - II - To make he i s expe imen al assay o s a es ing he hypo hesis ha i bo h he ne ous ganglia signalling and oes ogens play a modelling ole in he bi al es’ gonadal ma u a ion and spawning — and all indica es ha such impac s a e no equal ways in emales and males — hen wa e bo ne exposu e o xenoes ogens can cause mo pho unc ional impac s in he cen al ne ous sys em. This aim makes e en mo e sense i he wo ks p oposed o o he goals gene a e e idences suppo ing he hypo hesis ha sex may shape he s uc u e o he S. plana cen al ne ous sys em. Pu suing he ci ed goals will expand he knowledge o he bi al es’ ne ous sys em, in S. plana especially, and has po en ial o in oducing his species as a aluable model in neu oscience. - III - ABSTRACT This esea ch was ca ied ou on he bi al e peppe y u ow shell, Sc obicula ia plana, which al eady well- ecognized as one o he impo an species in en i onmen al moni o ing and o he ypes o esea ches. S. plana has i s na u al habi a in he in e idal so sedimen along he A lan ic coas om Eu ope o A ica. I s ne ous sys em is poo ly s udied, and hus we aimed he e o imp o e such lack o knowledge. A quali a i e s udy was done o examine he gene al his ology o he ne ous ganglia, including he neu al cell ypes a bo h ligh and elec on mic oscopy. Fo ligh mic oscopy, animals we e measu ed, anes he ized, dissec ed and ixed in 10% bu e ed o malin. They we e ou inely p ocessed o pa a in embedding, and sec ioned o a ied pu poses along he sagi al plane, using a ully mo o ized mic o ome. An immunohis ochemical su ey was also made o iden i ying neu ons ha could con ain se o onin and dopamine, as a i s s ep o ga he he knowledge abou he p esence and ole o neu oendoc ine neu ons in S. plana. Fo ansmission elec on mic oscopy, dissec ed ganglia we e ixed in 2.5% glu a aldehyde, pos - ixed in 1% osmium e oxide, all bu e ed, and ou inely p ocessed o epoxy embedding. As o se o one gic and dopamine gic cell bodies and neu i es, hey we e iden i ied in all he ganglia, in adul s o bo h sexes and in imma u e animals (wi h unde ined sex). Bo h quan i a i e and quali a i e me hods we e conduc ed o s udy h ee dimensional (3D) ea u es o he ganglia, aking he 3D models o ind ou di e ences be ween ganglia ypes and i he biome ic pa ame e s can be co ela ed wi h he ganglionic olumes and su ace a eas, which was he case o some pa ame e s. S e eology was applied la e one, using he Ca alie i’s p inciple o es ima ing he ganglion olume and he op ical disec o - ac iona o me hod o es ima ing cellula i y (numbe s o neu ons, glia and pigmen ed cells); namely in es iga ing he in luence o he sex and gonad ma u a ion on he quan i a i e s uc u al pa ame e s. The s a is ical app oach elied on mul i-way analysis o a iance. In summa y, he main indings we e as ollows: (1) 3D- econs uc ion shows ha each ype o ganglia has a peculia 3D-shape, and da a sugges a sligh le - igh asymme y as o he ce eb al ganglia shape. Rega ding o al su aces, co ela ions exis o he ce eb al and isce al ganglia, bu i is he isce al ha consis en ly shows s ong posi i e co ela ions wi h each biome ic pa ame e . Despi e he di e ences in olume/su ace among ganglia, he olume a io o co ex s. medulla is ai ly s able (≈ 1.5), sugges ing a unc ional op imum. In his i s app oach i seemed ha no majo di e ences exis be ween sexes; (2) His ological analysis using ligh and ansmission elec on mic oscopic analysis shows ha each ganglion has pe ineu ium (ou e mos laye ), ou e co ex, and inne medulla. The neu ons (smalle o - IV - la ge ) a e ypically unipola , gliocy es a e elonga ed, oundish o iangula , and he e a e pigmen ed cells. Gene ally, glial cells a e much smalle han neu ons, ha ing highe nucleus o cy oplasm a io; (3) Unbiased s e eology analyses we e conduc ed in h ee s udies. Fi s ly, o in es iga ing e en ual in luences o he sex and gonadal ma u a ion s a us o animals ha did no di e in size. Quan i a i e pa ame e s we e es ima ed in he ne ous ganglia and hei cells, in males, emales, and undi e en ia ed specimens. O e all, he e was a endency o he ganglionic olume o be g ea e in emales, ollowed by males, and undi e en ia ed animals. As o he ype o ganglia, he wo ce eb al ones a e simila in size, bu he olumes inc eased signi ican ly owa ds he pedal ganglia, which is g ea e han he ce eb al and much smalle han he isce al. The size di e ences be ween all ganglia ypes a e independen o he gende and o he gonad ma u a ion s a us a he ime. As o he ela i e olumes (VV) o he co ex and medulla, he co ex is ≈ 60% and he medulla ≈ 40% o he all ganglia. As o he numbe neu al cells, he e we e no signi ican di e ences among gende , bu signi ican di e ence we e ound among ganglia ypes. The isce al ganglion has he highes numbe o cells (≈ 68000) and he ce eb al ganglia ha e he lowes (≈ 12000). A second s e eological s udy was on hypo hesize impac s o age on he ne ous ganglia in ma u e males s. emales. Conside ing ha size is a p oxy o age, he animals we e spli in o wo-size classes, ha we named “Small” (age: 2+ yea s) and “Big” (age: 3+ yea s). We disclosed in e ganglionic, sex- ela ed and size- ela ed signi ican e ec s upon he ganglionic olumes, ela i e olumes o co ex and medulla, and o al numbe s o neu ons, glial cells, and pigmen ed cells. The e ec o size (age) was consis en ly ma ked, and s a is ically signi ican , wi h he olde specimens ha ing app oxima ely wice as bigge ganglia ( ega dless o i s ype and o he animals’ sex), ha con ained signi ican ly mo e neu al cells o all ca ego ies. The inc ease in cellula i y ook place i conside ing he en i e ganglia, o he co ex and medulla sepa a ely. Da a suppo ou hypo hesis ha neu ogenesis con inues o occu in adul S. plana, i espec i e o he animals’ sex. In his ein, S. plana can become a s imula ing model o neu ogenesis and age- ela ed s udies. Las ly, in iew ha ne ous sys em signalling and sex-s e oids bo h in luence bi al e ep oduc ion, acu e exposu e o e hinyles adiol (EE2) in wa e a wo nominal concen a ions (0.05 and 5 µg/L) was se up, o s a de e mining whe he o no endoc ine modula ion and/o dis up ion o he ne ous sys em occu s in S. plana. E en hough ou p elimina y da a did no e eal signi ican impac s, ei he in he ganglion olume o in cellula i y, he s udy se ed as a “kick o ” o u he es s, o ins ance using longe exposu es, selec ing o he o ganic a ge s, and expanding he echnical po olio, iz. ia s e eological es ima o s o cell olume changes. - V - RESUMO Es e es udo usou como modelo o bi al e lambujinha, Sc obicula ia plana, econhecida como uma espécie impo an e em moni o ização ambien al e em ou os ipos de pesquisas. A S. plana em como habi a na u al o sedimen o macio in e idal, ao longo da cos a a lân ica da Eu opa e a é ao no e de Á ica. O seu sis ema ne oso é pouco es udado e, assim, p ocu ou- se nes a Tese con ibui pa a mino a ais alhas de conhecimen o. Foi execu ado um es udo quali a i o sob e a his ologia ge al dos gânglios ne osos, incluindo sob e os ipos de células neu ais, an o em mic oscopia de campo cla o como mic oscopia ele ónica de ansmissão. Pa a a p imei a, os animais o am medidos, anes esiados, dissecados e ixados em o malina amponada a 10%. As peças o am p ocessadas de o ma o inei a pa a inclusão em pa a ina, co ando-se o animal segundo o plano sagi al, u ilizando-se mic ó omos mo o izados. En e ou os, ez-se um es udo imuno-his oquímico pa a se iden i ica em neu ónios que pudessem possui se o onina e dopamina, como um p imei o passo pa a aumen a o conhecimen o sob e a p esença e papel de neu ónios neu oendóc inos em S. plana. Pa a mic oscopia ele ónica, gânglios isolados o am ixados em glu a aldeído a 2,5%, pós- ixados em e óxido de ósmio 1%, ambos amponados, e p ocessados o inei amen e pa a inclusão em esina epóxi. Quan o aos soma a e neu i es se o oniné gicos e dopaminé gicos, eles o am iden i icados em odos os gânglios, em adul os de ambos os sexos e em animais ima u os (i.e., com sexo inde inido). Me odologias quali a i as e quan i a i as pe mi i am es uda ca ac e ís icas idimensionais (3D) dos gânglios, endo os modelos 3D pe mi ido elucida di e enças en e os á ios ipos de gânglios e se os pa âme os biomé icos e am co elacioná eis com os olumes gangliona es e á eas de supe ície; o que oi o caso pa a alguns pa âme os. Depois, oi usada es e eologia, a a és do p incípio de Ca alie i, pa a es ima o olume gangliona , e do mé odo “op ical disec o - ac iona o ” pa a es ima a celula idade (núme os de neu ónios, de células gliais e de pigmen adas); es udando-se a in luência do sexo e da ma u ação da gónada nos pa âme os es u u ais quan i a i os. A abo dagem es a ís ica baseou-se em análise de a iância múl ipla. Em esumo, os p incipais esul ados o am os seguin es: (1) a econs ução 3D mos ou que cada ipo de gânglio em o mas pa icula es e que os dados suge em uma ligei a assime ia esque da-di ei a na o ma dos gânglios ce eb ais. Em elação a á eas de supe ície, exis em co elações pa a gânglios ce eb al e isce al, mas é es e que mos a consis en emen e o es co elações, posi i as, com cada pa âme o biomé ico do animal. Apesa das di e enças de olume/supe ície en e gânglios, a p opo ção olume do có ex s. medula é bas an e es á el (≈ 1,5), suge indo um ó imo uncional. Nes a p imei a abo dagem, não su gi am di e enças assinalá eis en e sexos; (2) A análise his ológica e ul aes u u al mos ou que cada gânglio em pe ineu o (um in óluc o), có ex (ex e no) e medula (in e na). Há neu ónios (pequenos - XII - Labo a o y o His ology and Emb yology (a ICBAS) and o he His omo phology, Physiopa hology and Applied Toxicology G oup (a CIIMAR), o hei assis ance. I owe a special no e o g a i ude o P o esso Alexand e Lobo da Cunha (namely as supe iso o he Elec on Mic oscopy Uni , ICBAS), Elsa Oli ei a, Ângela Al es and Sónia Rocha o hei assis ance and sha ing hei expe ise in elec on mic oscopic echniques. I ha e sha ed he good and ha d imes wi h many Thai iends in Po ugal. Thank you o being wi h me o p ese e bo h o hose imes o ou li es, especially D . Ouaypo n Tang honchai, D . Po nchai Julama and D . Chommana d Sumnge n. I would especially like o hank my good iends D . Piya hip Tinnawo n, D . Su ade Bu a-chon, and D . Ra chanee Tang-on, no only o hei willingness o help and sha e hei pe sonal expe iences wi h me bu also o hei emo ional suppo which is e y much app ecia ed. I uly app ecia e o he suppo om he g ea iends D . Su hee Ploisawa chai, D . Banya Lekp ase , Rung i a Kapol and P aphani Suang ho. Also, my e y special hanks go o Pa a eeya Yawanopas Guima ães, and he husband, Emanuel Guima ães o hei ines imable help o he whole ime o my s ay in Po ugal. I is an hono o me o exp ess my g a i ude o P o esso Anake Kijjoa and P o esso Madalena Pin o o hei iendship, guidance and uncondi ional suppo . To he wo king eam o Chemis y Labo a o y (ICBAS), Júlia Bessa, Sónia San os, Isabel Sil a, and s uden s who always showed he in e es , kind conce n and a ailabili y o suppo . I would especially like o hank e e yone no men ioned he e who, in one way o ano he , always ga e me assis ance and iendship owa ds my s udies. Las bu no leas , wo d o dedica ion goes o my pa en s, all my amily membe s, uly g ea iends, as well as my belo ed eache s, who ha e been by my side, no ma e how a hey a e. Thei emo ional suppo , encou agemen , and endless lo e s and by me h ough e e y momen o my li e and ha is he ma elous suppo allowing my accomplishmen s. - XIII - CONTENTS OBJECTIVES………………………………………………………………....…. I ABSTRACT…………………………………………………………………....…. III RESUMO…………………………………………………………………………. V AUTHOR STATEMENT……………………………………………................... VII DEDICATION OF THE THESIS.…………………………………………........... IX ACKNOWLEDGEMENTS.……………………………………………...………. XI Chap e 1 In oduc ion — The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion............................................... 3 Chap e 2 The Peppe y Fu ow Shell (Sc obicula ia Plana (da Cos a, 1778)) — An O e iew ……………………………………………...….... 21 Chap e 3 Quali a i e And Quan i a i e Insigh s In o The 3D-Mic oana omy O The Ne ous Ganglia O The Peppe y Fu ow Shell Sc obicula ia Plana (Bi al ia, Tellinoidea, Semelidae)………...... 41 Chap e 4 O e iew O The Neu ocy ology O Ganglia And Iden i ica ion O Pu a i e Se o onin- and Dopamine-sec e ing Neu ons In he Bi al e Peppe y Fu ow Shell ( Sc obicula ia Plana )…….………... 65 Chap e 5 A S e eological S udy O The Volumes And Cellula i y O he Ne ous Ganglia O Males, Females And Undi e en ia ed Adul s O The Peppe y Fu ow Shell (Sc obicula ia Plana)....…………... 95 Chap e 6 Impac s O Age In The Ne ous Ganglia Volume And Cellula i y In Two Adul Size-Classes O The Bi al e Peppe y Fu ow Shell Sc obicula ia Plana………………..……………………………… 131 - XIV - Chap e 7 Do Es ogens In luence The Bi al e Ne ous Ganglia Size And Cellula i y? A S udy On The Pedal Ganglia O The Peppe y Fu ow Shell Sc obicula ia Plana Acu ely Exposed To E hinyles adiol…………………..……………………………...… 169 Chap e 8 Final Rema ks………………………...…………………...………. 189 CHAPTER1 INTRODUCTION — THE BIVALVE NERVOUS SYSTEM AND ITS RELEVANCE FOR THE PHYSIOLOGY OF REPRODUCTION 227 Volume 3 Numbe 4, Oc obe - Decembe 2014 Indian Jou nal o Ana omy Volume 3 Numbe 4, Oc obe - Decembe 2014Re iew A icle Au ho ’s A ilia ion: *PhD S uden , King Mongku ’s Uni e si y o Technology Thonbu i (KMUTT), Bangkok 10140, Thailand. Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), 4050-313, Po o, Po ugal. CIIMAR/CIMAR - In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, U.Po o, 4050-123, Po o, Po ugal., **Full P o esso , Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), 4050-313, Po o, Po ugal. CIIMAR/CIMAR - In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, U.Po o, 4050-123, Po o, Po ugal., ***Associa e P o esso , Depa men o Zoology, Facul y o Science, Kase sa Uni e si y, Bangkok 10900, Thailand., ****Associa e P o esso , Supe io Ins i u e o Heal h Sciences- No h (ISCS-N), 4585-116 Gand a-Pa edes, Po ugal. Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), 4050-313, Po o, Po ugal. CIIMAR/CIMAR - In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, U.Po o, 4050-123, Po o, Po ugal. Co esponding Au ho : Edua do Rocha, Full P o esso , Labo a o y o His ology and Emb yology, Depa men o Mic oscopy, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), Rua de Jo ge Vi e bo Fe ei a n.º 228, 4050-313 Po o, Po ugal. E-mail: e [email protected] The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion Sukanlaya Tan iwisawa uji*, Edua do Rocha**, U haiwan Ko i adhi***, Ma ia J. Rocha**** Abs ac Bi al es a e widesp ead in e eb a es ha a e mos ly ma ine and ben hic, wi h g ea impac s in he aqua ic sys ems ood chains. Thei so body is la e ally comp essed and co e ed wi h a ha d shell, o en ha ing bila e al symme y. S ong adduc o muscles help in he shell mo emen . Va ious species a e used as bioindica o s o en i onmen al quali y. Many, such as mussels, clams, scallops, o oys e s, a e hea ily ha es ed/ ea ed o human consump ion. Bi al es a ailabili y, adap abili y and simple ana omy make hem a ac i e o bo h undamen al and applied esea ch. One pa icula a ge o such s udies is he ne ous sys em. I is ypically made o a cen al ne ous sys em holding h ee ypes o ganglia (ce eb al, pedal, isce al), o ganized in o an ou e neu on- and glia- ich co ex and an inne axon- ich medulla. Ne es in e connec he ganglia as well as hese and pe iphe al ne ous sys em componen s, made o senso ial s uc u es such as eyes (man le, en acles), and osph adia (gills) and s a ocys s ( oo ); They a e in ol ed in pho o ecep ion o a e mechano o chemo ecep o s. Among o he oles, he ne ous sys em go e ns ep oduc ion, ia in luences in he sexual de elopmen , game ogenesis, e iliza ion and spawning. Such modelling is ia neu o ansmi e s and neu oho mones, in e playing wi h di ec / indi ec impac s o bio ic (eg, ood abundance) and abio ic (eg, empe a u e, pH, salini y) ac o s. We know now ha many pollu an s can dis up he ne ous sys em and gonads and hei poo ly known in e ac ion. Knowing he ne ous sys em unc ional mo phology is c i ical o unde s and such dis up ions and o eseen ep oduc i e consequences. Acco dingly, his wo k o e s a sys ema ic o e iew abou he bi al e ne ous sys em and ela ed ep oduc i e e en s. Keywo ds: Ana omy; His ology; Bi al es; Ne ous sys em; Ganglia; Neu ons; Glial cells; Neu ocy ology; Neu ophysiology; Rep oduc ion. In oduc ion In almos all me azoans, he coo dina ion is accomplished by wo main mechanisms, ho mones and ne ous sys em signals. These wo cen al sys ems in e ac wi h each o he o main ain he homeos asis o animals and o espond app op ia e in o ma ion o he en i onmen al s imulus [1-2]. In addi ion o hese basic i al unc ions, he ne ous sys ems o highe o ganisms a e able o pe cei e and eac o a g ea e ange o en i onmen al s imuli in in ica e and a ied way including esponsible o eeling, hinking, and lea ning [3]. In e eb a es he e a e mo e complica ed componen s o he ne ous sys em. Ana omically, he e a e wo sys ems: he cen al ne ous sys em (CNS) and he pe iphe al ne ous sys em (PNS). CNS consis s o b ain and spinal co d. The PNS comp ises he soma ic and Chap e 1 | The mo phology o he bi al es ne ous sys em - 3 - 228 Indian Jou nal o Ana omy Edua do Rocha e . al. / The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion. au onomic ne ous sys ems. Soma ic a e en s ca y senso y in o ma ion om he skin, muscle, and join s o he CNS, while mo o e e en ne es inne a e skele al muscle o cause he mo emen con ac ion. [4] The au onomic ne ous sys em can be hough o as a mo o sys em o isce al o gans, because i p ojec s o hese o gans o inne a e and con ol he unc ion o smoo h muscle, ca diac muscle, endoc ine, and exoc ine glands. The au onomic ne ous sys em is ypically u he di ided ana omically and unc ionally in o he sympa he ic and pa asympa he ic subdi isions. [2, 4, 5] In lowe in e eb a es o he animal kingdom, like Coelen e a es o Cnida ians, he ne ous sys em consis o specialized ne e cells o ec ode m called ne e ne ha consis s o senso y and muscle cells di usely dis ibu ed. [6, 7] The mos highly e ol ed g oups, like la wo ms, show he i s eal CNS because hei senso y cells a e g ouped in o special ana omical collec ion o ming a ne e ing o ganglia o ganized in he bila e ally symme ical longi udinal body axis as ne e co ds. Thei ganglia can assume a segmen -like s uc u e as a esul o he mo e o less egula a ay o c oss-connec ions inne a ing he whole body. [8] In he head egion, he e a e specialized s uc u es, such as p imi i e “eye o ocelli”. These s uc u es a e also be ound in annelids, in which in he an e io end he e is a dis inc b ain and segmen ed body plan, wi h ganglia o ganized in o a ladde -like chain in each segmen . [9] The do sal b ain is connec ed o he en al chain o segmen al ganglia ia ci cumesophageal connec i es. Each segmen al ganglion, which ypically is said o consis o abou 1000 neu ons, is o ganized in a bila e ally symme ical way. Bo h hal es a e linked o one ano he by commissu es and o neighbo ing ganglia by connec i es. Pe iphe al ne es, ypically h ee pai s, p ojec s om each ganglion and inne a e he segmen al body wall. [10] All ganglia ha e a s uc u e which is cha ac e is ic o highe in e eb a es; neu ons wi hin co ex and he neu onal p ocesses (dend i es and axons) lie in a neu opil in he co e o he ganglion. In some annelids, dis inc i e gian neu ons occu , and hese play an impo an ole in as escape esponses. [11] In some species he s uc u e o op ic ganglia is o med. [12, 13] In a h opods, howe e , he body o ganiza ion is di e en om ha o annelids wi h a icula ed appendages and he usion o o iginally uni a y, me ame ic segmen s in o he unc ional en i ies comp ising he head, ho ax, and abdomen. Fo such insec s and c us aceans, hei head egion ends o o m a complex b ain consis ing o ex ensi ely used ce eb al ganglia. These a e o en associa ed wi h he p ocessing o in o ma ion om specialized senso y o gans, o example, a p o oce eb um o insec s, which ecei es isual senso y inpu om bo h compound eyes and om he simple ocelli, a deu oce eb um which ecei es senso y inpu om he an ennae, and a i oce eb um which ecei es inpu om he head su ace. [14] These b ain s uc u es oge he con ain abou 90% o he neu ons in he cen al ne ous sys em, which in he la ge c us aceans sum abou one million ne e cells. In he highe a h opods, he e a e b ain egions which consis o associa i e neu opil cen es, cell body egions, and agg ega es o neu osec e o y cells. [15, 16] The equi emen o accu a e mo o con ol o he a icula ed body appendages, especially he ho acic legs, has led o an inc easing specializa ion o he en al segmen al ganglia. [17] The e a e he ho acic ganglia ypically con aining mo e in e neu ons, e e en p ojec ing mo o neu ons and a e en senso y ibe s han he abdominal ganglion. The la e , o en associa ed wi h specialized s uc u es, a e loca ed in he pos e io end o he animal. In addi ion, he e is a endency owa d usion o he segmen al ganglia in o ewe (in some cases single) ganglia. [18] One such ganglion, he subesophageal, i is o med by se e al ganglia and con ols he mou hpa s – his is gene ally ound enclosed in he head capsule. [19] The segmen al specializa ions o he a h opod ne ous sys em allow complex mo o ac i i y o be gene a ed. This includes lying, unning, jumping, manipula ion, and sound p oduc ion. [12, 18] In molluscs, he e a e a ia ions in he o ganiza ion o he ne ous sys em. In o de o ge sensa ions hey ha e a collec ion o neu ons in he en al co d which a e called ganglia. The basic o ganiza ion o hei CNS comp ises abou i e pai s o ganglia which a e a anged a ound he gu , no mally nea he head, and a e linked o one ano he by connec i es and commissu es. I is possible o dis inguish ce eb al, buccal, pleu al, pedal, and abdominal ganglia. [13, 20] The basic o ganiza ional plan can a y signi ican ly among indi idual molluscan species, o he ex en ha he a ious ganglia can change hei posi ion and e en use wi h one ano he . [12, 13, 20] In Gas opoda, Scaphopoda, Polyplacopho a, Monoplacopho a (slow-mo ing animals) and Cephalopoda (ac i e p eda o y li es yle), he e is cephalisa ion. [7, 11, 21] The e a e nume ous s udies on he ne ous sys em o he gas opod mollusc Aphysia cali o nica, which is an animal model o he neu obiologis s’ s udy o beha iou s, namely lea ning and memo y. [22, 23] In bi al es, such as, clams, mussels, and scallops, he e is bila e al symme y and so body. They ha e an in e es ing simple model o CNS, ecognized as Chap e 1 | The mo phology o he bi al es ne ous sys em - 4 - 229 Volume 3 Numbe 4, Oc obe - Decembe 2014 e y use ul o s udies anging om neu obiochemis y o neu ophysiology. [7, 24, 25] Howe e , li le seems o be known abou he de ailed ana omy o componen s in he bi al e ne ous sys em. This Chap e e iews wo majo pa s in bi al es. The i s conce ns he s uc u e and unc ion o ne ous sys em. The second desc ibes he neu al con ol o ep oduc ion. Gene al mo phology and unc ions o ne ous sys em in bi al es Knowledge abou he mo phology and unc ioning o cen al ne ous sys em in bi al es is s ill somewha sca ce and needing u he s udy. The easons o his limi a ion a e a ied. The his ology, hough s udied o a ce ain ex en , is di e en om ha o e eb a es; mos o he a ailable o ms a e small, and he ew expe imen al wo k has been pe o med using me hods ui ul in e eb a es bu , possibly, inadequa e o insu icien o bi al es. A g ea ea u e o he bi al e ne ous sys em is he small numbe o neu onal elemen s wi hin ganglia and ha con ibu e o he pe iphe al issue. [20, 26] This makes possible a ype o analysis ha is di icul o achie e in e eb a es. Also, in e es ing di ec co ela ions be ween he size o he ganglia and hei unc ion can be disclosed in bi al es. The cen al ne ous sys em Ana omy o he ganglia The basic plan o o ganiza ion o all bi al es ne ous sys em is bila e ally symme ic which each hal body segmen possessing a ganglion. In ypical bi al es, hey consis o h ee pai s o ganglia: ce eb opleu al (commonly called as ce eb al), isce al and pedal; along wi h wo pai s o long ne e co ds. Bo h ce eb al ganglia a e in e connec ed o isce al and pedal ganglia by bila e ally unning ne e co ds. Each ganglion gi es ise o ne e ibe s ha supply he o gans and issues in close p oximi y. [24, 26, 28] Fo ins ance, he ce eb al ganglia inne a e labial palps, an e io adduc o muscle, an e io pa o he man le, and senso y o gans, including s a ocys s (equilib ium o gans) and osph adia (a chemo- mechanical sense o gan). [29] The isce al ganglion inne a es he gills, hea , pos e io adduc o muscle, pos e io pa o he man le, siphons, and senso y o gans in he man le. [30] As in o he bi al e species, he isce al ganglion o Venus e ucosa comes om he usion o wo o iginal ganglia, hus showing bila e al symme y; pai s o symme ical ne es eme ge om each pole and di e ge. Las ly, he pedal ganglion, as he name indica es, inne a es he oo . [28, 31, 32] Ce eb al ganglia In mos bi al es, he pai ed ce eb al ganglia a e well sepa a ed om each o he (le and igh ) and hey a e usually iangula in shape, wi h he colo a ying om milky whi e o b igh ed. These ganglia a e si ua ed be ween he base o he labial palps and he i s esophageal subdi ision o he diges i e ac , being sho ly c oss-connec ed by a commissu e a ching o e he esophagus, as well as, longi udinal linked be ween pedal and isce al by connec i es. In eali y, hey a e o med by usion o he ce eb al and pleu al ganglia a ound he an e io pa , and ha is why hey a e commonly e e ed as ce eb opleu al ganglia o ce eb al ganglia in he li e a u e [20, 32]; and he ein we shall use he la e e m hence o h o consis ency. F om each ce eb al ganglion no only he p incipal wo pai o ne es co ds lead owa d he pos e io o he animal: one, ce eb o-pedal connec i es ha ex end pos e io and en ally o he pedal ganglia in he oo ; ano he , ce eb o- isce al connec i es, unning di ec ly back om he ce eb al ganglia o he isce al ganglion, which is loca ed on he su ace o he pos e io adduc o muscle. Bu , he e a e also he pallial ne es inne a ing he labial palp, an e io adduc o muscle, gill [33], and pa o man le ma gin, as well as he s a ocys s and osph adia. In he absence o cephalic sense o gans he ce eb al ganglia a e weakly de eloped and small. [33]In snails, he cen al ganglia a e mo e concen a ed and he isce al loop is so sho ha all o he p incipal ganglia a e in he an e io ne e ing abo e he esophagus. [20, 21, 34] Pedal ganglia In gene al bi al es, he pedal ganglia is posi ioned below he esophagus and is an e io o he base o he oo . They ha e he same ype o colo a ion bu a e la ge han he ce eb al ganglia and mo e ounded in appea ance. The pedal commissu es a e a e; in mos o ms he igh and le ganglia ha e me oge he in he middle line. Each ganglia ex end he ollowing ne es: 1) he pedal ne e, which inne a es he oo , o igina es om he en al pos e io su ace; 2) in genus My ilus, he en al byssus e ac o ne e, inne a ing he byssus o gan Edua do Rocha e . al. / The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion. Chap e 1 | The mo phology o he bi al es ne ous sys em - 5 - 230 Indian Jou nal o Ana omy ©Red Flowe Publica ion P .L d and muscle and a ising om he pos e io en al side o he ganglion; 3) he do sal byssus e ac o ne e, which also inne a es he uppe byssus muscles a ise om he pos e io do sal o he ganglion. In C assos ea i ginica, he e a e as well no pedal ganglia in line wi h he lack o a oo o mo ing. [24, 32, 35] Visce al ganglia In ypical bi al es he isce al ganglion is he la ges ganglia, being de i ed om he usion o wo o iginal ganglia. Visce al ganglia ei he appea as “ ounded iangles” o else ha ing mul iple lobules, wi h milky whi e o b igh ed in colou a he en al end o he isce al mass, on he an e o en al bo de nex o adduc o muscle. The isce al ganglia a e much la ge han he ce eb al and ne es emana ing om i inne a e he man le, gills, in es ine, anus, skin, pos e io pa o he geni al appa a us, kidney, he main diges i e gland and pos e io adduc o muscle. [32-36]In addi ion o hei usual au onomic unc ions, he isce al ganglia also ecei e senso y inpu s om he senso y en acles o he man le.The en acles a e pho o ecep i e, mechano ecep i e, and e en chemo ecep i e o gans. [24, 32] I is o in e es o no e ha he dis ibu ion o he ne es which o igina e om he isce al ganglia is no always iden ical o each ganglion. P ocesses could be seen o ex end om ne e cell bodies. Fib es could be seen in he ce eb o- isce al connec i e and in he o igin o he b anchial ne e. [36] The la ge whi e isce al ganglion can be e ealed by opening he exhalan chambe and cloaca and looking be ween he pylo ic p ocess and he pos e io adduc o muscle. [30, 37, 38] His ology o he ganglia I espec i e o he ganglia ypes, hey ypically consis o h ee laye s, an ou e mos pe ineu ium, he ou e co ex and he inne medulla, which can be called neu opil. [20, 39] Acco dingly, he ypical s uc u al o ganiza ion o he ganglia , bi al es like hose o mos in e eb a es, consis o a mul ilaye ed ind o neu onal cell bodies which send hei p ocesses o a cen al co e, a e shea hed by a connec i e issue pe ineu ium and con ain wo ypes o cells: ne e cells (neu ons) and glial cells.[13, 20] Pe ineu ium Ul as uc u al analysis o he V. e ucosa ganglia shows — om he ganglion pe iphe y he pe ineu ium — a limi ing en elope o med by a shea h o connec i e issue ha consis s o collagen ibe s and ib oblas s; hey a e a anged in a loose h ee-dimensional ne wo k, al e na ing wi h shea hs o dense mic o ilamen ous ma e ial wi h he appea ance o a basal lamina su ounding he ganglia. [36] As o i s unc ion, he pe ineu ium is likely o p o ide no only a p o ec i e en elope, bu also a pe meabili y ba ie , which may be pa icula ly impo an in bi al e ganglia which p obably lack a glial blood-b ain ba ie . [13, 20] Bu he pe ineu ium in e eb a es is di e en om ha in in e eb a es, because i is a concen ic laye o bundled ne es ha i is a p o ec i e laye o connec i e issue loca ed a ound ne es in he body and he in e nal o gans. Indeed, i is composed o concen ic laye s o connec i e issue ha o m a p o ec i e shea h a ound bundles o ne e ibe s. This s uc u e is a anspa en ube-shaped laye ha is easily pulled away om he bundled ne es. Pe ineu ium ne e co e ings a e a pa o he pe iphe al ne ous sys em (PNS), which is esponsible o ansmi ing messages om he cen al ne ous sys em (CNS) in he b ain o he e ec o s, like a ms, legs, and in e nal o gans. [2] The co ical pa o ganglia (co ex) The co ex, a mul ilaye ed a ea o neu on and sa elli e glial cells in V. e ucosa [36], is o be he complex ne wo k cen e o neu onal cell bodies and glial cells. The co ex is no only in ol ed in he con ol o many in e nal, homeos a ic egula o y p ocesses, bu also in he p oduc ion o complex beha iou s. Many o ne e cell bodies loca ed in he co ex we e adially o ien ed and closely associa ed wi h he connec i e issue shea h. Many o he neu ons send hei axons in o he neu opil ganglion (inne ) zone.[40] Medulla o neu opil egion As p e ious men ion, in he mos in e eb a e ganglia such as a h opods and annelids, he cell bodies o neu ons occu in a hin ind on he pe iphe y o he ganglion, and he co e ha con ains axons and dend i es is called he neu opil, a ganglionic co e con aining he axonal p ocesses o he co ical neu ons in V. e ucosa. [36] These ne e Edua do Rocha e . al. / The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion. Chap e 1 | The mo phology o he bi al es ne ous sys em - 6 - 231 Volume 3 Numbe 4, Oc obe - Decembe 2014 Edua do Rocha e . al. / The Bi al e Ne ous Sys em And I s Rele ance Fo The Physiology O Rep oduc ion. cell bodies appea ed o be ex ensi ely inne a ed, as indica ed by he speci ic s aining o endings on hei su ace hei p ocess o he ne e cell body ape ed as i ex ended om he body. The neu opil egion has a ib e o ganiza ion o axons in he ne e ac s ha o m clus e ed a eas o complex synapses, i.e., glome uli. [20, 37] Ganglionic s uc u e ollows a common pa e n in i ually all in e eb a es, wi h an ou e ind o neu onal soma a su ounding an inne co e o axons and dend i es. The soma a a e clus e ed in g oups. The axonal p ocesses o mo o neu ons lea e he ganglion h ough he la e al ne es o inne a e hei a ge s in he pe iphe y (o en muscles). Mos mo o neu ons ha e jus one axon lea ing he ganglion, bu a ew ha e axons in se e al ne es ha inne a e di e en a ge s. In his way a single mo o neu on can exe coo dina ed con ol o e se s o muscles ha need o ac oge he . Indi idual muscles a e gene ally inne a ed by jus one o a ew exci a o y mo o neu ons. [13, 20] Neu ons and glial cells (ganglionic cells) The e is no doub ha in all bi al es he numbe o cen al neu ons is smalle i compa ed o mo e complex animals. No wi hs anding, each neu on has a speci ic and o en complica ed ask o pe o m which in ol es ecei ing and making many synap ic connec ions.[41, 42] In ce ain ins ances, di e ences be ween he pai o neu ons in each hal o he cen al ne ous sys em a e sligh , so ha one can eplace he o he o a conside able ex en . Bu in many o he cases he loss o one ib e mus in ol e conside able loss o unc ion, which may be mi iga ed o a ce ain ex en by he o e lapping ields o di e en neu ons. The ne ous sys em also con ains cells ha su ound, nou ish, and suppo he neu ons and hei p ocess, and hese a e called glial cells. [40] Ne e cells o neu ons As in mos in e eb a es, unipola neu ons p edomina e, e en hough a ew bipola and e en mul ipola ne e cells ha e been desc ibed. [13, 20, 43] Neu onal cell bodies ha e o e all ul as uc u al ea u es simila o hose o mos e eb a e and in e eb a e neu on. They con ain a pale ound o o al nucleus wi h one o mo e p ominen nucleoli. The cy oplasm is ich in g anula and ag anula endoplasmic e iculum, ee ibosomes, mi ochond ia and glycogen deposi s. Some mi ochond ia ha e a pa ac ys alline s uc u e, simila o ha ound in he neu ons o Spisula solidissima [32, 44], which may be ela ed o he accumula ion o p o eins and lipid; as i is known o occu in a a ie y o e eb a e and in e eb a e cells. Mic o ubules and mic o ilamen s a e ba e. Golgi complexes a e nume ous and de eloped, being o med by long cu ed cis e nae illed wi h inely g anula elec on-opaque ma e ial and by esicula p o iles o a iable size and elec on densi y. In mos cell bodies, dense co e esicles a e an impo an componen and can be ound in la ge amoun s dispe sed in he cy oplasm. They display a g ea a iabili y o size, shape and elec on-opaci y and ep esen he only dis inc i e ea u e o he neu on, which a e compa able in o he ul as uc u al espec s. [37, 45] Mos neu onal bodies a e in he co ex and close o he pe ineu ium shea h o he ganglia. The e a e also he beginnings o he ne es ib es ha a e made o axons (i.e., neu i es in unipola neu ons) and e en ual dend i es. [46] Pigmen s can also be ound wi hin neu on, namely as g anules designa ed by cy osomes o lipochond ia, exac ly alike desc ibed in gas opods. [20, 37, 47] The cy oplasmic memb anes o neu onal cell bodies, which a e in ex ensi e ecip ocal con ac , do no show pa icula specializa ions, excep o he p esence o subsu ace cis e nae in pe iphe al neu ons o S. solidissima. [44] The neu onal cell p ocess o igina es om a la ge, cone-shaped ex ension o he soma which g adually ape . The cy oplasm con ains mic o ubules, neu o ilamen s, mi ochond ia and esicles displaying he same ul as uc u al he e ogenei y as hose in he cell bodies. The ganglionic co e is o med by a complex ne wo k o p ocesses o di e en diame e s. Ne e p ocesses con aining cy oskele al elemen s a e in e mingled wi h o he s illed wi h esicles. T ac s a e o med by wide axons o passage, while non-glome ula neu opil con ains ine p ocesses which a bo ise and es ablish synap ic con ac s. [20] Di e en ypes o neu ons can be iden i ied om hei b anched p ocess pa e n and in e ms o unc ion, and so hey can be g ouped in o h ee basic ca ego ies: a) neu ons wi h specialized endings ha espond o ene gy om he en i onmen a e called senso y neu ons; b) neu ons ha ha e axons e mina ing on muscle ibe s a e called mo o neu ons c) all o he neu ons, ha a e in e neu ons. [1, 21, 48] The majo i y o synap ic con ac s occu in he neu opil be ween ne e p ocesses, e en i a e, axoma ic synapses ha e also been ecognized wi hin he co ex. The p esynap ic sides can be iden i ied bo h he p esence o neu o ansmi e esicles and o Chap e 1 | The mo phology o he bi al es ne ous sys em - 7 - 238 Indian Jou nal o Ana omy ©Red Flowe Publica ion P .L d 15. Shiga S, Numa a H. The Role o neu osec e o y neu ons in he pa s in e ce eb alis and pa s la e alis in ep oduc i e diapause o he blow ly, P o opho mia e aeno ae. Na u wissenscha en. 2000; 87(3): 125-128. 16. Padmaja M, Deeca aman M, Jagana hbose MT. S udy o neu osec e o y cells in sand lobs e Thenus o ien alis o oyapu am coas -chennai. Wo ld J Fish & Ma ine Sci. 2010; 2(2): 82-85. 17. Ha zsch S, Glö zne J. An immunohis ochemical s udy o s uc u e and de elopmen o he ne ous sys em in he b ine sh imp A emia salina Linnaeus, 1758 (B anchiopoda, Anos aca) wi h ema ks on he e olu ion o he a h opod b ain. A h opod S uc De . 2002; 30(4): 251-270. 18. Ki sch R, Rich e S. The ne ous sys em o Lep odo a kind ii (B anchiopoda, Cladoce a) su eyed wi h con ocal scanning mic oscopy (CLSM), including gene al ema ks on he b anchiopod neu omo phological g ound pa e n. A h opod S uc De . 2007; 36(2): 143- 156. 19. Whi ing on PM, Maye G. The o igins o he a h opod ne ous sys em: Insigh s om he Onychopho a. A h opod S uc De . 2011; 40(3): 193-209. 20. Bullock TH. Mollusca: Pelecypoda and Scaphopoda. In: Bullock TH, Ho idge GA, edi o s. S uc u e and unc ion o he ne ous sys ems o in e eb a es. London: W. H. F eeman & Co; 1965. p. 1387-1431. 21. Chase R. Beha io and i s neu al con ol in gas opod Molluscs. Ox o d: Uni e si y P ess; 2002. 22. Glanzman DL. Habi ua ion in Aplysia: The Cheshi e ca o neu obiology. Neu obiol Lea n Mem. 2009; 92(2):147-154. 23. C oll RP. Ca echolamine-con aining cells in he cen al ne ous sys em and pe iphe y o Aplysia cali o nica.J Comp Neu ol. 2001; 441: 91-105. 24. Ba nes RD. 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Endoc ine dis up ion: Biological basis o heal h e ec s in wildli e and humans. New Yo k: Ox o d Uni e si y P ess; 2006. 99. Wo ld Heal h O ganiza ion (WHO). The wo ld heal h epo 2002: Reducing isks, p omo ing heal hy li e. Gene a (Swi ze land); 2002. 100. Ribei o C, Pa dal MA, Ma inho F, Ma galho R, Ti i an ME, Rocha E, e al. Dis ibu ion o endoc ine dis up o s in he Mondego i e es ua y, Po ugal. En i on Moni Assess. 2009; 149(1-4): 183-193. - 17 - Chap e 1 | The mo phology o he bi al es ne ous sys em CHAPTER2 THE PEPPERY FURROW SHELL (SCROBICULARIA PLANA (DA COSTA, 1778)) - AN OVERVIEW Chap e 2 | The peppe y u ow shell - 21 - The Peppe y Fu ow Shell (Sc obicula ia plana (da Cos a, 1778)) — An o e iew [Fo ma ed as a manusc ip o be submi ed o publica ion in an in e na ional jou nal. The e sion in his Thesis may change depending on u he imp o emen s om e iew by pee s] Sukanlaya Tan iwisawa uji a,b,c aKing Mongku 's Uni e si y o Technology Thonbu i (KMUTT), Bangkok, Thailand. bLabo a o y o His ology and Emb yology, Depa men o Mic oscopy, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), Po o, Po ugal. cHis omo phology, Physiopa hology and Applied Toxicology G oup, In e disciplina y Cen e o Ma ine and En i onmen al Resea ch (CIIMAR), Uni e si y o Po o (U.Po o), Po o, Po ugal. Running i le: The peppe y u ow shell Key wo ds: ecology, biology, bi al e, S. plana Co espondence o: Sukanlaya Tan iwisawa uji King Mongku 's Uni e si y o Technology Thonbu i (KMUTT) P achau id Road, Bangmod, Tungk u, Bangkok 10140 Thailand E-mail: sukanlaya_ an@ho mail.com Chap e 2 | The peppe y u ow shell - 22 - Chap e 2 | The peppe y u ow shell - 23 - Abs ac Sc obicula ia plana is one o he mos impo an ma ine bi al e ha habi a s widely in in e idal so sedimen along he A lan ic coas om Eu ope o A ica. The e ha e been a ious esea ches on his species such as dis ibu ion, g ow h, ep oduc ion. S. plana is p ima ily gonocho is ic, and he gonadal ep oduc i e sequence can be classi ied in ou s ages: indi e en ( es ing s age); de elopmen (p e-ac i e s age); ma u e (ac i e s age); and spawning. Ex e nal e iliza ion occu s wi hin an hou a e ma u e game es o bo h sexes a e eleased in he wa e . While in he ea ly de elopmen s ages, he la ae li e as a pa o he plank on and, a e one mon h, i becomes pedi elige and hen a ju enile. The pos -la a s age hus chooses a sui able su ace o se le on and g ow in o adul s. The pa e n o b eeding cycle can p esen di e ence acco ding o he la i ude o habi a , which is a ibu ed mos ly o he mal di e ences and ood a ailabili y. S udies o he ne ous and senso y sys ems o S. plana a e s ill a e. Beside comme cial alue (p o ein sou ce), om ishe y and aquacul u e in a ious coun ies, he e is a g owing in e es in s udying and using ha bi al es as a bioindica o , i.e., o moni o aqua ic en i onmen s, because o se e al cha ac e is ic, such as widesp ead loca ion (namely in a eas p one o pollu ion), abundance, easy o collec , conside able adap abili y, and pollu ion ole ance. Chap e 2 | The peppe y u ow shell - 30 - Table 1. The s ages o gonadal de elopmen o Sc obicula ia plana. S ages Male Female 1: Res ing The e a e ew ge m cells. The e a e some esidual la ge oocy es and la ge lumens. 2: P e-ac i e Ge m cells a e ela i ely a e and usually consis o a small pe iphe al laye o spe ma ogonia. The e a e no esidual oocy es a all and only a limi ed numbe o oogonia and small oocy es occu pe iphe ally. 3: Ac i e Cen al lumens inside la ge acini a e small o absen . The e a e ew nu i i e pa icles and ollicula cells. 4: Spawning Spe m a e o en isible lying loose wi hin hese spaces, in adial ow in he cen e o he lumens, and a e eady o be eleased. The dis al walls o many acini a e in e up ed and de ached ma u e oocy es o en occu wi hin acini. Nu i i e pa icles a e ound in some acini. Pos -spawning e iliza ion occu s ex e nally, wi hin an hou a e ma u e game e elease (Hughes 1971; Sola 1997). Emb yos de elop in he la ae ochopho e and hen in he elige s age (a plank on-ea ing p eda o ). Du ing he ea ly de elopmen s ages, he la ae li e as a pa o he plank on, and his may happen o a long ime in his species (Raleigh and Keegan 2006). In pa icula , a 18ºC, i was es ima ed ha elige s ook one mon h o become pedi elige s, which s ill needed some weeks be o e comple ing me amo phosis o become ju eniles wi h pe ec ly unc ioning siphons (F enkiel and Mouëza 1979). The pos -la a s age chooses a sui able su ace o se le on and g ow in o adul s. The S. plana b eeding cycle can p esen di e en pa e ns acco ding o he la i ude o i s habi a ; his is a ibu ed mos ly o he mal di e ences and ood a ailabili y (Sola 1997; Wanink and Zwa s 2001; San os e al. 2011). Fo example, in F ench es ua ies, he spawning pe iod o S. plana can be obse ed om May o July, hen a long pause occu s un il he end o he yea , and game ogenesis s a s again om Janua y, in an indi e en s age, comple ing he cycle again in May. The same can be obse ed in I eland (Raleigh and Keegan 2006). A lowe la i udes (e.g., Spain), he spawning pe iod s a s ea lie (Ma ch), he ep oduc i e pause las ing only om Oc obe o Decembe (Rod ıguez-Rúa 2003). Chap e 2 | The peppe y u ow shell - 31 - 2.4.2 Ne ous and senso y sys ems The e a e sca ce s udies on he ne ous and senso y sys ems o S. plana. The s udy o Odei e (1978b) in es iga ed he ine s uc u e o neu ons in he mid-do sal lobes o he isce al ganglia. I was ound ha neu ons con ain nume ous glycogen g anules and o ganelles such as mi ochond ia, endoplasmic e iculum, and “Golgi bodies”. Fu he mo e, he e a e la ge masses o o ange o eddish mul i-globula bodies in he ex acellula space su ounding neu al and glial cells and in he connec i e issue associa ed o he ganglia. The man le and siphon possess chemo ecep o s ha espond o ex e nal and in e nal wa e lows. Thei s imula ion may elici siphonal wi hd awal and al e closu e as a gene al s ess a oidance esponse. S. plana is an osmocon o ming bi al e, in which he al e closu e is media ed by he de ec ion o osmo ic p essu e change (F eeman and Rigle 1957). Cilia ed u s on he man le and siphons beha e as senso y o gans ha a e in ol ed in mechano- o chemo ecep ion. P e ious s udy sugges s ha he c uci o m muscle and he papilla sense o gan a e chemo ecep o s esponsible o blood p essu e egula ion in he siphons. Mo eo e , i was p o ed ha he papilla is a chemo ecep o and wo ks oge he wi h he c uci o m muscle (Odie e 1978a; Ha ison and Kohn 1997). 2.5 The ele ance o peppe y u ow shell o human consump ion and in oxicology 2.5.1 Economic impo ance The e a e many a ie ies o bi al es and a la ge numbe a e comme cially aluable om ishe ies and aquacul u e p oduc ion. They a e a cheap sou ce o p o ein and mine als in opical and wa m empe a e a eas o o e 13 million ons in he ecen yea (FAO 2014; Idayachandi an e al. 2014). On he A lan ic coas o Wes e n Eu ope, Medi e anean, and Wes e n coas o A ica S. plana is one o he key species o he in e idal communi y, being ha es ed and exploi ed on an in ensi e comme cial scale in a ious coun ies (Casag anda and Boudou esque 2005). 2.5.2 Bioindica o s species The e has been a long con inued in e es in he impac s o con aminan s on aqua ic sys ems, and nume ous s udies ha e been conduc ed ocusing on aqua ic animals as bioindica o s (And ew e al. 2008; Po e e al. 2006; Solé e al. 2009). S. plana is widely known as a aluable species o s udies biomoni o ing me allic con amina ion in es ua ine Chap e 2 | The peppe y u ow shell - 32 - sedimen s (B yan and Humme s one 1978; Cheggou e al. 2000; Coelho e al. 2006), ibu yl in (TBT) bioa ailabili y as deposi - eeding bi al e in sedimen s (Ruiz e al. 1995; No is and Ca 2006), es ogens as endoc ine dis up ion compounds (Langs on e al. 2007). These in es iga ions ecommend S. plana as a sui able species o helping o unde s and and assess he biological impac o aqua ic con aminan s acco ding o he ypical c i e ia o ideal bioindica o o ganisms as ollowing (Goldbe g 1986; Mouney ac e al. 2008): a) They a e seden a y and abundan in he Eu ope and A ica; b) Thei li e span may be mo e han 7 yea s, which is su icien o allow he sampling o mo e han one-yea -class; c) They a e good bioaccumula o s o me als and do signal changes in he en i onmen ; d) They a e easy o sample and s ong enough o su i e in he labo a o y; e) Thei size allows su icien issue collec ion o he mos di e se ypes o analyses. As ea lie men ioned, in es ua ine English and Po uguese habi a s some S. plana popula ions exhibi ed in e sex (Chesman and Langs on 2006; Gomes e al. 2009). This makes hem po en ially use ul o measu ing gene ic changes ela ed o in e sex induc ion. Such condi ion is ac ually inducible wi h expe imen al exposu e o endoc ine dis up o s (Ciocan e al. 2012). Acknowledgmen s The au ho exp esses he g a i ude o he schola ship ha gi en by he Thai Go e nmen Science and Technology, and o he commen a ies o P o esso Edua do Rocha, P o esso Ma ia João Rocha and P o esso U haiwan Ko i adhi, ha helped in imp o ing his ex . Chap e 2 | The peppe y u ow shell - 33 - 3. Re e ences And ew, M. N., Duns an, R. H., O’Conno , W. A., Van Zwie en, L., Nixon B. & MacFa lane, G. R. (2008) E ec s o 4-nonylphenol and 17α-e hynyles adiol exposu e in he Sydney ock oys e , Saccos ea glome a a: Vi ellogenin induc ion and gonadal de elopmen . Aqua ic Toxicology 88, 39-47. Akbe ali, H. B. & Da enpo , J. (1981) The esponses o he bi al e Sc obicula ia plana (da Cos a) o g adual salini y changes. Jou nal o Expe imen al Ma ine Biology and Ecology 53, 251-259. Boche , P., Pie sma, T., Dekinga, A., K aan, C., Ya es, M., Guyo , T., Folme , E. & Radenac, G. (2007) Si e- and species-speci ic dis ibu ion pa e ns o molluscs a i e in e idal so -sedimen a eas in no hwes Eu ope du ing a single win e . Ma ine Biology 151, 577-594. Boldina-Cosque ic, I., Amia d, J.-C., Amia d-T ique , C., Dedou ge-Ge a d, O., Mé ais, I., Mouney ac, C., Mou el, B. & Be he , B. (2010) Biochemical, physiological and beha iou al ma ke s in he endoben hic bi al e Sc obicula ia plana as ools o he assessmen o es ua ine sedimen quali y. Eco oxicology and En i onmen al Sa e y 73, 1733-1741. B yan, G. W. & Humme s one, L. G. (1978) Hea y-me als in bu owing bi al e Sc obicula ia plana om con amina ed and uncon amina ed es ua ies. Jou nal o he Ma ine Biological Associa ion o he Uni ed Kingdom 58, 401-419. Casag anda, C. & Boudou esqe, C. F. (2005) Abundance, Popula ion S uc u e and P oduc ion o Sc obicula ia plana and Ab a enuis (Bi al ia: Sc obicula idae) in a Medi e anean B ackish Lagoon, Lake Ichkeul, Tunisia. In e na ional Re iew o Hyd obiology 90, 376–391. 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(1981) The siphons o Sc obicula ia plana (Bi al ia, Tellinacea) - Obse a ions on mo emen and ex ension. Jou nal o Zoology 194, 445- 459. Hughes, R. N. (1969) A s udy o eeding in Sc obicula ia plana. Jou nal o he Ma ine Biological Associa ion o he Uni ed Kingdom 49, 805-823. Hughes, R. N. (1971) Rep oduc ion o Sc obicula ia plana, da Cos a (Pelecypoda: Semelidae) in No h Wales. Velige 14, 77-81. Idayachandi an, G., Mu hukuma , A., Kuma esan, S. & Balasub amanian, T. (2014) Nu i ional alue o ma ine bi al e, Donax cunea us (Linnaeus, 1758) om Cuddalo e coas al wa e s, sou heas coas o India. In en i Impac : Li e S yle 2014, 15- 19. ITIS (2013) In eg a ed Taxonomic In o ma ion Sys em. A ailable online a h p://www.i is.go . [Accessed on 7 Sep embe 2013.] Langs on, W. J., Bu , G. R. & Chesman, B. S. (2007) Feminisa ion o male clams Sc obicula ia plana om es ua ies in Sou hwes UK and i s induc ion by endoc ine- dis up ing chemicals. Ma ine Ecology P og ess Se ies 333, 173-184. Lino, F. C. (2010) E ec o empe a u e in oxygen consump ion and body mass condi ion du ing a s a a ion pe iod in he peppe y u ow shell - Sc obicula ia plana (da Cos a). Enginee Zoology. Mas e disse a ion. MA LIN (2013) The Ma ine Li e In o ma ion Ne wo k. A ailable online a h p://www.ma lin.ac.uk/. [Accessed on 7 Sep embe 2013.] Mo on, B. (1983) The Mollusca : Feeding and diges i e in bi al ia. Academic P ess. New Yo k. Mouney ac, C., Lino , S., Amia d, J. C., Amia d-T ique , C., Mé ais, I., Du ou, C., Minie , C. & Pelle in, J. (2008) Biological indices, ene gy ese es, s e oid ho mones and sexual ma u i y in he in aunal bi al e Sc obicula ia plana om h ee si es di e ing by hei le el o con amina ion. Gene al and Compa a i e Endoc inology 157, 133- 141. No is, D. O. & Ca , J. A. (2006) Endoc ine Dis up ion. Ox o d Uni e si y P ess. Ox o d. Chap e 2 | The peppe y u ow shell - 36 - Odie e, W. O. (1978a) C uci o m muscle and i s associa ed sense o gan in Sc obicula ia plana (da Cos a). Jou nal o Molluscan S udies 44, 180-189. Odie e, W. O. (1978b) Fine-s uc u e o he neu ons in he mid-do sal lobes o he isce al ganglion o he Lamellib anch mollusc Sc obicula ia plana (da Cos a). Jou nal o Molluscan S udies 44, 305-321. Odie e, W. O. (1979) Cen al ne ous con ol o he adduc o beha io o Lamellib anch mollusks. Malacologia 18, 499-506. Paes-da F anca, M. L. (1956) Va iação sazonal das gónadas em Sc obicula ia plana (da Cos a). A qui os do Museu Bocage 27, 107-130. Pizzolla, P. (2002) Sc obicula ia plana. Peppe y u ow shell. Ma ine Li e In o ma ion Ne wo k: Biology and Sensi i i y Key In o ma ion Sub-p og amme. Plymou h: Ma ine Biological Associa ion o he Uni ed Kingdom. A ailable online a h p://www.ma lin.ac.uk/speciesin o ma ion.php?speciesID=4316>. [Accessed on 7 Sep embe 2013.] Raleigh, J. & Keegan, B. F. (2006) The game ogenic cycle o Sc obicula ia plana (Mollusca: Bi al ia) in Mweeloon Bay (Galway, wes coas o I eland). Jou nal o he Ma ine Biological Associa ion o he Uni ed Kingdom 86, 1157-1162. Rod ıguez-Rúa, A., P ado, M. A., Rome o, Z. & B uzón, M. (2003) The game ogenic cycle o Sc obicula ia plana (da Cos a, 1778) (Mollusc: Bi al e) in Guadalqui i es ua y (Cádiz, SW Spain). Aquacul u e 217, 157-166. Ruiz, J. M., B yan, G. W., Wigham, G. D. & Gibbs, P. E. (1995) E ec s o ibu yl in (TBT) exposu e on he ep oduc ion and emb yonic de elopmen o he bi al e Sc obicula ia plana. Ma ine En i onmen al Resea ch 40, 363-379. San os, C. D., G anadei op, J. & Palmei im, J. M. (2005) Feeding ecology o Dunlin Calid is alpina in a sou he n eu opean es ua y. A deola 52, 235-252. San os, S., Ca doso, J. F. M. F., Ca alho, C., Lu ikhuizen, P. C. & an de Vee , H.W. (2011) Seasonal a iabili y in soma ic and ep oduc i e in es men o he bi al e Sc obicula ia plana (da Cos a, 1778) along a la i udinal g adien . Es ua ine, Coas al and Shel Science 92, 19-26. Sinde mann, C. J. (2006) Coas al Pollu ion: E ec on li ing esou ces and humans. Taylo & F ancis G oup, Ox o d. Chap e 2 | The peppe y u ow shell - 37 - Sola, J. C. (1997) Rep oduc ion, popula ion dynamics, g ow h and p oduc ion o Sc obicula ia plana da Cos a (Pelecypoda) in he Bidasoa es ua y, Spain. Ne he land Jou nal o Aqua ic Ecology 30, 283-296. Solé, M., Kopecka-Pila czyk, J. & Blasco, J. (2009) Pollu ion bioma ke s in wo es ua ine in e eb a es, Ne eis di e sicolo and Sc obicula ia plana, om a Ma sh ecosys em in SW Spain. En i onmen In e na ional 35, 523-531. Wanink, J. H. & Zwa s, L. (2001) Ra e-maximizing op imali y models p edic when oys e ca ches exploi a coho o he bi al e Sc obicula ia plana o e a 7-yea ime span. Jou nal o Animal Ecology 70, 150-158. WORMS (2013) Wo ld Regis e o Ma ine Species. [ci ed 7/09/2013]. A ailable online a h p://www.ma inespecies.o g/aphia.php?p= axde ails&id=141424. [Accessed on 7 Sep embe 2013.] CHAPTER3 QUALITATIVE AND QUANTITATIVE INSIGHTS INTO THE 3D- MICROANATOMY OF THE NERVOUS GANGLIA OF THE PEPPERY FURROW SHELL SCROBICULARIA PLANA (BIVALVIA, TELLINOIDEA, SEMELIDAE) Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 46 - Ou aim he ein was o conduc he i s h ee-dimensional (3D) econs uc ion o he S. plana ganglia, while es ima ing hei o al olumes and su ace a eas in he 3D models, and he ela i e olumes o he co ex and medulla. We also in es iga ed whe he sexual di e ences exis ed in ganglion size and in e nal composi ion. One a ionale o he hypo hesis elies on he key oles o he neu osec e o y neu ons in go e ning gonadal ma u a ion, pa icula ly in emales. Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 47 - Ma e ials and me hods We used adul S. plana ob ained om he es ua y o he Mondego Ri e , Po ugal. Samples we e aken in Ap il, when animals ha e ma u e gonads. A e ield collec ion, he animals we e kep o 24 hou s in glass aqua ia, wi h well ae a ed seawa e (salini y 30‰), and a a cons an empe a u e o 15°C. Fo his s udy, 6 males and 6 emales we e sampled and used o he 3D-dimensional econs uc ions. The shell leng h (cm), wid h (cm) and heigh (cm) we e measu ed wi h a Ve nie calipe . Be o e dissec ion, he animals we e anes he ized in a seawa e solu ion o magnesium chlo ide (6%). The bodies we e emo ed ca e ully om hei shells and hen immedia ely ixed in 10% bu e ed o malin, a oom empe a u e. A e ixa ion o 24 hou s, he samples we e washed in 70% e hanol, and u he p ocessed h ough 90% and 100% e hanol, and xylene, using an au oma ic issue p ocesso (Leica TP1020, Ge many). The samples we e hen embedded in high quali y pa a in (Pa aplas Plus, Sigma-Ald ich), using a modula issue embedding cen e (Leica EG 1140H, Ge many). Each animal was en i ely and se ially sec ioned in he sagi al plane. A ully mo o ized o a y mic o ome (Leica RM2155, Ge many) was used o p oduce 12 µm hick sec ions, which we e s ained wi h hema oxylin and eosin, clea ed in xylene, and moun ed wi h DPX (Sigma-Ald ich). All he sec ions con aining he ganglia we e selec ed o so wa e assis ed 3D-dimensional econs uc ion. In e e y selec ed slide each ganglion was pho og aphed unde a ligh mic oscope (Olympus BX50, Japan), equipped wi h a digi al came a (Olympus Camedia C-5050, Japan). Each pho og aph p o ided a high esolu ion image (JPEG, 2560x1920 pixels), and was aken wi h he 10 X objec i e lens o cap u ing each en i e ganglion p o ile. The h ee-dimensional econs uc ions we e made digi ally om he o iginal s acks o images, using he BioVis3D so wa e (Ve . 3.0, BioVis3D, U uguay). Final econs uc s p esen ed in Resul s we e expo ed as TIFF iles. Es ima es o su ace a eas, olumes and linea dimensions we e compu ed by he so wa e, a e calib a ing o magni ica ion. The s a is ical analysis was conduc ed wi h he so wa e STATISTICA 12 (S a so ). Two- way ANOVA was made (conside ing he sex and ype o ganglia as ixed a iables) o e e y quan i a i e pa ame e . Da a no mali y was con i med by he Shapi o-Wilks es . Homogenei y o a iances was es ed wi h he Le ene es . The Newman–Keuls es pos - hoc was used a e a signi ican ANOVA, conside ing he in e ac ion (sex s ganglia ype) and he sex and ganglia ype sepa a ely, acco ding o he ANOVA p ima y ou pu . We also Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 48 - conduc ed pa ame ic co ela ion analyses, be ween he body size pa ame e s o he animals and he o al olumes and su ace a eas o each ganglion. The le el o signi icance adop ed was he usual s anda d o 0.05. Resul s Body mo phome y and gene al mic oana omy o he ne ous sys em. Da a in Table 1 illus a es ha he shell size o he males and emales used we e app oxima ely he same size, bo h as o he mean alues and o e all in e indi idual a iabili y. Consequen ly, no s a is ically signi ican di e ences we e ound ega ding leng h, heigh o wid h. All he h ee pa ame e s we e highly linea ly co ela ed, wi h coe icien s o co ela ion ( ) showing e y s ong posi i e associa ions: leng h s heigh ( = 0.95; p < 0.001); leng h s wid h ( = 0.94; p < 0.001); heigh s wid h ( = 0.928; p < 0.001). The his ological analysis in ol ed he h ee ypes o neu al ganglia (ce eb al, pedal and isce al), in e connec ed by connec i es (Figu e 1). Table 1. Body mo phome y (cm) o he S. plana used in he s udy. Leng h Heigh Wid h Males 2.8 (0.18) 2.1 (0.17) 0.9 (0.19) Females 2.6 (0.20) 2.0 (0.18) 0.8 (0.25) All oge he 2.6 (0.19) 2.0 (0.25) 0.8 (0.24) A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion). The e a e no signi ican di e ences. Figu e 1. A diag amma ic ep esen a ion o he ne ous sys em o Sc obicula ia plana. A) Sagi al iew showing he posi ion o ganglia and connec i es wi h he le al e emo ed. B) Do sal iew o neu al elemen s. AAM - an e io adduc o muscle; CG - ce eb al ganglia; H - hea ; I - in es ine; LCG - le ce eb al ganglion; N - ne es; PG - pedal ganglion; PAM - pos e io adduc o muscle; RCG - igh ce eb al ganglion, S - s omach, U - umbo, VG - isce al ganglion. Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 49 - 3D ana omy o he ce eb al ganglia The ce eb al ganglia we e loca ed jus la e ally o he mou h, be ween he la e and he pos e odo sal end o he an e io muscle. The 3D econs uc ions e ealed ha he ce eb al ganglia we e pea -shaped, somewha ellip ical o e en oundish (Figu e 2). In iguingly, we ound ha in 90% o he analyzed animals he igh ganglion could be conside ed pea - shaped, whe eas in 10% o he cases he 3D shape was o he ellip ical ype. To he con a y, he le ce eb al ganglion was consis en ly oundish. Also, in all he specimens ha had he igh pea -shaped ganglia, he “ ip” o he “pea ” poin ed o he median posi ion, as depic ed in bo h images in Figu e 2. Figu e 2. A) F on al iew o a 3D- econs uc ion o he le (LCG) and he igh ce eb al ganglion (RCG) o Sc obicula ia plana, showing he ce eb al commissu e (IC), oge he wi h he eme gence o se e al ne es, which a e shown unca ed nea he ganglia. B) A sligh ly o a ed on al iew o he 3D- econs uc ion o he pai o ce eb al ganglia shown in A. In his semi- anspa en mode he shape o medulla (M) (in blue) can be seen inside he ganglia. APN - an e io pallial ne es; CPC - ce eb opedal connec i es; CVC - ce eb o isce al connec i es; IC - in e gangliona commissu e; N - ne es. In addi ion o he o e all 3D ou line, he econs uc ion o he medulla e ealed he p ecise ana omical spa ial posi ioning be ween he co ex and medulla. We no iced he medulla gene ally ollowed he o e all global shape o he ganglia (Figu e 2B), bu no exac ly in all cases, so ha he co ex did no ha e he same hickness along each ganglion. Also, he medulla bulged a imes owa ds he neu onal capsule, whe e i connec ed wi h he eme ging ne es (Figu e 2B). Acco dingly, o assis in unde s anding he shape a ia ions o he medulla, we ha e shown he i e o six ne es eme ging om each ganglion in he econs uc ion and con i m ha all majo bulges coincided wi h he poin o eme gence o he ne es. Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 50 - 3D ana omy o he pedal ganglia The wo pedal ganglia we e used in o one, being loca ed in he median plane o he body be ween he gonad and he oo . The econs uc ion shows a cylind ical o o oid-ba el shape, usually wi h a sligh ly undula ing con ou along he long axis (Figu e 3A). This long axis was always ans e se wi h espec o he an e io -pos e io axis o he animal. The ips o his ganglion we e ypically la a he igh side and poin ed a he le (Figu e 3A). The econs uc ed medulla esembled a ube wi hin a la ge ube (Figu e 3B). As seen in he ce eb al ganglia, hin and la ened o hinne and oundish p ojec ions un owa ds he co ex. Such medulla p ojec ions ul ima ely anas omose wi h ne es (Figu e 3C). Usually he e a e six ne es eme ging om he ganglion bu in a ew specimens he e we e se en o e en eigh . Gene ally, h ee o ou ne es eme ge om bo h he an e io and he pos e io su aces o each ganglion. Some imes medulla p ojec ions pie ced in o he co ex, eaching he capsule bu no ac ually pe o a ing i (Figu es 3C, 3D). Thus, in such egions, immedia ely below he capsule he e was medulla issue ins ead o ( he expec ed) co ex. Despi e such kind o subcapsula medulla egions did no di ec ly anas omose wi h an eme ging ne e, hey could be con inuous wi h nea by medulla p o usions — ha ul ima ely connec ed wi h a ne e. Figu e 3. A) Ven al iew o a 3D- econs uc ion o he pedal ganglia o Sc obicula ia plana. This specimen has an o oid-ba el shape, wi h he mo e poin ed ip loca ed a he le . B) An e io iew o he 3D- econs uc ion o he pedal ganglion shown in A. In his semi- anspa en mode he Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 51 - de ails o he medulla (M) (appea ing in pu ple) can be obse ed, which has a ai ly cylind ical shape. The capsule (C) is shown as a e e ence o he inne mos medulla loca ion. C) An e io iew o he 3D- econs uc ion o he pedal ganglia shown in B, also in semi- anspa en mode, bu in which he pu ple colou is now es ic ed o he medulla a eas (MA) connec ed wi h ne es. On he supe io pa o he medulla wo medulla oundish sp ou s (RS) can be seen ha we e no connec ed wi h any eme ging ne e. C - co ex; M - medulla. D) Le iew o a 3D- econs uc ion o a pedal ganglion. In his case he medulla (M) ( eddish) clea ly pie ced he all ull hickness o he co ex (C ) (in deep blue), hus eaching he ganglion capsule (C) (ligh g ey). ApdN - an e io pedal ne e; CPC - ce eb opedal connec i es; PdN - pedal ne e; PPdN - pos e io pedal ne es. 3D ana omy o he isce al ganglia The isce al ganglia we e loca ed e y close o he pos e io adduc o muscle. These ganglia we e also o ally used in o one, ha ing an i egula lobula shape (Figu e 4A) o a “de o med ec angle” (Figu e 4B). The e was no disce nible le - igh symme y o shape consis ency, excep ha he majo axis o he used ganglion was always pe pendicula o he an e io -pos e io axis o he animal. In con as wi h he o he ganglia, he medulla he e did no ollow so closely he o e all shape o he ganglia, being pa icula ly i egula in 3D. As seen in he o he ganglia, he e we e medulla p ojec ions ha pie ced he capsule and me ged wi h he ne es. Occasionally, he medulla ouched he capsule, bu did no ac ually c oss i (Figu e 4C). Usually, six o se en ne es a e associa ed o a isce al ganglion, mos o en eme ging om en ola e al posi ions (Figu e 4D). Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 52 - Figu e 4. A) An e io iew o a 3D- econs uc ion o a isce al ganglion o Sc obicula ia plana showing he somewha i egula shape. The semi- anspa en mode e eals ha he medulla (M) (pu ple) is somewha i egula , and does no exac ly ollow he shape and con ou o he co ex (C ) (blue). B) An e io iew o a 3D- econs uc ion o a isce al ganglion. The o e all shape is somewha i egula , bu now esembling a de o med, mo e o less ec angula box. The semi- anspa en mode e eals ha he medulla (M) ( eddish-o ange) is qui e i egula , no ollowing he exac same shape and con ou o he co ex (C ) (g een). C) An e io iew o a 3D- econs uc ion o he isce al ganglion shown in B. In his semi- anspa en iew i can be seen on he op o he ganglion ha he medulla (M) ( eddish-o ange) pie ced he co ex (C ), and on hose loca ions he medulla di ec ly ouched he e y hin ex e nal capsule (C) (ligh es g een, no shown a hose places whe e he medulla ouches he capsule). D) Pos e io iew o a 3D- econs uc ion o a isce al ganglion. In addi ion o he i egula shape, he en ola e al eme ging ne es (N) can be seen, wo mo e pos e io ly (in blue) and h ee mo e an e io ly (in da k pu ple). The co ex (C ) s ands ou in g een, while he da ke yellowish g een co esponds o po ions o he medulla (M). CVC - ce eb o isce al connec i es, PAN - pos e io adduc o ne es; PPN - pos e io pallial ne es. Mo phome y o he ce eb al, pedal and isce al ganglia The quan i a i e da a de i ed om he econs uc ions a e gi en in Tables 2-5. As o he o al olumes (Table 2) he e a e no in e sex di e ences. O e all, he isce al ganglion is signi ican ly la ge han he o he wo ganglia, wi h he ce eb als he smalles . The mean Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 53 - olume o he pedal ganglion di e s signi ican ly om bo h om he isce al and each ce eb al ganglion. These display an almos pe ec quan i a i e le - igh symme y as o he o al olume (and also in he ela i e olumes o co ex and medulla; Tables 3-4). Con as ing wi h he o he ganglia, he pedals ha e clea signi ican ly linea co ela ions be ween he o al olume and each o he body size pa ame e s: leng h s olume ( = 0.65; p < 0.05); heigh s olume ( = 0.66; p < 0.05); wid h s olume ( = 0.74; p < 0.01). The isce al ganglia olumes ha e lowe mode a e posi i e co ela ions, which a e only s a is ically signi ican (despi e ma ginally) wi h he heigh ( = 0.65; p = 0.04), and no signi ican ega ding he leng h ( = 0.58; p > 0.05) and wid h ( = 0.59; p > 0.05). The ce eb al olumes ha e esidual posi i e co ela ions wi h each o he biome ic pa ame e s. Table 2. To al olumes (µm3) o he ce eb al, pedal and isce al ganglia o S. plana. Ce eb al Pedal Visce al Righ Le Males 31.7x106 (0.51) 30.2x106 (0.17) 63.5x106 (0.14) 154.5x106 (0.51) Females 39.1x106 (0.41) 36.7x106 (0.53) 62.0x106 (0.18) 139.4x106 (0.28) All oge he 35.4x106 (0.44)A 33.8x106 (0.43)A 62.7x106 (0.16)B 146.9x106 (0.45)C A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion).Wi hin a ow, mean alues wi h di e en supe sc ip le e s di e s a is ically (p < 0.05). The e a e no signi ican di e ences be ween sexes (ANOVA sex e ec : p > 0.05). As o he ela i e olumes (Table 3-4), he ce eb al and pedal ganglia display a e y simila s uc u e, wi h he co ex comp ising ≈ 60% o he ganglion olume and he medulla ≈ 40%. Despi e no signi ican di e ences in ela i e olumes, including by sex, ganglion ype, o he in e ac ion be ween hem, i seems ha he olume a io o co ex s medulla shows some small di e ences: ≈ 1.6 (ce eb al), ≈ 1.5 (pedal), ≈ 1.3 ( isce al). Table 3. Rela i e olumes (%) o he ce eb al, pedal and isce al ganglia co ex o S. plana. Ce eb al Co ex Pedal Co ex Visce al Co ex Righ Le Males 60 (0.12) 61 (0.03) 62 (0.09) 54 (0.07) Females 64 (0.04) 64 (0.14) 59 (0.09) 60 (0.14) All oge he 62 (0.09) 62 (0.11) 60 (0.09) 57 (0.12) A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion).The e a e no signi ican di e ences. Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 54 - Table 4. Rela i e olumes (%) o he ce eb al, pedal and isce al ganglia medulla o S. plana. Ce eb al Medulla Pedal Medulla Visce al Medulla Righ Le Males 40 (0.18) 39 (0.05) 38 (0.15) 46 (0.08) Females 36 (0.07) 36 (0.25) 41 (0.14) 40 (0.21) All oge he 38 (0.15) 38 (0.18) 40 (0.14) 43 (0.16) A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion). The e a e no signi ican di e ences. As o he o al su ace a ea (Table 5), and in ag eemen wi h he olume ic ends, in bo h sexes he e is a signi ican endency o a smalle su ace a ea in he ce eb al ganglia, wi h he pedal in e media e and he isce al ganglion clea ly ha ing he la ges su ace a ea. The ce eb al ganglia ha e ma ching a e ages o he su ace a ea. As seen o bo h he o al and ela i e olumes, he e a e no in e sex di e ences, bu he e ec o ganglion ype in he ANOVA is signi ican . Table 5. To al su ace a ea (µm2) o he ce eb al, pedal and isce al ganglia o S. plana. Ce eb al Pedal Visce al Righ Le Males 1.3x106(0.29) 1.2x106(0.12) 1.8x106(0.23) 4.1x106(0.40) Females 1.3x106(0.32) 1.2x106(0.48) 1.7x106(0.20) 3.5x106(0.28) All oge he 1.3x106(0.29)A 1.2x106(0.34)A 1.7x106(0.21)B 3.8x106(0.35)C A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion). Wi hin a ow, mean alues wi h di e en supe sc ip le e s di e s a is ically (p < 0.05).The e a e no signi ican di e ences be ween sexes (ANOVA sex e ec : p > 0.05). Table 6. Su ace- olume a io (µm2/µm3) o he ce eb al, pedal and isce al ganglia o S. plana. Ce eb al Pedal Visce al Righ Le Males 3.4x10-2 (0.25) 3.9x10-2 (0.08) 2.8x10-2 (0.21) 2.8x10-2 (0.12) Females 3.4x10-2 (0.48) 3.0x10-2 (0.19) 2.9x10-2 (0.17) 2.4x10-2 (0.27) All oge he 3.4x10-2 (0.36) 3.3x10-2 (0.20) 2.8x10-2 (0.18) 2.6x10-2 (0.21) A o al o 6 males and 6 emales we e used. Da a gi en in mean (coe icien o a ia ion). The e a e no signi ican di e ences. Howe e , he co ela ion analyses o he o al su ace a ea in ela ion o he o al olumes (Table 6) showed ha he isce al ganglion was consis en ly s ongly and signi ican ly posi i ely co ela ed wi h each o he biome ic pa ame e s: leng h s su ace a ea ( = 0.84; p < 0.01); heigh s su ace a ea ( = 0.82; p < 0.01); wid h s su ace a ea ( = 0.83; p < 0.01). Bo h ce eb al ganglia p o ide some mode a e (mainly in he igh side) o s ong (in he le side) co ela ions, as ollows: 1) igh ce eb al – leng h s su ace a ea ( = 0.65; Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 55 - p < 0.05); heigh s su ace a ea ( = 0.73; p < 0.05); wid h s su ace a ea igh ce eb al ( = 0.68; p < 0.05); 2) le ce eb al – leng h s su ace a ea ( = 0.79; p < 0.01); heigh s su ace a ea ( = 0.84; p < 0.01); wid h s su ace a ea ( = 0.87; p < 0.01). Discussion The alue o compu e assis ed h ee-dimensional econs uc ions o unde s anding ei he he mac o o he mic oana omy o cells, o gans and e en en i e iny o ganisms, based on se ial his ological sec ions, is well illus a ed in he li e a u e (e.g., Neusse e al. 2006, 2011; Da Cos a e al. 2007; Ge e al. 2012; Geiselb ech and Melze 2013; Ne es e al. 2013). The impo ance and wide ange o po en ial applica ions o his s a egy o ana omical esea ch has long been ecognised (Salisbu y 1994), including he po en ial o de i ing (besides quali a i e aspec s) unbiased/ aluable mo phome ic da a (namely objec coun ing and olume ic measu emen s) om he econs uc s; some imes e en wi h ad an ages o e o he es ablished app oaches, like s e eology. The p og am we used in his s udy o he 3D econs uc ions om his ological se ial sec ions allowed us, wi h p ope calib a ion, o addi ionally ake bo h wo dimensional (likes su ace a ea) o h ee dimensional (namely olume) measu emen s, as well as de ailed 3D ea u es. Typically, he 3D ana omy o he ganglia o bi al es has been desc ibed om g oss obse a ions om dissec ions and mic oscopic obse a ion. Tha app oach can p o ide ealis ic in o ma ion, bu is p one o ca ea s, such as: i is di icul o e en impossible o p ope ly app ecia e de ails when he ganglia a e small: he possibili y o inducing damage o de o ma ions du ing collec ion: he inabili y o assess he inne 3D ana omy. The inal esul ( ypically a d awing) may depend much on he a is ic skills o he obse e . Ca e ul, de ailed compa isons a e some imes necessa y, o example, he e alua ion o e- e alua ion o he axonomy o a species (e.g., Rome a e al. 2013) o o p ope ly look a he ne ous sys em du ing la al de elopmen (e.g., Ellis and Kemp 2011). D awings o he whole ganglia a e a ely accompanied wi h pho omic og aphs, a leas wi h espec o bi al es. Real images o ganglia a e hus a e and no mally om la ge bi al es e.g., he isce al ganglia in he eshwa e mussel Hy iopsis biala us (Simpson, 1900) (Meechonki e al. 2012). F om ou expe ience wi h Sc obicula ia plana i is di icul no only o ully isola e all he ganglia om one animal o app ecia e i s s anda d 3D ana omy and also a iabili y, bu Chap e 3 | 3D- econs uc ion o S. plana ne ous ganglia - 62 - Siniscalchi, A., Ca allini, S., Sone i, D., Sb enna, G., Capuano, S., Ba bin, L., Tu olla, E. & Rossi, R. (2004) Se o one gic neu o ansmission in he bi al e Venus e ucosa (Vene idae): a neu ochemical and immunohis ochemical s udy o he isce al ganglion and gonads. Ma ine Biology 144, 1205-1212. Smaal, A. C. (1991) The ecology and cul i a ion o mussels: new ad ances. Aquacul u e 94, 245-261. Solé, M., Kopecka-Pila czyk, J. & Blasco, J. (2009) Pollu ion bioma ke s in wo es ua ine in e eb a es, Ne eis di e sicolo and Sc obicula ia plana, om a Ma sh ecosys em in SW Spain. En i onmen In e na ional 35, 523-531. S e ano, G. B., Cade , P., Sinis e a, J., Cha les, R., Ba ne , J., Ku u illa, S. & Aiello, E. (1990) Func ional neu al ana omy o My ilus edulis: Monoamine gic and opioid localiza ion. In: S e ano, G. B. (Ed.), Neu obiology o My ilus edulis. Manches e Uni e si y P ess, Manches e , pp. 38-56. Tanabe, T., Osada, M., Kyozuka, K., Inaba, K. & Kijima, A. (2006) A no el oocy e ma u a ion a es ing ac o in he cen al ne ous sys em o scallops inhibi s se o onin- induced oocy e ma u a ion and spawning o bi al e mollusks. Gene al and Compa a i e Endoc inology 147, 352-61. CHAPTER4 OVERVIEW OF THE NEUROCYTOLOGY OF GANGLIA AND IDENTIFICATION OF PUTATIVE SEROTONIN-AND DOPAMINE- SECRETING NEURONS IN THE BIVALVE PEPPERY FURROW SHELL ( SCROBICULARIA PLANA ) Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 65 - O e iew o he neu ocy ology o ganglia and iden i ica ion o pu a i e se o onin- and dopamine-sec e ing neu ons in he bi al e peppe y u ow shell ( Sc obicula ia plana ) [Fo ma ed as a manusc ip o be submi ed o publica ion in an in e na ional jou nal. The e sion in his Thesis may change a e he e ision o be made by all p ospec i e au ho s.] Sukanlaya Tan iwisawa ujia,b,c, Fe nanda Malhãoa,b, Célia Lopesa,b, Ana Sil ab, U haiwan Ko i adhid, Miguel A. Pa dale, Ma ia J. Rochaa,b and Edua do Rochaa,b aLabo a o y o His ology and Emb yology, Depa men o Mic oscopy, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), Po o, Po ugal. bHis omo phology, Physiopa hology and Applied Toxicology G oup (PATH), In e disciplina y Cen e o Ma ine and En i onmen al Resea ch (CIIMAR), Uni e si y o Po o (U.Po o), Po o, Po ugal. cKing Mongku 's Uni e si y o Technology Thonbu i (KMUTT), Bangkok, Thailand. dDepa men o Zoology, Facul y o Science, Kase sa Uni e si y, Bangkok, Thailand. eCen e o Func ional Ecology (CFE), Uni e si y o Coimb a, Coimb a, Po ugal. Running i le: Neu ocy ological aspec s o ne ous ganglia o S. plana Key wo ds: bi al e, glial cells, his ology, ul as uc u e, monoamines, ne ous sys em, neu ons Co espondence o: Edua do Rocha Depa men o Mic oscopy Labo a o y o His ology and Emb yology Ins i u e o Biomedical Sciences Abel Salaza (ICBAS) Uni e si y o Po o (U.Po o) Rua de Jo ge Vi e bo Fe ei a n.º 228 4050-313 Po o Po ugal E-mail: e[email p o ec ed] Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 66 - Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 67 - Abs ac The cy ology o he h ee ganglia ypes o Sc obicula ia plana was ne e s udied in ine de ail, excep in some egions o he isce al ganglia. Howe e , o suppo undamen al and applied s udies using his species ne ous ganglia, i appea s cons uc i e o us ha a baseline s uc u al desc ip i e s udy is made a ligh and elec on mic oscopy. To s a illing he knowledge gaps we made a gene al his ological (including special s ainings) and ul as uc u al s udy in S. plana. Adul s o bo h sexes and imma u e specimens we e collec ed om he Ria Fo mosa lagoon, Po ugal. Fo ligh mic oscopy obse a ions, he animals we e measu ed, anes he ized, dissec ed and hen ixed in 10 % bu e ed o malin. Then a e hey we e ou inely p ocessed o pa a in embedding and sec ioned o di e se pu poses along he sagi al plane, using a ully mo o ized mic o ome. A sys ema ic su ey was made o a i s iden i ica ion o neu ons ha con ain se o onin and dopamine, as a i s s ep o ga he o knowledge o he p esence and ole o neu oendoc ine neu ons in S. plana. Fo ansmission elec on mic oscopy (TEM) dissec ed ganglia we e ixed in 2.5 % glu a aldehyde, pos - ixed in 1 % osmium e oxide, and p ocessed o epoxy embedding. Sec ions we e p oduced wi h a diamond kni e using an ul amic o ome. As we expec ed, he gene al his ology o he ganglia ma ched wha is known o bi al es in gene al, wi h an ou e co ex and an inne medulla. In he co ex he e a e wo basic ca ego ies o neu ons: la ge and small. Almos all a e unipola neu ons; only a ew bipola we e iden i ied. We ound no e idences o myelin like ma e ial. In all ganglia he e is a dec easing size o he neu ons as hey a e loca ed close o he medulla. Glial cells appea a ound neu ons, and also amids he axons ha ou e in o he medulla. They ypically ha e smalle sizes and g ea e nucleus-cy oplasm a io when compa ed wi h neu ons. Fo desc ip ion, we conside ed h ee basic cy ological ypes: usi o m, oundish and iangula . Facing hei shapes and di e se ul as uc u e we can hypo hesize ei he hey a e di e en sub- ypes o glia, o a leas hey co espond o di e se unc ional s a us — a ma e o deba e in bi al es. Facing ou immedia e objec i es ou ocus was on ligh mic oscopy, bu as o he ul as uc u e we no ed di e ences in ela ion o ea lie indings, mainly in ype o inclusions and acuoles. This disc epancies seem also wo h explo ing in u u e, co ela ing wi h ganglia ype and bio ic and abio ic ac o s. A las , se o one gic and dopamine gic cell bodies and neu i es we e iden i ied in all ganglia. Ou da a sugges ed an iden ical exp ession pa e n in adul s o bo h sexes and in he imma u e animals (wi h unde ined sex). Howe e , he ex en /in ensi y o se o onin posi i i y in isce al ganglia suppo ha ma u e animals ha e s onge exp ession han undi e en ia ed ones, Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 68 - which may be connec ed he ole o ha ammine in helping bi al e gonad ma u a ion and spawning. O e all, ou s udy o e s a ange o baseline da a ha a e use ul o u he s udies in S. plana. Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 69 - In oduc ion The ne ous sys em o in e eb a es is composed o wo main ca ego ies o cells, neu ons and glial cells (Bullock and Ho idge 1965; Coles and Abbo 1996; Ha ison 1997; Oikonomou and Shaham 2011; Hidalgo e al. 2011), in line wi h wha is seen in e eb a es (B odal 1992; Galbia i e al. 2003; Laming e al. 2000). Also as in he la e g oup, he e a e neu osec e o y cells in in e eb a es, such as desc ibed in a h opods (Bha a hi 2014; Padmaja e al. 2010; Pe ić-Ma a uga 2011) and in molluscs (Meechonki e al. 2010; Sleem 2003; Wijdenes e al. 1980). Rega ding cell peculia i ies, he e a e cy ological cha ac e is ics ha he neu ons ha e when hey a e sec e o y, pa icula ly lipid d ople s and g anules illed wi h sec e ion, which p oduc s — e.g., dopamine, no ad enaline and se o onin — can ac as neu oho mones (Ca oll and Ca apane 2007; Gagné and Blaise 2003). Being p oduced in soma a, he neu oho mones a e packed in g anules ha a e ul ima ely mo ed in o he axonal endings, like he synap ic esicles (Bullock and Ho idge 1965; Roubos 1975; Tho ndyke and Goldswo hy 1988). Neu osec e o y cells ecei e inpu s om o he neu ons, bu , unlike he egula neu onal cells (i.e., non-sec e o y), which display cell- o-cell communica ions o e e y sho dis ances, a he synapses, neu osec e o y neu ons elease hei p oduc s in o an ex acellula space, which may be a some dis ance om hei a ge . In an o ganism wi h a closed ci cula o y sys em, he neu oho mones a e ypically sen h ough he ascula ou e up o hei si e o ac ion. Ye , in lowe in e eb a es, which lack an o ganized ci cula o y sys em, he neu oho mones seem o simply di use om he elease si e up o he a ge (Ke a a e al. 2008). In molluscs he e a e many e idences o neu osec e o y ac i i y — o example, in he cen al ganglia o he sea ha e, Aplysia oculi e a, and he sea snail, Nep unea a h i ica, h ee and ou ypes o neu osec e o y cells, espec i ely, we e cha ac e ized (Sleem 2003; Yaha a and Takahashi 1972). Also in bi al es co ela ions ha e been ound be ween, on he one end, he neu osec e o y ac i i y and, on he o he end, he neu onal lipid s o es and he b eeding cycle — o ins ance, in he ganglia o he mussel, C enomy ilus g ayanus, whe eas nume ous neu onal lipid d ople s we e p esen in he spawning pe iod, e y low amoun s o hose inclusions we e ound in he p e-spawning pe iod; i.e., he lipid con en s we e in e sely p opo ional o he ex en o neu osec e ion (Reuno a e al. 1997). In he scallop, Nodipec en subnodosus, he e is an inc eased concen a ion o monoamines (including no epineph ine, dopamine and 5-HT) in he gonad, gill, and man le issues du ing he ma u ing s age, wi h a d op a e spawning, sugges ing ha he animals used a ied neu o ansmi e s du ing he Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 70 - ep oduc i e cycle (López-Sánchez e al. 2009). In bi al es, as in o he in e eb a es, he monoamines, dopamine and se o onin a e conside ed neu oho mones, because hey a e known o ac in se e al issues/o gans, ei he as a neu o ansmi e o as a neu omodula o o di e se p ocesses (Bullock and Ho idge 1965; Gagné e al. 2007). His ological and ul as uc u al s udies abou he neu al cells (inc. neu osec e o y) a e e y limi ed in he bi al e peppe y u ow shell, Sc obicula ia plana. Conside ing ha his species is o economic impo ance and also ha i is used as pollu ion bioindica o — ha is p one o neu onal and ep oduc i e dis up ion — a be e knowledge o i s ne ous sys em is no only o a undamen al and compa a i e in e es , bu i also can be used o p ac ical pu poses. Fo ins ance, his opa hological app oaches o app ecia ing he impac s o pollu an s do need a good baseline cha ac e iza ion o he no mal his ological and cy ological ea u es. In iew o his backg ound, we aimed: 1) o e iew he his ology and cy ology o he neu al cells in S. plana, egis e ing i s main aspec s and e en ually looking a e any s ill undesc ibed ea u e; and 2) o iden i y he neu osec e o y neu ons — iz. hose pu a i ely p oducing se o onin and dopamine — ac oss he ce eb al, pedal and isce al ganglia, and conside ing he gonadic sex. Ma e ials and me hods Animals Adul ma u e and imma u e S. plana we e collec ed a ebb ide om he in e idal zone o he Ria Fo mosa Lagoon, sou h o Po ugal. A e cap u e, he animals we e ans e ed o in house acili ies in he same day, and main ained in glass aqua ia (10 L) illed wi h ae a ed seawa e (salini y 30 ‰), a a empe a u e o 15 oC. In he nex day, he animals we e anes he ized by imme sion in a solu ion o MgCl2 (6 %) un il o al elaxa ion o he al es. Leng h, wid h, and heigh we e measu ed using a Ve nie calipe . The medium animals sized animals (3.4 ± 0.2 mm in leng h) we e selec ed o ligh mic oscopy (including immunohis ochemis y), and he bigge ones (3.9 ± 0.02 cm in leng h) we e used o ansmission elec on mic oscopy (TEM). Ligh mic oscopy — Fixa ion o pa a in embedding Each animal was emo ed ca e ully om he shell and ixed in o o o 1-2 h, using 10 % neu al bu e ed o malin, a oom empe a u e. Subsequen ly, mos o he specimens we e sliced wi h a sha p azo blade and 3 c oss-sec ioned slabs we e aken a ound he body zones whe e he ne ous ganglia a e known o be posi ioned. The smalles animals we e ixed only Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 71 - in o o. In ma u e specimens he p ocedu e allowed an immedia e con i ma ion o he sex, due o he dis inc i e g oss aspec o he gonad. A e slicing, all he pieces we e kep in he ixa i e o 24 h. The ixed o ganic agmen s we e washed in 70 % e hanol, dehyd a ed in inc easing concen a ions o e hanol (up o 100 %), clea ed in xylene, and hen in il a ed wi h pa a in (His osec, Me ck) in an au oma ic issue p ocesso (TP1020, Leica, Ge many). Embedding o he pieces in plas ic casse es was made in a pa a in s a ion (EG 1140H, Leica, Ge many). Ligh mic oscopy — Rou ine and special s aining me hods Animals we e cu on a ully mo o ized o a y mic o ome (RM2155, Leica, Ge many), ei he in non-se ial (5 µm hin) o in se ial sec ions (30 µm hick), in c oss o in sagi al planes. The sec ions we e depa a inized in xylene, ehyd a ed in dec easing concen a ions o e hanol (absolu e up o 70 %), ollowed by wa e , and inally ei he subjec ed o ou ine hema oxylin and eosin (H&E) s ain, Klu e -Ba e a me hod (wi h c esyl iole coun e s aining – a classic nucleic acid s ain o highligh ing he soma), Weil’s s ain (usually used o de ec ing myelin), and, inally, o Bielschowsky's sil e s ain ( o highligh ing neu onal ib es). Fo obse a ion o slides and eco ding o images we used a ligh mic oscope (BX50, Olympus, Denma k), equipped wi h a digi al came a (Camedia C-5050, Olympus, Japan). T ansmission elec on mic oscopy (TEM) Fo TEM, ganglia we e isola ed om adul (bigges ) animals and cu unde a s e eomic oscope (LSM 510 Me a, Zeiss Inc., Ge many) in o iny pieces (app oxima ely 1 mm3). The cells we e ixed in 2.5 % glu a aldehyde, in 0.2 M sodium cacodyla e- hyd ochlo ic acid bu e , pH= 7.2, o 2 h, a 4ºC, and hen washed wice in he same bu e , 10 min each. Pos - ixa ion was made in 1 % osmium e oxide, also in 0.2 M sodium cacodyla e-hyd ochlo ic acid bu e a pH = 7.2, o 2 h, a 4ºC. The ixed pieces we e hen dehyd a ed in inc easing se ies o e hanol ( om 50 % o absolu e e hanol p.a.), imme sed, 2 imes, in p opylene oxide o 15 min, and hen in mix u es o p opylene oxide and epoxy esin (3:1; 1:1; 1:3, in his o de , each o 1 hou ). Embedding was made in he same esin. A e 2 days o polyme iza ion in he o en, a 60ºC, he ha dened blocks we e immed and cu wi h a diamond kni e (Dia ome, Swi ze land), in an ul amic o ome (Reiche Supe no a, Leica, Ge many). One µm hick semi hin sec ions we e s ained wi h a mix u e 1:1 o 1 % me hylene blue and 1 % azu e II. Nine y nm hick ul a hin sec ions we e placed in o 200 mesh hexagonal coppe g ids, and con as ed wi h u anyl ace a e and lead ci a e. Sec ions o Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 78 - deg ee o he e ogenei y as o he signal s eng h wi hin each ganglion, i.e., no all neu ons s ain equally. Quali a i ely, no global pa e ns o immunos aining a e seen as o he e ec o sex s a us o o ganglia ype. G ading da a o he immunoma king is gi en on Table 2. The median g ades we e ei he 2.5 o 3, despi e alues a ied om 1.5 o 3. No animal was a ed wi h he lowes possible sco e o 1. The mos common scena io ound was qui e s able, wi h 60% o 80%, o mo e, o he sec ioned neu ons being labelled, and he s aining in ensi y being s ong o e y in ense. Only one signi ican di e ence was ound, and in he isce al ganglion, wi h he emales ha ing a highe median sco e o se o onin when compa ed o males and undi e en ia ed specimens. Table 2. Immunologic in ensi y o posi i e neu on in ce eb al, pedal and isce al ganglia. Ganglia and sex condi ion Se o onin Dopamine Ce eb al Median (Min-Max) Median (Min-Max) Males 3.0 1.5-3.0 3.0 3.0-3.0 Females 2.5 2.5-3.0 3.0 2.5-3.0 Undi e en ia ed animals 3.0 2.5-3.0 3.0 2.5-3.0 Pedal Median (Min-Max) Median (Min-Max) Males 3.0 2.0-3.0 3.0 2.5-3.0 Females 3.0 2.5-3.0 2.5 2.0-3.0 Undi e en ia ed animals 3.0 3.0-3.0 3.0 2.5-3.0 Visce al Median (Min-Max) Median (Min-Max) Males 3.0a 3.0-3.0 2.5 2.0-3.0 Females 3.0a 2.5-3.0 3.0 2.5-3.0 Undi e en ia ed animals 2.5b 2.5-3.0 2.5 2.0-2.5 * Median alues bea ing di e en supe sc ip le e di e signi ican ly (P <0.05). Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 79 - Discussion Gene al ligh and elec on mic oscopy aspec s The gene al his ological o ganiza ion o he ganglia was he expec ed one, in acco dance wi h ha al eady s udied in a 3D pe spec i e (in he Chap e 3 o his Thesis). Ou obse a ions hus se ed he pu pose o no e any speci ici y as o he s uc u e o he neu al cells and/o o hei dis ibu ion wi hin he ganglia. As a as we know, in S. plana only Odie e (1978) s udied he neu ocy ology o he isce al ganglia, epo ing aspec s o hei ine s uc u e. Howe e , ha s udy nei he e ealed he gene al his ology no p o ided da a abou e en ual pheno ypes o neu ons (pa icula ly ega ding he size) and glial cells (namely as o size o pa icula shapes). In cu en s udy, we ound ha he neu ons con as much in size — o he sake o simplici y we jus classi ied hem as la ge and small — and ha mos o la ge neu ons a e loca ed a he pe iphe y o all h ee ganglia. I espec i e o he size and posi ion o he neu ons in he co ex, soma a ypically ha e a single p ocess di ec ed owa ds he medulla. Occasionally we spo ed wo p ocesses eme ging om he soma o la ge neu ons. This inding is in acco dance wi h he li e a u e, as bipola neu ons a e consis en ly ound o be he a less dominan neu on ypes in ganglia o mos in e eb a es (O ha and Mulle 2005; C oll 2001). The nomencla u e abou neu onal p ojec ions is o en used in he li e a u e in a non-sys ema ic and unanimous o m. Acco ding wi h he e iew ecommenda ions o Rich e e al. (2010) o in e eb a es, “all cell p ocesses o neu ons” should “collec i ely be e e ed o neu i es”, and, impo an ly in ou con ex , “The single main p ocess eme ging om he soma o unipola neu ons and connec ing hem o dend i es and axons is hen called p ima y neu i e”. These aspec s a e wo h ci ing he e because i has been ecognized o long ha in in e eb a es i is di icul o dis inguish axons o m dend i es, bo h in s uc u al and unc ional poin s o iew (Bullock and Ho idge 1965). He e and elsewhe e we use he e m neu i e and axon as neu i es can be sepa a ed in o p ima y neu i es, axons (he e mos ly om unipola neu ons), and dend i es (Rich e e al. 2010). The special s aining p ocedu es and he elec on mic oscopy obse a ions did no e idenced he p esence o myelin-like ma e ial a ound neu i es. Howe e , Odie e (1978) illus a ed in his species a so o concen ically lamella igu es ha we e deemed as myelina ed axons (bu his only in small sized ones), s a ing, howe e , ha mos axons en e ing he medulla we e ypical unmyelina ed. Ou elec on mic oscopy app oach was no an ex ensi e s udy, and, e en ually, we may ha e missed such kind o s uc u es. This is a ma e wo h u he Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 80 - s udy, namely in ligh o he di e gen da a and con inuous esea ch abou he p esence o ea ly- o- ecen o ms o myelina ion h oughou i s complex e olu ion p ocess (Zalc 2006; Cas el anco and Ha line 2015). Ap opos, i mus be s essed ha axonal shea hs o ue myelin (i.e., as in e eb a es) seem no o exis in in e eb a es, including o ins ance he mul i-lamella e glial shee s desc ibed in bi al es, c us aceans and annelids (Ha line 2008; Roo s 2008). As o neu ons cy oplasm, especially o la ge ones, he e a e acuoles/ esicles, which appea as ligh oundish spaces a ligh and as memb ane bounded bodies o a ying elec on-densi y when seen a elec on mic oscopy. Some o hese s uc u es had la ge dense g anules inside, much g ea e han he elec on-dense o elec on-lucen small esicles ha appea ed a a icosi ies o p e-synap ic egions o he axons. These esicles a e hough o se e mul iple pu poses, om accumula ion o p o eins and lipids o s o age o neu oho mones and neu o ansmi e s, and hey a e known o appea in a ious in e eb a es (Golding and Pow 1988; Siniscalchi e al. 2004; Meechonki e al. 2010). The s uc u e o neu onal esicles is qui e di e se in bi al es, and a emp s we e made o ad ance some sys ema iza ion. Fo example, o My ilus edulis he neu onal esicles we e desc ibed as small g anula , la ge g anula , la ge opaque, and pleomo phic esicles, wi h a ia ions (Vi ella o-Zucca ello and Biasi 1990). The di e si y o s uc u e is known o long o ma ch a a ie y o neu o ansmi e s, in bi al es and o he molluscs (Endean 1972), and s udies ha e been made o know he esicles/neu al zones ha ha e each neu ochemical (e.g., Ka hunen e al. 1993; Ka hunen e al. 2004; Meechonki e al. 2010). We did no ind he so-called “yellow globules” o “cy osomes” ha Odie e (1978) desc ibed in some neu ons o he mid- do sal lobes o he isce al ganglia o S. plana; such globules we e hen cha ac e ized by displaying an elec on-dense co ex and elec on-lucen cen um. Mos likely, such cy osomes occu in pa icula egions o he ganglia, and ou elec on s udy was qui e gene al, based on andom samples o he co ex ha a e no enough o disc imina e be ween pa icula egions. As o o he ea u es o S. plana neu onal soma a, his ology and ul as uc u e e ealed neu ons wi h one euch oma ic ound- o-o al nucleus wi h one salien nucleolus, and cy oplasm wi h he usual key o ganelles, s anding ou mi ochond ia; o e all in line wi h he o he bi al e species (Bullock and Ho idge 1965; S e ano, 1990; Mo se and Za dus 1997; Odie e 1978). As o glial cells, hey we e iden i ied he ein by hei mo phology and posi ioning, appea ing in he co ex and medulla. In bi al es, he glial cells his ology/cy ology has dese ed much less a en ion han neu ons, bu hei compa a i ely smalle cell size and scan y cy oplasm has Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 81 - been desc ibed, pa icula ly in ganglia o M. edulis (Vi ella o-Zucca ello and Biasi 1990; Paemen e al. 1992). In S. plana, Odie e (1978) ocused he a en ion on he di e si y o glial g anules. Ou ope a ional de ini ion o he glial cells in o usi o m, oundish, and iangula , is one i s a emp o a leas sys ema ize hei desc ip ion, and i may no ha e unc ional implica ions. I is possible ha hese o ms can be me e s ages o he same cell ype o i may co espond o di e en oles. Only mo e in dep h s udies can answe his ques ion, bu in ou pos e io s udies we will main ain his ope a ional de ini ion, namely when making di e en ial cell coun ing. The di icul y in cha ac e izing he in e eb a e glia based on clea consis en c i e ia is a well- ecognized ca ea , and he e is no unique se o “mo phological glial ma ke s” (Ha line 2011). In he pa icula case o bi al es, i was ecognized ha he ul as uc u e (no o men ion unc ion) o he glia wi hin he ganglia is a om unde s ood, and ha andem ques ions exis , such as i he e is one o mo e glial sub ypes and i en i onmen al ac o s in luence he o en seen s uc u al a ia ions (Vi ella o-Zucca ello and Biasi 1990). Despi e all his, he e a e some consis en ea u es appea ing in di e en bi al es, such as he exis ence in he ganglia o glial cells ha ha e an elec on-dense cy oplasm and hin p ojec ions, o ins ance in M. edulis Vi ella o-Zucca ello and Biasi 1990 and Spisula solidissima (P io and Lip on 1977), as we saw in S. plana. Wha e e he a iabili y and poo ly cha ac e ized mo pho unc ional sub ypes, i is well- ecognized ha glial cells a e p ominen in in e eb a es, including molluscs, whe e hey con ac and co e neu ons, and a e absolu ely c i ical o as inc easing numbe o known unc ions, esembling hose much be e known in mammalian glial cells (Coles 2009). In all ypes o ganglia, we consis en ly ound neu onal cells ha showed a b own-yellowish colou a ligh mic oscopy. We labelled hem as pigmen ed cells. They appea mos ly in he co ex, bu also in he medulla. In he as majo i y o occasions hese cells we e o small size — compa ible wi h ha o many glial cells bu also wi h ha o small neu ons — and showed an o oid o ellipsoidal con ou . Fo ope a ional easons and acing a deg ee o unce ain y abou i s ue na u e, we op ed o p esen ing hese cells as a sepa a e neu al cell en i y. One o he easons is he ac ha pigmen s can be ound in bi al es wi hin bo h neu ons and glial cells, namely as o ganelles. A di e si y o pigmen ed cy oplasmic s uc u es may appea in bi al es and o he molluscs, such as he yellow globules o cy osomes o neu ons and he mul iglobula bodies/ used yellow globules o glial cells, as desc ibed in he mid do sal lobes o S. plana (Odie e, 1978). Memb ane bound bodies wi h yellowish pigmen s in neu al cells a e no exclusi e o bi al es. Fo example cy osomes (also named lipochond ia) ha e been Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 82 - s udied in gas opods (e.g., Lay and Roge s 1956; Zs-Nagy 1971; Sugaya and Onozuka 1978; Robles e al. 1986). A ligh mic oscopy, he mo phology o he pigmen ed cells we spo ed we e consis en ly mo e compa ible wi h glial cells, and no la ge neu ons displayed such yellowish inclusions/ acuoles. Despi e his, u he s udies a e needed o unde s and he di e ences in he pigmen ed acuoles/globules we obse ed he ein and hose epo ed by Odie e (1978). Such di e gences can be he e lex o bio ic and abio ic ac o s, namely when knowing ha pigmen (lipoch ome) se es a ious uses, such as anoxic endogenous oxida ion ha helps su i ing low oxygen ension (Zs-Nagy 1971, 1974). In iew o o he unc ions, as o lipo uscin and/o lipoch ome pigmen s in neu ons o he gas opod Aplysia cali o nica (Henka 1975; Schwa z e al. 1979), mo e s udies a e wa an in S. plana. The se o onin- and dopamine-sec e ing neu ons Al hough se o onin and dopamine ha e been epo ed wi hin he ganglia o some o he bi al es (Gagné e al. 2007; López-Sánchez e al. 2009; Meechonki e al. 2010), ou wo k is he i s o epo hei immuno eac i i y in ma u e (males and emales) s. undi e en ia ed S. plana. We obse ed cells wi h consis en posi i i y o se o onin and dopamine in all ganglion ypes. Quali a i ely he e we e no pe cei able in e ganglionic di e ences, bu knowing ha neu o-signalling is c i ical o he success o adul bi al e ep oduc ion, we used semi-quan i ica ion o he s aining as a pilo app oach o sea ch o clues o in e sex and age- ela ed di e ences. A e implemen ing ou p e-de ined g ading sco es, bo h se o onin- and dopamine sec e ing neu ons we e always p esen and showing wha seems a con inuous e iden exp ession o he wo pep ides. Howe e , one signi ican di e ence was ound in isce al ganglia, whe e se o onin exp ession o undi e en ia ed animals (as measu ed by he ex en and s eng h o immunos aining) was lowe han in ma u e males and emales. Such di e ences we e no seen ega ding dopamine. Assuming ha he signi ican di e ence is no a me e occasional e en , i sugges s ha adul isce al neu ons o S. plana p oduce (use, elease) g ea e quan i ies o se o onin, e en ually connec ed wi h gonad ma u a ion e en s. This in e es ing possibili y is wo h explo ing in he u u e, using mo e e ined echniques, including chemical de e mina ions o neu o ansmi e s. Indeed, in e ac ions o se o onin and dopamine in ne ous issue a e ecognized o be impo an o ep oduc ion and o he unc ions in bi al es, including muscle con ac ion in spawning and espi a ion (Bei as and Widdows 1995; López-Sánchez e al. 2009; Meechonki e al. 2012). Ou esul s make pa icula sense conside ing ha se o onin is known o induce spawning in bi al es (Gibbons and Cas agna, 1984); which ag ees wi h lowe le els in imma u e S. plana. Ne e heless his Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 83 - explana ion is pa ial, as i does no i in he no di e ence be ween adul and imma u e specimens as o dopamine. Indeed, he la e monoamine is also e iden ly connec ed wi h gonadal ma u a ion and spawning e en s (Osada e al. 1987; Klouche e al. 2015). S udies on dis ibu ion o monoamine p oduc ion neu ons in bi al es epo ed di e se pa e ns. Fo example, in M. edulis, his o luo escen localiza ion in he ce eb al ganglia showed ha hey enclose dopamine and se o onin posi i e cells, ha he pedal con ained mainly se o onin, and ha he isce al possesses smalle amoun s o dopamine; no da a abou he sex o he animals used was gi en (S e ano and Aiello 1975). In con as , he mo e in ense s aining o se o onin was de ec ed in he ne e ib es and e mini o he isce al ganglia o Hy iopsis biala a (Meechonki e al. 2010). In he la e s udy, he la ge neu ons we e he ones ha s aining s onge bo h wi h pa aldehyde uchsin and agains se o onin, which is in acco dance wi h ou obse a ion in neu ons o S. plana. We can an icipa e ha in e species di e ences can be due ei he o bio ic (e.g., Bu ell and S e ano 1981) and/o abio ic ac o s (e.g., Hi ipi e al. 1982), bu we lack mo e analyses o be able o pe cei e global pa e ns. In iew o he absence o s udies, esea ch on neu oamines in S. plana is wa an because hey should play oles as neu o ansmi e s and neu oho mones, expec able in acco dance wi h hose seen in bi al es so biodi e se as Mya a ena ia, Misuhopec en yessoensis, Hy iopsis biala us, and o he (Ca oll and Ca apane 2007; Kho imchenko and De ido ich 1989; Meechonki e al. 2012). We ound he e ha immunos aining o se o onin and dopamine was no es ic ed o he soma bu ha i was also p esen in neu i es. Thus, i can be concluded ha S. plana has a ne wo k o dopamine gic and se o one gic neu i es dis ibu ed h oughou he ganglionic medulla. Se o one gic neu i es we e also no iced in isce al ganglia and gonad o he bi al e Venus e ucosa (Siniscalchi e al. 2004). Despi e he ew s udies, we know ha se o one gic and dopamine gic ne wo ks can be o med soon in he bi al e emb yonic de elopmen , as ea ly as 24 h pos - e iliza ion, as es ablished in S. solidissima (K eyling e al. 2001). Such knowledge on monoamine gic sys ems and he cu en new da a o S. plana, o e la ge po en ial o basic and applied pu poses, bo h in aquacul u e (Kho imchenko and De ido ich 1991; FAO 2004) and oxicology (Klouche e al. 2015). Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 84 - Acknowledgmen s The i s au ho was suppo ed by a Thai Go e nmen Science and Technology Schola ship. This wo k was pa ially suppo ed by he Eu opean Regional De elopmen Fund (ERDF) unds h ough he Compe i i eness and T ade Expansion P og am (COMPETE) and by Na ional Funds as p o ided by he Fundação pa a a Ciência e a Tecnologia (FCT), ia he esea ch p ojec s PEs -C/MAR/LA0015/2013 and UID/Mul i/04423/2013. The s udies a elec on mic oscopy le el addi ionally bene i ed om he p ojec EUCVOA (NORTE-07- 0162-FEDER-000116), co- inanced by he No h Po ugal Regional Ope a ional P og am (ON.2 O No o No e), unde he Na ional S a egic F amewo k (NSRF), ia he ERDF. Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 85 - Re e ences Bei as, R. & Widdows, J. (1995) E ec o he neu o ansmi e s dopamine, se o onin and no epineph ine on he cilia y ac i i y o mussel (My ilus edulis) la ae. Ma ine Biology 122, 597-603. Bha a hi, A., Sa ojini, N. & Padmaja, M. (2014) Compa a i e s udy o neu osec e o y cells in emale Penaeus indicus a e unila e al eyes alk abla ion. 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In: Squi e, L. R. (ed.) Encyclopedia o Neu oscience. Else ie : Ams e dam, pp. 749-759. Coles, J. A. & Abbo , J. N. (1996) Signalling om neu ones o glial cells in in e eb a es. TINS 19, 358-362. C oll, R., Boudko, D. & Had ield, M. (2001) His ochemical su ey o ansmi e s in he cen al ganglia o he gas opod mollusc Phes illa sibogae. Cell and Tissue Resea ch 305, 417 - 432. Endean, R. (1972) Aspec s o Molluscan Pha macology. In Flo kin, M. & Shee , B. T. (Eds.), Chemical Zoology Vol. 8 pp. 421-466. Academic P ess, New Yo k & London. Food and Ag icul u e O ganiza ion o he Uni ed Na ions (2004) The S a e o Food Insecu i y in he Wo ld, Rome, I aly. Galbia i, M., Ma ini, L. & Melcangi, R. C. (2003) Role o glial cells, g ow h ac o s and s e oid ho mones in he con ol o LHRH-sec e ing neu ons. Domes ic Animal Endoc inology 25, 101-108. Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 86 - Gagné, F. & Blaise, C. (2003) E ec s o municipal e luen s on se o onin and dopamine le els in he eshwa e mussel Ellip io complana a. Compa a i e Biochemis y and Physiology Pa C 136, 117–125. Gagné, F., Blaise, C., Pelle in, J. & And é, C. (2007) Neu oendoc ine dis up ion in Mya a ena ia clams du ing game ogenesis a si es unde pollu ion s ess. Ma ine En i onmen al Resea ch 64, 87-107. Gibbons, M. C. & Cas agna, M. (1984) Se o onin as an induce o spawning in six bi al e species. Aquacul u e 40, 189-191. Golding, D. W. & Pow, D. V. (1988) The new neu obiology – ul as uc u al aspec s o pep ide elease as e ealed by s udies o in e eb a e ne ous sys ems. In: Tho ndyke, M.C. &and Goldswo hy, G.J. (Eds.) Neu oho mones in In e eb a es. Camb idge Uni e si y P ess. pp. 7-18. Ha ison, W. F. & Kohn, J. A. (1997) Mic oscopic ana omy o in e eb a es. Wiley-Liss,Inc., New Yo k. Ha line, D. K. (2008) Wha is myelin? Neu on Glia Biology 4, 153-163. Ha line, D. K. (2011) The e olu iona y o igins o glia. Glia 59, 1215-1236. Henka , M. (1975) Ligh -induced changes in he s uc u e o pigmen ed g anules in Aplysia neu ons. Science 188, 155-157. Hidalgo, A., Ka o, K., Su cli e, B., McIl oy, G., Bishop, S. & Alahmed, S. (2011) T ophic neu on-glia in e ac ions and cell numbe adjus men s in he ui ly. Glia 59, 1296- 1303. Hi ipi, L., D. E. Bu ell, M. B own, P. Assanah, S anec, A. & S e ano, G. B. (1982) Analysis o monoamine accumula ions in he neu onal issues o My ilus edulis and Anodon a cygnea (Bi al ia)—III. Tempe a u e and seasonal in luences. Compa a i e Biochemis y and Physiology Pa C: Compa a i e Pha macology 71, 209-213. Ka hunen, T., Ma i, S., Ai aksinen, Tuomis o, L. & Panula, P. (1993) Neu o ansmi e s in he ne ous sys em o Macoma bal hica (Bi al ia). The Jou nal o Compa a i e Neu ology 334, 477-88. Ke a a, I., Denie , X., Hamza-Cha ai, A. & Minie , C. (2008) Endoc ine- ela ed ep oduc i e e ec s in molluscs. Compa a i e Biochemis y Physiology Pa C: Toxicology and Pha macology 147, 261-270. Chap e 4 | Neu ocy ological aspec s o ne ous ganglia o S. plana - 87 - Kho imchenko,Y. S. & De ido ich I. I. (1989) The e ec o dopamine and galope idol on cyclic AMP in he gonad o he bi al e mollusc Mizuhopec en yessoensis and he sea u chin S ongylocen o us in e meius. Compa a i e Biochemis y and Physiology- Pa C: Toxicology and Pha macology 92, 23-26 Kho imchenko, Y. S. & De ido ich, I. I. (1991) Monoamine gic and choline gic mechanisms o ep oduc ion con ol in ma ine bi al e molluscs and echinode ms: a e iew Compa a i e Biochemis y and Physiology- Pa C: Toxicology and Pha macology 100, 311-31 Klouche, M. S., De Deu wae dè e, P., Dellu-Hagedo n, F., Lakhda -Ghazal, N. & Benoma , S. (2015) Monoamine con en du ing he ep oduc i e cycle o Pe na pe na depends on si e o o igin on he A lan ic Coas o Mo occo. Scien i ic Repo s 5, 13715. K eiling, J. A., Jessen-Elle , Mille , K., J., Seegal. R. F. & Reinisch, C. L. (2001) Ea ly de elopmen o he se o one gic and dopamine gic ne ous sys em in Spisula solidissima (su clam) la ae. Compa a i e Biochemis y and Physiology Pa A: Molecula & In eg a i e Physiology 130, 341-351. Lacy, D. & Roge s, G. E. (1956) Recen obse a ions by ligh and elec on mic oscopy on he cy oplasmic inclusions o he neu ons o Pa ella ulga e. Jou nal o he Royal Mic oscopical Socie y 75, 172-175. Laming, P.R., Kimelbe g, H., Robinsonc, S., Salmd, A., Haw ylakd, N., Mulle e, C. & Roo s , K.B.N. (2000) Re iew: Neu onal–glial in e ac ions and beha iou . Neu oscience and Biobeha io al Re iews 24, 295–340. López-Sánchez, J. A., Maeda-Ma ínez, A. N., C oll, R. P. & Acos a-Salmón, H. (2009) Monoamine luc ua ions du ing he ep oduc i e cycle o he Paci ic lion's paw scallop Nodipec en subnodosus. Compa a i e Biochemis y and Physiology Pa A: Molecula & In eg a i e Physiology 154, 425-428. Meechonki , P., Ko i adhi, U., Cha cha al anich, K., S e a ugsa, P. & Wee acha yanukul, W. (2010) Localiza ion o se o onin in neu onal ganglia o he eshwa e pea l mussel, Hy iopsis (Hy iopsis) biala us. Jou nal o Molluscan S udies 76, 267-274. Meechonki , P., Asu apongpa ana, S., Jum omn, W., Ko i adhi, U. & Wee acha yanukul, W. (2012) Sexual di e ences in se o onin dis ibu ion and induc ion o synch onous la al elease by se o onin in he eshwa e mussel Hy iopsis biala us. Jou nal o Molluscan S udied 78, 297-303. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 96 - Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 97 - Abs ac Neu o ansmi e s do play key oles as o he ma u a ion o gonads in bi al es. Also, sex s e oids we e sugges ed o ha e also c i ical unc ions in he ep oduc i e con ol. Ye , i emains i ually unknown wha kind o in e play may exis wi h hose wo kinds o con ols, and i he e a e unde lying di e ences in he ne ous sys em s uc u e be ween sexes. To help in es iga ing his issue, a quan i a i e s e eological s udy was made on he ne ous ganglia o adul peppe y u ow shell, Sc obicula ia plana; a bioindica o species wi h ele ancy o local economies. Ma u e males, ma u e emales, and undi e en ia ed animals we e collec ed a Ria Fo mosa Lagoon (Po ugal), ixed wi h 10% bu e ed o malin, and p ocessed o pa a in embedding, o s udies a ligh mic oscopy. The animals we e se ially cu in o 35 µm hick sec ions ha we e ou inely s ained wi h hema oxylin-eosin. Sec ions ha ing he ce eb al, pedal, and isce al ganglia we e s udied. The pa ame e s o in e es we e he absolu e olumes o he ganglia and he o al and ela i e olumes o hei co ex and medulla, and, inally, he o al numbe o cells (neu ons, glial, and pigmen ed) in he whole ganglia and each compa men . The olumes and he cell numbe s we e es ima ed, espec i ely, by he Ca alie i’s p inciple, and by he op ical ac iona o me hod. Da a we e analyzed by ANOVA and pos -hoc es s. Apa om he ac ha emales ha e a highe glia- o-neu on nume ical a io, we do no ind o he majo di e ences be ween ma u ing males and emales. These ha e a g ea e ganglionic olume when compa ed wi h undi e en ia ed adul s; wi h males showing in e media e alues. These ac s poin ha size o he ganglia is ela ed somehow wi h he sex and gonad ma u a ion. The nume ical da a sugges ha cell size di e ences may be a he basis o he di e ences, because he e a e no signi ican di e ences in he o al cellula i y among he gende s udied. The h ee ypes o ganglia di e in o al olumes and olume a io o co ex e sus medulla. The signi ican ly g ea e olumes o he pedal ganglia (in ela ion o he ce eb al ones) and o he isce al ganglia (in ela ion o all o he ) imply mo e oluminous co exes and medullae, bu mo e neu onal and non-neu onal cells only in he isce al. We disclose o he i s ime ha a small bi al e as S. plana can ha e a mean o al numbe o neu al cells ha may each o e 68000 in he isce al ganglia. The new undamen al da a he ein hope ully can help sus aining be e in e p e a ions as o he bi al e neu ophysiology, and how i ela es wi h unsol ed issues in malacology, such as hose ela ed o nocicep i e beha io and i s implica ions in animal wel a e. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 98 - Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 99 - In oduc ion Neu oscience esea ch has shown ha emale and male b ains can be di e en in a ious ways (Leenaa s e al. 2013; Leong and Packa d 2014; Cosg o e e al. 2007), and ha gonadal s e oidal ho mones no only a e in ol ed in he egula ion o ep oduc ion, bu hey can also induce sexually dimo phic b ain de elopmen and o ganiza ion (A nold 2003). All hese ac s make he b ain – and by p oxy he whole gene al cen al ne ous sys em – a key a ge o s udy he basic ques ions ha p e ail as o in e sexual di e ences. Fo example, i is s ill in doub i he b ains (cells) wo k di e en ly o speci ic skills be ween sexes (Bu gale a e al. 2012). Mo eo e , neu onal su i al and degene a ion a e ela ed wi h sex-s e oids such as es ogens, p oges e one, and es os e one (Ga cia-Segu a and Bal haza 2009; Gillies and Mca hu 2010). These s e oid ho mones a e hough o link beha io s ei he wi h an in e nal mechanism, like o ula ion, o wi h an ex e nal ac o , such as a nu i ional condi ion (Gillies and Mca hu 2010). In some animals he sexual dimo phism is o en de ini ely obse ed. The e a e s udies co e ing a ange o animals, o ins ance bi ds, lamb, and a , in which speci ic zones o he b ains (indi idual beha io ) o di e en sexes we e in es iga ed, namely by compa ing he o gan’s olume and he neu on numbe (A nold 2003; Sahin e al. 2001). Fo in e eb a es, he ela ion be ween he sex o indi iduals and di e ences in i s ne ous sys em s uc u e and unc ion is much less clea . Howe e , some indings show ha di e ences may exis ; o ins ance, ui lies show signs o he sex di e ences on decision- making beha io in hei ma ing – hese modula ions may occu by exp ession o neu ons and ne wo ks in he ly’s b ain (Dickson 2008). Simila indings as o ol ac o y p e e ences we e ound, in ela ion o beha io o mo emen and ep oduc ion o nema odes, in which sexual dimo phism is ela ed o speci ic g oups o neu ons wi hin a co e ne ous sys em sha ed by bo h sexes. This speci ic senso y beha io seems o ake place om unc ional modula ion o common neu al ci cui s con olled by sex ch omosomes (Lee and Po man 2007). The e a e al eady a ew examples o ne ous sys em dimo phism in bi al es oo. Fo ins ance, in he Paci ic lion’s paw scallop, he de ec ion o speci ic monoamines – including dopamine (DA), se o onin (5HT), and no epineph ine (NA) – was highe in male o emales depending on he pe iods o he ep oduc i e cycle (López-Sánchez e al. 2009). These monoamines we e ound in gonad, diges i e gland, gill, and man le, whe e i may modula e mechanisms in ol ed in mo o beha io s. Mos o he 5HT was egis e ed in he male gonad, Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 100 - a nea ly all he ma u a ion s ages, excep a he spen s age, whe eas no epineph ine was abundan in he emale gonad (López-Sánchez e al. 2009). In he New Zealand mussel’s isce al ganglia, a ew selec i ely a ge ed monoamines and neu opep ides we e iden i ied by immunohis ochemis y, wi h peculia exp essions ound ei he in small o in la ge neu ons o bo h males and emales (Mahmud e al. 2008). Al hough he la e s udy did no sea ch o di e ences be ween sexes, i poin ed he p esence o subs ances labeled as “ esponsible o di e en aspec s o ep oduc ion and spawning”, and s essed he need o know he e ec s o seasons/gonad s ages; dialogues be ween he ne ous and gonad sys ems may logically occu . I should be no ed ha , as seen in o he animal axa, bi al e sex-s e oid ho mones a e mos ly p oduced in he gonads (Pazos and Ma hieu 1999; C oll and Wang 2007; Yan e al. 2011). In addi ion o he neu al mechanisms unde lying he sexual di e en ia ion/ma u a ion, we know ha many oxican s can dis up he ne ous sys em (including o bi al es), and his is a eason why much be e desc ip ions o ha sys em’s no mal mo phology and physiology in bi al es a e in need. These will su ely con ibu e o be e diagnose, app ecia e and p edic he neu o oxic impac s ha ha e been desc ibed in hese o ganisms (e.g., Ma ozzo e al. 2005; Ma in e al. 2008). O he easons a e mo e undamen al in na u e, such as unde s anding he e olu ion o he ne ous sys em in e sexual di e en ia ion, and he po en ial use o bi al es as expe imen al o ganisms, e en in biomedicine, o ge new mechanis ic insigh s (Nelson e al. 2010). In line wi h he all he abo e conside a ions, we hypo hesize he ein ha because o he key in ol emen o he ne ous ganglia in he gonad di e en ia ion and ma u a ion in bi al es, he mic oscopic mo phology o such ganglia can a y be ween sexes o ma u a ion s a us. Also, because each ganglia ype seems o ha e speci ic unc ions, we u he heo ize ha he loca ion/ unc ion o he ganglia shapes bo h i s size and cellula i y. To s a ackling hese hypo heses we did a s e eological s udy on he ne ous ganglia o gonad ma u ing and o exhaus ed peppe y u ow shell, in simila ly sized adul animals, looking in o di e ences be ween gende and ganglia ypes. We elec ed hei global and compa men olumes and he cellula i y, using up- o-da e gold-s anda d (design-based, unbiased, and e icien ) s e eology (Mayhew and Lucocq 2015). The species is o conside able ecological and economical alue (Wo all e al. 1983), and has been used as a bioindica o o ganism o a ious pollu an s (Chesman and Langs on 2006; Gomes e al. 2009; Pe idis e al. 2009; Ahmad e al. 2011). Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 101 - Ma e ials and me hods Animals and his ological p ocedu es Wild adul peppe y u ow shell (Sc obicula ia plana) we e collec ed a Ria Fo mosa Lagoon, Po ugal. The animals we e ans e ed o in house acili ies in he day o cap u e, and main ained in glass aqua ia (10 L), wi h ae a ed seawa e (salini y 30 ‰) and a a empe a u e o 15oC. In he day a e , a bi a ily sampled animals we e anes he ized by imme sion in a seawa e solu ion o magnesium chlo ide (6%), kep a oom empe a u e (≈ 20ºC). Thei leng h, wid h, heigh , and esh and o al mass we e measu ed be o e p ocessing. Each sampled animal (la e iden i ied, h ough his ology, as 6 males, 6 emales, and 6 animals wi h undi e en ia ed/spen gonad) we e emo ed ca e ully om he shell and ixed in o o o 24 h, using 10% bu e ed o malin, a oom empe a u e. A e ixa ion, he samples we e washed in 70% e hanol, dehyd a ed wi h inc easing concen a ions o alcohol (70% o 100%), clea ed in xylene, and in il a ed wi h pa a in. Dehyd a ion o in il a ion was ca ied ou using an au oma ic issue p ocesso (Leica TP1020, Ge many). Pa a in embedding used a s a ion (Leica EG 1140H, Ge many). Each animal was cu in o se ial sec ions (35 µm in hickness), on a mo o ized o a y mic o ome (Leica RM2155, Ge many), and kep on o 3-aminop opyl ie hoxysilane coa ed slides be o e hema oxylin-eosin (H&E) s aining, xylene clea ing, and DPX moun ing. Sec ions ha ing neu al ganglia we e used o s e eology (o he we e occasionally used o sexing he animal). The le ce eb al, igh ce eb al, pedal, and isce al ganglia we e he a ge s. Gene al quali a i e obse a ions we e made wi h a ligh mic oscopy (BX50, Olympus). S e eological analyses The Ca alie i’s p inciple was used o es ima ing he olume (V) o each ganglion (and sepa a ely o i s co ex and medulla), based on he o mula: V = ∙∑ A, whe e is he mean dis ance be ween analyzed sec ion planes, and A he sec ional a ea o he a ge o in e es (Gunde sen and Jensen 1987). The olume o he ganglia was de e mined semi-au oma ically, using he s e eological wo ks a ion CAST-G id ( e sion 1.5, Olympus Denma k), unning wi h a ligh mic oscope (BX50, Olympus), equipped wi h a mic oca o (Heidenhain MT-12), a mo o ized s age wi h 1 µm X-Y mo emen accu acy (P io ), and a CCD ideo came a (Sony) displaying li e image in a 17’’ CRT moni o (Sony). Analyses we e done unde he x10 Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 102 - objec i e lens. Fo each ganglion, he a eas o he co ex and medulla we e egis e ed in e e y sec ion ac oss hem, so o la e apply he abo e ci ed o mula. The o a ganglion was con i med by measu ing he sec ion hickness wi h he mic oca o (see below). The o al olumes we e used o es ima e he ela i e olumes (VV) o co ex and medulla in he ganglion: VV (compa men , ganglion) = V (medulla o co ex) ÷ V (ganglion). The numbe o ne ous ganglia cell was es ima ed ia he op ical disec o - ac iona o combina ion (Gunde son 1986) he o al numbe (N), making use o he gene al o mula: N = Q ∙ (1 / ss ) ∙ (1 / as ) ∙ (1/ hs ), whe e Q e e s o he o al numbe o cells ac ually coun ed in all he op ical disec o s; hs is he heigh sampling ac ion, cap u es he a io o he sec ion hickness ha was sc eened; as is he a ea sampling ac ion, i.e., he a io be ween he a ea o he coun ing ame and he a ea co e ed by each x,y mo emen ; ss is he sec ion sampling ac ion, i.e., he ac ion o o al sec ions sampled. He ein, hal o o al sec ions o each ganglia we e sampled and a minimum o 100 neu ons and 100 glia cells we e coun ed pe ganglia. The p ocedu e was also en o ced semi-au oma ically, wi h he abo e s e eological wo ks a ion. Coun ing was done unde he x100 (NA = 1.35) oil imme sion lens, in sys ema ically sampled ields. To check and accoun o any e en ual non-uni o m de o ma ion, was measu ed in e e y ield, and as we did no no ice such de o ma ion he a e aged was used o hs = h/ (Do ph-Pe e sen e al. 2001). He e, he a e age was 33 µm and disec o h was 25 µm. We se a minimal op gua d zone o 3 µm as he e is no he e ogeneous dis ibu ion o cells ac oss he z-axis ( on Ba held 2002). As o cellula i y, da a is gi en in a ious o ms, including spli ing he N in numbe s pe cellula con ingen s de ined by mo phology, iz. la ge and small neu ons, also usi o m, oundish, and iangula ly shaped glial cells, and, inally, pigmen ed neu al cells. Despi e he concep o la ge and small may seem dubious, unde he mic oscope i is easy o ensu e; each coun ed cell is agged as a “la ge” (o al/ ound, o en wi h la ge cy oplasmic g anules, Ø o ≈ 18 x 25 µm) o as a “small” neu on (o al/ ound, hick he e och oma in im, Ø ≈ 7 x 15 µm). S a is ical analysis The s a is ical analyses we e pe o med using he so wa e STATISTICA ( e sion 12.0 S a So Inc.). Da a se s we e checked o no mali y and homogenei y o a iances — using he Shapi o–Wilk’s W- es and Le ene’s es , espec i ely — p io o implemen a wo-way analysis o a iance ( wo-way ANOVA). A e a signi ican ANOVA, mul iple compa isons Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 103 - we e made simul aneously using he Tukey’s and Newman-Keuls’ es s; in case only one es would indica e signi icance, he esul would be conside ed as ma ginally signi ican . In some cases, loga i hmic and squa e oo ans o ma ions we e ca ied ou o no malizing and/o homogeniza ion o a iances o he aw da a. When ans o ma ion was unsuccess ul, a non- pa ame ic K uskal–Wallis ANOVA was used, ollowed by Mann-Whi ney U es s o pai s, wi h a sequen ial Bon e oni co ec ion. The signi icance le el was se a he usual 5%. Da a in Tables a e gi en ei he as mean (CV – coe icien o a ia ion = s anda d de ia ion / mean) and da a in g aphs a e gi en as mean connec ed o he espec i e 95% con idence in e al. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 104 - Resul s Quali a i e his ological obse a ions The gene al s uc u e o h ee ne ous ganglia ypes is p esen ed in Figu e 1, being easily dis inguishable he ou e basophilic and highly cellula co ex, which con as s wi h he e y eosinophilic inne medulla, essen ially composed o neu al cell p ocesses. Figu e 1. T ans e sely sec ioned ne ous ganglia o S. plana, picked andomly o illus a ion aims. A) Le ce eb al ganglion. B) Righ ce eb al ganglion. C) Pedal ganglion. D) Visce al ganglion. The ou e cellula co ex and he con as ing inne medulla a e labeled. H&E s aining. Scale ba = 200 μm. The neu ons a e ecognizable o hei bigge size, ha ing a single oundish nucleus, ypically wi h one nucleolus (Fig. 2A). The la ge neu ons usually a e a he ou e mos co ex and he smalle ones a e agg ega ed in be ween he la ge neu ons, and p edomina e in he inne co ex. Typical neu ons and pigmen ed cells appea in he medulla as well, sca cely bu Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 105 - sys ema ically. As o glial cells, hey a e sca e ed ac oss he ganglia, amids neu ons and medulla neu i es (Fig. 2B). They show h ee pheno ypes: usi o m, oundish, and iangula . Figu e 2. Pho omic og aphs aken om his ological hick sec ions o a ne ous ganglia o S. plana. A) Co ex (C ), wi h a s anding ou la ge neu on (LN). B) Medulla (M), wi h neu onal and glial eosinophilic p ojec ions, and soma a o glial cells (GC). C) De ail o co ex, whe e smalle neu ons (SN), an elonga ed glial cell (GC) and a pigmen cell (Pi) a e seen. H&E S aining. Scale ba : 10 μm. Fo de e mining he sex/gonadal s a us o each animal, we looked a he oo zones nea he pedal ganglion. The se ial sec ions allowed us o de ini ely pin-poin o each case which kind o game es, i any, we e di e en ia ing, a ing he animals as males, emales o as undi e en ia ed (Fig. 3); in he la e , he gonadal acini do no exhibi ac i e game ogenesis. Figu e 3. Pho omic og aphs aken om his ological hick sec ions o a ne ous ganglia o S. plana. A) Male: spe ma ogenesis is e iden wi hin he gonadal acini (Gn); a uppe and lowe le co ne s o he image. B) Female: gonadal acini (Gn) a e illed wi h oundish ma u ing oocy es. C) Image om one undi e en ia ed animal, wi h a ophic acini de oid o ma u ing game es, occasionally appea ing wi hin he connec i e issue (CT). PG – Pedal ganglion. H&E s aining. Scale ba : 200 μm. The quali a i e obse a ions did no allow o disce n cellula i y di e ences be ween animals o di e en sexes (de ined as pe hei gonad ma u a ion/di e en ia ion deg ee), o ac oss he h ee ne ous ganglia ca ego ies — le ce eb al ganglion (LCG); igh ce eb al ganglion (RCG); pedal ganglion (PG); isce al ganglion (VG) —, and so we could conclude only A B F Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 112 - Table 5. To al mean numbe (N) o neu al cells, by ganglia ype and gende in S. plana. Ganglia Gende Neu ons Glial cells Pigmen ed cells Small La ge To al Fusi o m Roundish T iangula To al LCG Males 2,636 (0.3) 4,056 (0.3) 6,692 (0.3) 1,613 (0.1) 2,461 (0.4) 1,672 (0.3) 5,746 (0.2) 997 (0.4) Females 2,886 (0.3) 4,977 (0.4) 7,863 (0.2) 2,047 (0.6) 3,904 (0.2) 3,584 (0.2) 9,535 (0.1) 1,595 (0.8) Undi e en ia ed 3,524 (0.4) 5,061 (0.2) 8,585 (0.3) 3,088 (0.4) 5,960 (0.6) 4,915 (0.5) 13,963 (0.5) 2,785 (1.3) RCG Males 2,430 (0.1) 3,052 (0.3) 5,482 (0.1) 1,379 (0.3) 2,416 (0.2) 1,926 (0.4) 5,721 (0.2) 817 (0.9) Females 2,268 (0.1) 3,555 (0.1) 5,823 (0.1) 2,866 (0.3) 4,248 (0.6) 2,365 (0.3) 9,479 (0.3) 1,458 (0.4) Undi e en ia ed 3,149 (0.6) 3,647 (0.5) 6,796 (0.6) 2,151 (1.0) 2,697 (0.9) 3,099 (0.2) 7,947 (0.6) 2,785 (1.3) PG Males 4,692 (0.4) 3,897 (0.2) 8,589 (0.2) 2,871 (0.4) 5,503 (0.4) 3,182 (0.4) 11,556 (0.3) 1,336 (0.5) Females 3,805 (0.2) 3,388 (0.3) 7,193 (0.2) 1,682 (0.3) 3,182 (0.5) 7,195 (0.2) 14,709 (0.3) 1,688 (0.5) Undi e en ia ed 4,745 (0.5) 3,534 (0.5) 8,279 (0.5) 2,894 (0.8) 7,438 (0.7) 3,914 (0.3) 14,246 (0.5) 2,906 (0.8) VG Males 7,408 (0.2) 8,578 (0.5) 15,986 (0.3) 6,200 (0.7) 11,455 (0.4) 8,670 (0.4) 26,325 (0.4) 3,241 (0.8) Females 8,964 (0.1) 9,339 (0.4) 18,303 (0.3) 7,833 (0.3) 14,407 (0.2) 10,207 (0.3) 32,447 (0.2) 3,554 (0.6) Undi e en ia ed 12,437 (0.6) 8,736 (0.4) 21,173 (0.5) 7,531 (0.7) 22,138 (0.8) 11,070 (0.4) 40,739 (0.7) 6,251 (1.3) Six animals pe gende we e used. Da a gi en as mean (coe icien o a ia ion). LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 113 - Table 6. To al mean numbe (N) o neu al cells in he co ex, by ganglia ype and gende in S. plana. Ganglia Gende Neu ons Glial cells Pigmen ed cells Small La ge Sum Fusi o m Roundish T iangula Sum LCG Males 2310 (0.3) 4001 (0.3) 6311 (0.3) 1218 (0.1) 1848 (0.4) 1335 (0.4) 4401 (0.2) 785 (0.4) Females 2745 (0.3) 4798 (0.4) 7543 (0.2) 1354 (0.6) 2806 (0.3) 2390 (0.2) 6550 (0.1) 1373 (0.8) Undi e en ia ed 3125 (0.4) 4887 (0.2) 8012 (0.3) 2173 (0.4) 4075 (0.6) 3345 (0.5) 9593 (0.4) 3274 (0.7) RCG Males 2171 (0.3) 2975 (0.3) 5146 (0.2) 1017 (0.3) 1513 (0.3) 1303 (0.4) 3833 (0.2) 498 (0.7) Females 2059 (0.1) 3273 (0.1) 5332 (0.1) 2112 (0.4) 2871 (0.6) 1571 (0.2) 6554 (0.4) 1027 (0.4) Undi e en ia ed 2801 (0.6) 3296 (0.4) 6097 (0.5) 1313 (1.1) 1477 (1.1) 2025 (0.4) 4815 (0.8) 2269 (1.3) PG Males 4382 (0.4) 3798 (0.3) 8180 (0.2) 2085 (0.4) 4358 (0.5) 2196 (0.4) 8639 (0.4) 978 (0.3) Females 3628 (0.3) 3206 (0.3) 6834 (0.2) 2467 (0.6) 5496 (0.2) 3188 (0.4) 11151 (0.3) 1407 (0.6) Undi e en ia ed 4346 (0.5) 3409 (0.5) 7755 (0.5) 2207 (0.8) 5230 (0.8) 2865 (0.4) 10302 (0.6) 2224 (0.9) VG Males 6903 (0.2) 8326 (0.5) 15229 (0.3) 4362 (0.7) 7279 (0.4) 5091 (0.4) 16732 (0.5) 2178 (0.7) Females 8371 (0.1) 9062 (0.4) 17433 (0.2) 5045 (0.3) 9595 (0.2) 6355 (0.3) 20995 (0.2) 2719 (0.5) Undi e en ia ed 10809 (0.5) 8322 (0.4) 19131 (0.5) 5398 (0.7) 12424 (0.6) 7752 (0.4) 25574 (0.6) 5000 (1.2) Six animals pe gende we e used. Da a gi en as mean (coe icien o a ia ion). LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 114 - Table 7. To al mean numbe (N) o neu al cells in he medulla, by ganglia ype and gende in S. plana. Ganglia Gende Neu ons Glial cells Pigmen ed cells Small La ge Sum Fusi o m Roundish T iangula Sum LCG Males 325 (0.6) 55 (1.0) 380 (0.4) 394 (0.1) 613 (0.5) 337 (0.6) 1344 (0.2) 212 (0.9) Females 141 (1.7) 180 (0.9) 321 (1.3) 693 (0.6) 1098 (0.1) 1194 (0.5) 2985 (0.3) 222 (0.7) Undi e en ia ed 398 (0.5) 174 (1.2) 572 (0.6) 916 (0.4) 1885 (0.6) 1569 (0.6) 4370 (0.5) 623 (0.8) RCG Males 259 (1.5) 77 (0.9) 336 (1.3) 362 (0.5) 903 (0.4) 623 (0.9) 1888 (0.4) 319 (1.3) Females 209 (0.9) 282 (0.5) 491 (0.5) 754 (0.2) 1377 (0.6) 794 (0.5) 2925 (0.2) 431 (0.4) Undi e en ia ed 347 (1.0) 351 (1.5) 698 (1.2) 837 (0.8) 1221 (0.6) 1075 (0.3) 3133 (0.4) 516 (1.3) PG Males 310 (1.2) 99 (1.3) 409 (1.2) 786 (0.6) 1145 (0.6) 986 (0.6) 2917 (0.4) 358 (1.4) Females 178 (1.1) 182 (1.3) 360 (1.1) 715 (0.4) 1700 (0.7) 1143 (0.3) 3558 (0.3) 280 (0.3) Undi e en ia ed 399 (0.7) 126 (1.3) 515 (0.8) 687 (0.9) 2208 (0.8) 1049 (0.3) 3945 (0.6) 682 (1.2) VG Males 505 (0.7) 252 (1.2) 757 (0.8) 1838 (0.7) 4176 (0.3) 3579 (0.4) 9593 (0.4) 1063 (1.1) Females 592 (0.8) 276 (0.8) 868 (0.6) 2789 (0.4) 4812 (0.5) 3851 (0.5) 11452 (0.4) 835 (0.9) Undi e en ia ed 1628 (1.0) 414 (1.0) 2042 (0.9) 2133 (0.7) 9714 (1.1) 3318 (0.5) 15165 (0.8) 1251 (1.8) Six animals pe gende we e used. Da a gi en as mean (coe icien o a ia ion). LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion. Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 115 - Table 8. Glia- o-neu on (numbe ) a io in he ce eb al, pedal and isce al ganglia medulla o S. plana. Ganglia Gende Glia- o-neu on a io Whole ganglion Co ex Medulla LCG Males 0.9 (0.11) 0.7 (0.17) 3.7 (0.24) Females 1.3 (0.25) 0.9 (0.25) 26.7 (0.83) Undi e en ia ed 1.3 (0.40) 1.2 (0.23) 8.9 (0.56) RCG Males 1.1 (0.14) 0.7 (0.16) 6.9 (0.54) Females 1.2 (0.50) 1.2 (0.34) 16.9 (0.98) Undi e en ia ed 1.1 (0.40) 0.7 (0.30) 9.7 (1.13) PG Males 1.5 (0.17) 1.0 (0.23) 17.2 (0.91) Females 2.2 (0.37) 1.7 (0.21) 18.8 (1.05) Undi e en ia ed 1.7 (0.15) 1.3 (0.14) 6.8 (0.14) VG Males 1.6 (0.16) 1.1 (0.26) 13.7 (0.58) Females 1.8 (0.13) 1.2 (0.10) 22.6 (0.96) Undi e en ia ed 1.8 (0.22) 1.3 (0.10) 12.1 (1.00) Six animals pe gende we e used. Da a gi en as mean (coe icien o a ia ion). LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion. Figu e 9. Glia- o-neu on (numbe ) a io in he ne ous ganglia o S. plana, conside ing he whole ganglia. A) Da a pe ganglion ype, i espec i e o gende . B) Resul s om all ganglia, g ouped pe gende . Di e en le e s mean signi ican di e ences. Da a as mean and 95% con idence in e al. LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion; M: males; F: emales; U: undi e en ia ed. *Based on ou c i e ia, he e is a ma ginal s a is ical di e ence be ween M and U (Newman-Keuls’ es , p = 0.031). * Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 116 - Figu e 10. Glia- o-neu on (numbe ) a io in he co ex o he ne ous ganglia o S. plana. A) Da a pe ganglion ype, i espec i e o gende . B) Resul s om all ganglia, g ouped pe gende . Di e en le e s mean signi ican di e ences. Da a as mean and 95% con idence in e al. LCG: le ce eb al ganglion; RCG: igh ce eb al ganglion; PG: pedal ganglion; VG: isce al ganglion; M: males; F: emales; U: undi e en ia ed. *Based on ou c i e ia, he e is a ma ginal s a is ical di e ence be ween VG and bo h LCG and RGC (Newman-Keuls’ es , p = 0.014 and p = 0.011, espec i ely). **Based on ou c i e ia, he e is a ma ginal s a is ical di e ence be ween M and U (Newman-Keuls’ es , p = 0.026). * ** Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 117 - Discussion To ou knowledge his is he i s s udy ha makes quan i ica ions o bi al e cells and o gans wi h design-based (o unbiased) s e eological ools, which do no ely on he olde me hods’ un ealis ic and uncon olled assump ions abou he s uc u e’s geome ic shape o spa ial o ien a ion and dis ibu ion o s uc u es in 3D space. The ad an ages o he unbiased me hods ha e been es ablished, illus a ed and e ined o e he las h ee decades (Gunde sen 1986; Gunde sen and Jensen 1987; Do ph-Pe e sen e al. 2001, Mayhew and Lucocq 2015). In bi al es we ind only one a icle ha used he op ical ac iona o , no o es ima ing any componen o he animal, bu a he o e ealing he exac numbe o he in ec ing p o ozoan pa asi e Pe kinsus ma inus in he man le o C assos ea i ginica (Remacha e al. 2008). The la e s udy and ou s illus a e well how he same s e eological echniques can ackle so a ied ques ions, no o men ion he la e ly discussed key po en ial o be pa o he new ield o “mo phomics”, in line wi h o he “omics” (Mayhew 2015; Mayhew and Lucocq 2015). The s e eology ools we used o s udy he ne ous ganglia o S. plana a e well ecognized in e eb a e neu ocy ology, and ha e been pa amoun o sus ain ad ances (Schmi z and Ho 2005; Walløe e al. 2014). Ou s udy is signi ican no only because i es s he echnology and un eils new da a o S. plana, sui ed o ackle hypo heses and sus ain mo pho unc ional in e ences, bu also since i encou ages u he use o unbiased s e eology in bi al e esea ch. The quan i a i e app oach he ein aimed o compa e a 3D- ele an size o he ne ous ganglia o S. plana (absolu e and addi ionally ela i e olumes o he whole ganglia and o i s co ex and medulla) and o he cellula i y, measu ed as he numbe o hei cons i uen cells. The hypo he ical backg ound was a plausible undamen al in luence o he animal sex (when i can be disclosed) in he mic oana omy o he bi al e ne ous sys em. We s udied animals ha we e ei he males o emales, as explici ly iden i ied by hei ma u ing game es, and also specimens ha could no be sexed because hei gonads we e spen and a ophic. Facing he key physiological modelling ac ions o he ne ous sys em on he game ogenesis o bi al es (Siniscalchi e al. 2004; Gagné e al. 2007; López-Sánchez e al. 2009), and e en ual (bu no well es ablished) eedback loops, a leas in heo y we s udied animals ha should be as unc ionally dissimila as S. plana adul s o a di e en sex could be. We hus op ed o analyze he h ee “gende ypes” as a way o p omo e he odds o cap u ing a di e ence, i i exis ed. Also, his s a egy helped o inc ease powe o s udying di e ences be ween ganglia ypes, Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 118 - wi hin he wo-way ANOVA, pa icula ly in case o a no signi ican e ec o gende – his was ele an he e as we wished o back/ex end ea lie sugges ed in e -ganglionic di e ences. As o gende di e ences, one ha is s a is ically con i med conce ns he o al olume o he ganglia, which is g ea e in emales han in he undi e en ia ed, wi h he males do no di e ing om ei he o he o he g oups. The co ex and medulla e idence basically he same di e ences, wi h one addi ional ma ginal being ound in co ex; wi h males ha ing a smalle olume (Fig. 5B). The no signi ican di e ence be ween males and emales conco ds wi h ou p e ious da a, ha despi e based on ano he echnic (3D- econs uc ion), o e ed es ima es in he same o de o magni udes and close o hose he ein (Tan iwisawa uji e al. 2015). I would be specula i e o poin one pa icula eason o he di e ence be ween emales e sus undi e en ia ed, bu i is a ac in pe ec acco dance wi h ou hypo hesis ha he sex/gonadal s a us “shapes” he bi al e ne ous sys em s uc u e — ei he because o he ac i i y o he la e in in luencing gonads (e.g., Siniscalchi e al. 2004) o by e ec s o ac o s o igina ed in he gonad (e.g., sex-s e oids) and impac ing on neu al elemen s (e.g., S e ano e al. 2003). I espec i e o he unc ional implica ions, wha makes emales ha ing g ea e olumes han he undi e en ia ed and enden iously mo e han males? A e he e mo e neu onal cells and/o bigge ones? Finally, a e he e any di e ences in he amoun /size o neu al p ocesses? This s udy was no designed o answe all hese ques ions, bu la e in his Discussion we will go back o hose so in e es ing and puzzling di e ences, a e deba ing he da a on cell numbe s. Despi e he e is no in e ac ion be ween gende and ganglia ype, he e is a s a is ically signi ican e ec o he la e in he olumes o he ganglia and o hei compa men s. The wo CG a e simila in size, bu olumes o e all inc ease signi ican ly owa ds he PG, which is g ea e han he ce eb al and much smalle han he VG. The co ical and medulla pa s do signi ican ly ollow he ends o he whole ganglia. Once mo e, he ac s nicely ag ee wi h ou p io wo k, in which we un eil he same pa e n in males and emales (Tan iwisawa uji e al. 2015). We can hus con iden ly sugges ha he size di e ences be ween all ganglia ypes a e independen o he gende and o he condi ion o being in a p ocess o gonad ma u a ion. In addi ion o he absolu e olumes, we look a he ela i e olumes (VV) o he co ex and medulla, quan i ying ha o e all he co ex is ≈ 60% and he medulla 40%. Ye , i gende does no seem o ma e o he co ex o medulla a io, he e is a s a is ically signi ican Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 119 - e ec o he ype o ganglia in VV, wi h he less oluminous CG showing he highes mean alues o he co ex VV, wi h PG being in e media e, and he VG ha ing he smalles alues; con e sely, he VV o he medulla ollowed a ma ching opposi e pa e n (Fig. 6). I is wo h men ioning ha we no ed an app oxima e end in ou p e ious s udy, bu we could no p o e signi icance (Tan iwisawa uji e al. 2015). Such ine s uc u al di e ences be ween he VV o he ganglia likely a e no a andom e en and should ha e a a ional and a unc ional impac . One possible eason can be ela ed wi h he numbe o neu i es ha eme ging om he co ex go in o he medulla, ha in absolu e e ms is expec ed o be g ea e a leas in VG, acing he highe o al cellula i y his ganglion has when compa ed wi h he o he s. A highe numbe and/o size o neu onal and glial p ojec ions would p omo e a ela i ely g ea e VV (medulla, ganglion), when compa ed wi h o he ganglia ypes. The lowes cellula i y o he LCG and RCG, logically wi h less p ojec ions going in o he medulla neu opil, would also explain he smalles VV (medulla, ganglion). On he o he hand, his so o a ionale does no explain he in e media e alue o he PG, as in his case he o al cellula i y is no g ea e han ha in ei he ype o CG (see discussions on cellula i y below). So, a mix u e o mo pho unc ional ac o s mus con ibu e o he di e ences in VV. Among hem, we can also hink abou s ill uns udied di e ences as o he neu on and glia cell olumes, also he deg ee o complexi y in hei in e connec ions, pa icula ly in he co ex o he ganglia, which can ha e impac s on he olume a io o co ex o medulla, be ween each ganglion ype, in iew o hei unc ions. To suppo he a ional o ou discussion we ecall ha each ganglia ypes has speci ic deg ees o o ganiza ion and unc ion. Fo ins ance, he VG is iewed as he mos di e en ia ed cen al ne ous sys em s uc u e in bi al es (Bullock 1965; Ha ison and Kohn 1997). Some e idences poin ed ha his ganglion is esponsible o in luencing he ca diac hy hm and mo ili ies o he shell, man le, siphons and gills (S e ano 1945; Bullock 1965; Ca oll and Ca apane 2007). The PG esponds o s imula ions o he oo , wi h local con ac ions, bu equi es he ce eb al connec ion o allow digging (Bullock, 1965). The CG play oles in he an e io adduc o con ol, in coo dina ion o isce al and pedal ac ions, and i is dominan on beha io al hy hms (Bullock 1965); he e can a dominance o he ce eb al unc ion (Wilkens, 2006). Along wi h he VG, he CG ha e oles in espi a o y me abolism (Mane e al. 1990; Jadha e al. 2012). An upda e iew o he ganglia unc ions can be ound in Gosling (2015). Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 120 - As o cellula i y, despi e he di e ences be ween gende s as o he o al V o ganglia, and also he di e ences be ween he olumes o he di e en ganglia ypes i espec i e o he gende , he e we e no majo dissimila i ies as o he N o neu ons, glial and pigmen ed cells, when compa ing he LCG and RCG wi h he PG; despi e he la e being signi ican ly bigge . By o he hand, he VG consis en ly — i.e., in whole ganglia, co ex and medulla — showed a signi ican ly highe N o neu ons and glial cells; bu no o pigmen s cells. The highe o al N in he VG is mos su ely di ec ly ela ed wi h he ac ha hey di ec ly/ unc ionally con ol a as a ea, as ecen ly s essed by Gosling (2015), which mus be based on mo e neu al cells. Abo e in he Discussion, a p opos o he di e ences in he absolu e olume o ganglia be ween emales, iz. wi h he undi e en ia ed specimens — wi h emales ha ing g ea e olumes — we ose ques ions abou wha could s uc u ally sus ain he dissimila i ies. As we did no ound di e ences in he absolu e numbe s, his ac means ha emales mus ha e a highe ela i e cellula i y, o ela i e olume o cells pe uni olume, ypically ep esen ed in s e eology as he NV. By di iding he V o a ganglion (o one o i s compa men ) by he N o cells i con ains we ge an es ima e o he NV (cell, con aining space). I we in es iga e his, in he neu ons o glia in he co ex, we ge a NV in he undi e en ia ed (o ≈ 4.3 ×105 neu ons /mm3 and 5.5×105 glia cells/mm3) ha mo e han doubles he alues o emales (o ≈ 2.0×105 neu ons/mm3 and 2.5×105 glia cells/mm3); wi h males si ua ed in be ween bo h o he gende . I we make his exe cise wi h all neu al cells, in all ganglia, we ind ha he undi e en ia ed animals ha e ≈ 2.3 mo e cells pe uni o ganglionic olume (≈ 11×10 6 cells/mm3), when compa ed wi h ei he emales (≈ 4.9×106 cells/mm3) o males (≈ 4.7×106 cells/mm3), ha a e globally qui e simila . These in e ences sugges ha , o e all, undi e en ia ed animals ha e a simila N o cells in hei ganglia i ed in o less olume, implying ha bo h he neu ons and glial cells a e mo e “concen a ed”, and so likely smalle in size; o else he ganglia olume would no be smalle . O e all, ou da a s ongly poin ha in S. plana he e a e gende /gonad s age ela ed undisclosed di e ences in he mean olume o neu ons and/o glial cells, and/o o hei p ojec ions — a ma e o coun less s udies in e eb a e neu oscience (Schmi z and Ho 2005) bu o ally “un ouched” in bi al es. Thus, looking a cell sizes is wo h s udying in he u u e o be e unde s and he cy ology and physiology o he bi al e ne ous sys em. S ill abou cellula i y, i is wo h poin ing ha his is he i s s udy in a bi al e ha p o ides es ima es o glia- o-neu on a ios, a undamen al aspec ha has been ho ly deba ed in Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 121 - e eb a es and mos ly o he human b ain, wi h he once well-es ablished 10:1 a io being ecen ly challenged wi h igo ous es ima es poin ing o a 1:1 a io, suppo ing he conclusion ha humans ha e an “isome ically scaled-up p ima e b ain” (Aze edo e al. 2009; Hilge ag and Ba bas 2009). Ou global da a o S. plana (i.e., all ganglia and gende combined) sugges a ≈ 1:1 glia- o-neu on a io in he co ex, and when joining he medulla he a io ises o ≈ 1:1.5. Howe e , he exac a io depends on he gende and on he ganglia ype — ei he ac o ac ing independen ly — wi h ce eb al ganglia ha ing signi ican ly lowe a ios, and emales showing he highes a io. In iew o he neu al suppo i e unc ions glial cells ha e ac oss phylogeny, ou da a likely ha e unc ional e ec s. Specula ions abou he new ac s would be a e ched, bu i is e y in e es ing o no e ha di e ences be ween sexes as o glia- o-neu on a io we e kep along e olu ion, up o humans, and ha hey may be also dependen on neu al egions (Pel ig e al. 2008; Oli ei a-Pin o e al. 2014). Ou new indings add one mo e piece o he puzzle o he e olu iona y o igins o he glia and o hei always acqui ed news oles (Ha line 2011), and o e “ancien oo s” in line wi h he no ion ha , once he e, b ains gained non-neu onal cells in pa allel wi h neu onal addi ions, esul ing in ai ly cons an ela i e densi ies/ a ios o non-neu onal cells (He culano-Houzel 2011). Rega dless o he in e es ing ag eemen among ou s udy and o he ecen ones as o ha cell a io, and al hough we used a “gold-s anda d “ echnically coun ing p ocedu e, we mus iew ou da a wi h cau ion, namely because we a e iden i ying neu ons e sus glial cells based only on hei mo phology as seen a ligh mic oscopy. Despi e we made a p eceding his ological and ul as uc u al s udy o ge u he mo phological insigh s abou S. plana neu al cells (see Chap e 4 o his Thesis), we canno disca d he possibili y ha a ma ginal numbe o e y small neu ons could be iden i ied/coun ed as glial cells; being he opposi e si ua ion much mo e unlikely o occu , in ou opinion. The i s design-based s e eological s udy ( echnically ha de o do a he ime) ha es ima ed neu ons and glial cells in humans deal wi h his ca ea oo (Pakkenbe g and Gunde sen 1988). Only mo e ecen ly he e a e an ibodies ha allow an unambiguous dis inc ion be ween hose cells ypes in humans and in commonly used oden s (He culano-Houzel and Len 2005; Lyck e al. 2008). Despi e a ew con inuous a emp s o p oduce and/o es an ibodies o disclosing neu onal subpopula ions in bi al es (C oll e al. 1993; Mahmud e al. 2008; Meechonki e al. 2010), he cu en lack o es ed and accep ed speci ic immunoma ke s o neu ons and glial cells in bi al es is one Chap e 5 | S e eology o he neu al ganglia o S. plana conside ing sex and gonadal s age - 128 - on Ba held, C. (2002) Coun ing pa icles in issue sec ions: choices o me hods and impo ance o calib a ion o minimize biases. His ology and His opa hology 17, 639-648. Yan, H., Li, Q., Liu, W., Ke, Q., Yu, R. & Kong, L. (2011) Seasonal changes o oes adiol-17β and es os e one concen a ions in he gonad o he azo clam Sinono acula cons ic a (Lama ck, 1818). Jou nal o Molluscan S udies 77, 116-122. Walløe, S., Pakkenbe g B. & Fab icius, K. (2014) S e eological es ima ion o o al cell numbe s in he human ce eb al and ce ebella co ex. F on ie s in Human Neu oscience 8, 1-9. Wilkens, L. A. (2006) Neu obiology and beha iou o he scallop. Chap e 5. In: Shumway, S. E. & Pa sons, G. J. (Eds.), Scallops: Biology, Ecology and Aquacul u e. Else ie , pp. 317-356. Wo all, C. M., Widdows, J. & Lowe, D. M. (1983) Physiological ecology o h ee popula ions o he bi al e Sc obicula ia plana. Ma ine Ecology P og ess Se ies 12, 267-279. CHAPTER6 IMPACTS OF AGE IN THE NERVOUS GANGLIA VOLUME AND CELLULARITY IN TWO ADULT SIZE-CLASSES OF THE BIVALVE PEPPERY FURROW SHELL ( SCROBICULARIA PLANA ) Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 131 - Impac s o age in he ne ous ganglia olume and cellula i y in wo adul size-classes o he bi al e peppe y u ow shell (Sc obicula ia plana) [Fo ma ed as a manusc ip o be submi ed o publica ion in an in e na ional jou nal. The e sion in his Thesis may change a e he e ision o be made by all p ospec i e au ho s.] SukanlayaTan iwisawa ujia,b,c, Ma ia J. Rochaa,b, Ana Sil ab, U haiwan Ko i adhid, Maja Jo dano ae, Miguel A. Pa dal and Edua do Rochaa,b aIns i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o (U.Po o), Po o, Po ugal. bIn e disciplina y Cen e o Ma ine and En i onmen al Resea ch (CIIMAR), Uni e si y o Po o (U.Po o), Po o, Po ugal. cKing Mongku 's Uni e si y o Technology Thonbu i (KMUTT), Bangkok, Thailand. dDepa men o Zoology, Facul y o Science, Kase sa Uni e si y, Bangkok, Thailand. eIns i u e o Biology, Facul y o Na u al Sciences and Ma hema ics, Ss. Cy il & Me hodiusUni e si y, Skopje, Macedonia. Cen e o Func ionalEcology (CFE), Uni e si yo Coimb a, Coimb a, Po ugal. Running i le: Does size/age ma e s o neu al ganglia o he peppe y u ow shell? Key wo ds: age, bi al e, cell numbe , g ow h, glia, ganglia, neu ons, s e eology, olume Co espondence o: Edua do Rocha Depa men o Mic oscopy Labo a o y o His ology and Emb yology Ins i u e o Biomedical Sciences Abel Salaza (ICBAS) Uni e si y o Po o (U.Po o) Rua de Jo ge Vi e bo Fe ei a n.º 228 4050-313 Po o Po ugal E-mail:e[email p o ec ed] Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 132 - Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 133 - Abs ac Scien is s a e now ce ain ha he ne ous ganglia o bi al es con ol many o he animal’s li e essen ial aspec s, including g ow h and gonadal ma u a ion and spawning- ela ed e en s. Basic ques ions on he s uc u e and unc ion o he bi al e ne ous sys em emain unanswe ed, such as e en ual in insic in luences and changes ela ed wi h de elopmen and age and also gende . Wi hin he scope o he s uc u al cha ac e iza ion we a e conduc ing on Sc obicula ia plana, we s a s udying in e ganglionic and sex- ela ed di e ences, and he ein we p opose o expand ou in es iga ion scope by looking a he size o he ganglia and neu al cellula i y wi h age. In iew o he gene ally highe plas ici y o he in e eb a e ne ous sys em, we hypo hesise ha i he adul animal con inues o g ow i s ganglia could con inue o de elop oo, inc easing o example i s cellula i y, i.e., ge ing mo e neu al elemen s, pa icula ly neu ons and glial cells. Twen y ou adul specimens o S. plana, wi h well- de ined gonads, sampled in he es ua y o he Mondego Ri e es ua y, we e used; six animals pe size-class and pe gende . Conside ing ha size is a p oxy o age, he animals we e spli in o wo-size classes, ha we named “Small” (age 2+ yea s) and “Big” (age 3+ yea s). Fo malin- ixed and pa a in-embedded animals we e cu in o 35 µm hick sec ions ha we e s ained in hema oxylin-eosin and used o s e eological analyses. These es ima ed o al olumes o ganglia, hei co ex and medulla ela i e olumes, and o al numbe o ea lie cha ac e ised neu al cells: neu ons, glial cells and pigmen ed cells. In animals no di e ing in size/leng h and mass, we ound in e ganglionic, sex- ela ed and size- ela ed signi ican e ec s upon he ganglionic olumes, ela i e olumes o co ex and medulla, and o al numbe s o bo h neu ons, glial cells, and pigmen ed cells. Unde mul i-way analysis o a iance, he e ec o size (age) was consis en ly ma ked, and s a is ically signi ican , wi h he olde specimens ha ing app oxima ely wice as bigge ganglia (i espec i e o i s ype and o sex), ha con ained signi ican ly mo e neu al cells o all ypes. The inc ease in cellula i y ook place in each whole ganglion, and in bo h co ex and medulla. Da a suppo ou hypo hesis ha neu ogenesis con inues o occu in adul S. plana, i espec i e o he animals’ gende . New ques ions a ise om ou esul s, namely abou he na u e and sou ces o neu al cell p ogeni o s. In line wi h ecen in e es o esea che s as o he u ili y o bi al es as models in neu osciences, we hink ha S. plana can also be a s imula ing model o neu ogenesis and age- ela ed s udies. Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 134 - Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 135 - In oduc ion Aging is a key e en o li ing o ganisms, and ypically en ails con inuous changes along li e, om bi h o ma u i y and hen a e by g adual declining owa ds senescence (Campisi and d'Adda di Fagagna 2007), which can ul ima ely lead o c i ical de e io a ion ollowed by dea h — conside ing he wide ange o physiological mechanisms and ou comes, a ple ho a o dele e ious changes ypically occu in neu al ac i i ies wi h he passage o ime (Ta dy 2003; Be s e al. 2005). Mainly because o he equen heal h p oblems ela ed wi h Human aging, mammals ha e been pa icula ly s udied in wha conce ns he p o usion o ne ous sys em changes wi h aging. Such s udies include innume ous mo phological e alua ions, including he use o quan i a i e app oaches (such as s e eological echniques), mos ly made in a s, mice, monkeys and humans (e.g., Samo ajski and Rols en 1973; Mon ei o e al. 1991; Hen ique e al. 2001). Resea ch on neu oscience o molluscs has been mainly made and inc eased based bo h on gas opods (C oll e al. 1993; F anchin e al. 1985; Janse e al. 1996; Simmons and Young 1999; To ska e al. 1968) and cephalopods (Amano e al. 2008; Di C is o 2013; Takuwa-Ku oda e al. 2003). Ye , he e a e a ew s udies wi h bi al es, such as one wi h My ilus edulis showing ha aging is linked o a decline o dopamine-s imula ed adenyla e cyclase ac i i y in he pedal ganglion (S e ano 1982), and al e he monoamine accumula ion in he neu onal issue (Bu ell and S e ano 1983). So, aging makes a di e ence in wha espec s he ne ous sys em o bi al es, and he e o e hese o ganisms can be much be e unde s ood in ha espec , wi h po en ial gains o he o e all unde s anding o aging. Indeed, i is ecognized by esea ch communi y ha in e eb a es ha e been e y impo an o ge new insigh s abou he physiology o aging, wo king bo h wi h eme ging and es ablished models o he s udy o human aging (Yeoman and Fa aghe 2001; Yeoman e al. 2012) Despi e he esea ch po en ial o molluscs, and bi al es in pa icula , o s udying he ne ous sys em aging and plas ici y, he e a e p ac ical p oblems oo. Pa icula ly, he e a e di icul ies o es ima ing he bi al e’s exac age when using inc emen al changes in g ow h lines o annual ma ks on he shell (Gosling 2004); e en so, inc eases in shell size a e well co ela ed wi h age. Fo ins ance, in he genus P o o heca animals eached a mean leng h o 3.7 cm a he age o app oxima ely 3.5-4 yea s, in Columbia (Shaw 1986), and in he genus Sc obicula ia i was in he ange o 2.2 cm a he age o abou 1.4 yea , in Bidasoa es ua y (Sola 1997). On he side o ad an ages, i should be s essed ha bi al es display a wide ange o li espans, om 1 o awesome ± 400 yea s as e iewed by Abele e al. (2009), which Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 136 - makes hem po en ially p one o ackle qui e di e en ques ions. To his aspec we mus add he ac ha he bi al es ne ous sys em has a pe cei ed (al hough no ye quan i ied) low numbe o neu al cells, making hem e y a ac i e o ganisms o s udying neu al ne wo ks (Ko syuba and Ko syuba 2002). Despi e ele ancy and oppo uni ies, a he ew s udies we e conduc ed in he bi al es’ ne ous sys em, and i ually none conce ning i s quan i a i e mo phology. I espec i e o de ails, i is well known ha he basic ypes o bi al e ganglia (ce eb al, pedal, and isce al) con ain neu ons ha a e c i ical o con olling he po olio o undamen al esponses needed o he indi idual, and ul ima ely o he species, o su i e, iz. ep oduc ion (Kho imchenko 1991), eeding (Ma ga e e al. 2007), mo emen (Hodgson and T ueman 1981), and ca diac unc ions (Kodi o 2011). I is also e iden ha he ganglia co ex ( he ganglionic ex e nal egion) includes he as majo i y o neu al cells (S e ano e al. 1990). Bo h he neu ogenesis and neu oplas ici y along li e a e s ill poo ly s udied opics in bi al es, and he ew exis ing publica ions a e de o ed no so much o wha happens in he adul s bu ocus ins ead he e y ea lie ma u ing s ages (Flyachinskaya 2000; Raine i 2009). In ue, adul neu ogenesis (including b ain egene a ion) has been neglec ed, con a ily o he g ea a en ion ha has been paid o his issue in in e eb a e and e eb a e animal models (Chen e al. 2013; Kizil e al. 2012). In iew o ca ea s, Vo onezhskaya e al. (2008) emphasized ha in spi e o “unde s anding o neu onal de elopmen in T ochozoa has p og essed subs an ially in ecen yea s, ela i ely li le a en ion has been paid o he bi al e molluscs in his ega d”. In a p e ious wo k we de o ed ou a en ion o look a e he size and cellula i y o he ne ous ganglia o he peppe y u ow shell (Sc obicula ia plana), quan i a i ely de ailing and compa ing each ganglia ype in ma u ing males and emales, and in animals ha did no ha e a di e en ia ed/ma u ing gonad o allow sexing hem. He ein, we con inue hose new esea ch e o s, by ad ancing he hypo hesis ha no only he sex bu also he age ac o , in adul s, may in luence he mic oscopic ana omy o he ne ous sys em o his species; pa icula ly he cellula i y o he ganglia, wi h e en ual implica ions o he size ( olume) o he ganglia. Ou a ionale elies o he ac ha neu al plas ici y exis sin he ne ous sys ems o in e eb a es and e eb a es, bo h along de elopmen and in adul s (Mo e 1996; Kizil e al. 2012; Ash on 2013; Chen e al. 2013). The ques o knowing i adul s loose (o i gain) Chap e 6 | Does size/age ma e s o neu al ganglia o he peppe y u ow shell? - 137 - neu al cells along li e has been a g ea e ocus o a en ion o neu oscien is s, and e en he once epu able iews o neu ogenesis in humans ha e been con inuously o e u ned (Cu is e al. 2011). As mos bi al es g ow con inuously, pe haps S. plana and o he s can gene a e new neu al cells as ju eniles and du ing adul li e; a leas be o e senescence. To s a s udying ou ques ions, we did a s e eological s udy on ganglia o wo size-coho s o adul ma u ing males and emales, knowing ha he body size in bi al es co ela es well wi h longe i y (Ridgway e al. 2011).