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Neu oendoc ine pa hways a isk? Sim as a in induces in e and ansgene a ional dis up ion in he
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keys one amphipod Gamma us locus a
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T. Neupa ha,£,⁎, N. Al esa,b,£, A.M. Machadoa,b, M. Pinhei oa,b, R. Mon esd, R. Rodild, S. Ba osa,c, R.
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Rui oa, L. Filipe C. Cas oa,b, J.B. Quin anad, M.M. San osa,b,⁎
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a CIIMAR—In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, Uni e si y o Po o,
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A enida Gene al No on de Ma os, S/N, 4450-208 Ma osinhos, Po ugal
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b FCUP – Depa men o Biology, Facul y o Sciences, Uni e si y o Po o, Po o, Po ugal
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c CITAB – Cen e o he Resea ch and Technology o Ag o-En i onmen al and Biological Sciences,
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Quin a de P ados – Ed. Blocos Labo a o iais C1.10, 5000-801, Vila Real, Po ugal
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d Depa men o Analy ical Chemis y, Nu i ion and Food Sciences, IAQBUS – Ins i u e o Resea ch on
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Chemical and Biological Analysis, Uni e sidade de San iago de Compos ela, R. Cons an ino Candei a
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S/N, 15782 San iago de Compos ela, Spain
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* Co esponding au ho s: CIIMAR - G oup o Endoc ine Dis up o s and Eme ging Con aminan s,
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Uni e si y o Po o, A enida Gene al No on de Ma os S/N, 4450-208, Ma osinhos, Po ugal (T.
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Neupa h) and FCUP - Depa men o Biology, Facul y o Sciences, Uni e si y o Po o, Po o, Po ugal
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(M.M. San os).
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E-mail add esses: neupa [email p o ec ed] (T. Neupa h), miguel.san[email p o ec ed] (M.M. San os)
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£ T. Neupa h and N. Al es con ibu ed equally o his s udy.
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This is he pos p in (accep ed manusc ip ) e sion o he a icle published in Aqua ic Toxicology
h p://dx.doi.o g/10.1016/j.aqua ox.2022.106095, © 2022, Else ie .
This manusc ip e sion is made a ailable unde he CC-BY-NC-ND 4.0 license: h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
Highligh s
-Sim as a in In e and ansgene a ional e ec s a e no well unde s ood in aqua ic o ganisms
-I ´s unexplo ed i sim as a in e ec s a e associa ed wi h neu oendoc ine sys em dis up ion
-Sim as a in in e / ansgene a ional exposu e se e ely impac s G.locus a neu oendoc ine egula ion
-Ecdys e oid, Dopamine, NO/cGMP/PKG, GABA and Choline gic signaling pa hways a e a ec ed
- These indings imp o e isk assessmen o biological ac i e compounds, such as Sim as a in
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ABSTRACT
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The p ima y ocus o en i onmen al oxicological s udies is o add ess he di ec e ec s o chemicals
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on exposed o ganisms (pa en al gene a ion – F0), mos ly o e looking e ec s on subsequen non-
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exposed gene a ions (F1 and F2 – in e gene a ional and F3 ansgene a ional, espec i ely). He e, we
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add essed he e ec s o Sim as a in (SIM), one o he mos widely p esc ibed human pha maceu icals
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o he p ima y ea men o hype choles e olemia, using he keys one c us acean Gamma us locus a.
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We demons a e ha SIM, a en i onmen ally ele an concen a ions, has signi ican in e and
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ansgene a ional (F1 and F3) e ec s in key signalling pa hways in ol ed in c us aceans´
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neu oendoc ine egula ion (Ecdys e oids, Ca echolamines, NO/cGMP/PKG, GABAe gic and Choline gic
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signalling pa hways), concomi an ly wi h changes in apical endpoin s, such as dep essed ep oduc ion
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and g ow h. These indings a e an essen ial s ep o imp o ing haza d and isk assessmen o biological
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ac i e compounds, such as SIM and highligh he impo ance o s udying he ansgene a ional e ec s
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o en i onmen al chemicals in animals’ neu oendoc ine egula ion.
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Keywo ds: Sim as a in; Gamma us locus a; Neu oendoc ine signalling pa hways; T ansc ip omic; In e
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and T ansgene a ional e ec s; Regula o y agencies.
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1.INTRODUCTION
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Aqua ic en i onmen s a e he ul ima e ese oi s o mos an h opogenic chemicals. Acco dingly,
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aqua ic o ganisms a e ch onically exposed du ing c i ical pe iods in ea ly li e s ages o e en o an
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en i e li e cycle (Capela e al., 2016; G oh e al., 2015; Majo e al., 2020). Knowledge o he haza ds
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and isks associa ed wi h ch onic exposu e o an h opogenic chemicals has g own conside ably o e
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he las decades. Howe e , con as ing wi h he ex ensi e li e a u e dealing wi h he di ec e ec s o
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chemicals on pa en al exposed o ganisms (F0), li le is known abou he e ec s ha pa en al exposu e
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(F0) exe s on indi ec ly exposed (F1 and F2) o uly non-exposed (F3) gene a ions – in e gene a ional
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and ansgene a ional e ec s (Bhanda i e al., 2015; Kalichak e al., 2019; Neupa h e al., 2020a).
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Ye , gi en he po en ially b oad ecological impac s o such e ec s in aqua ic ecosys ems, a g owing
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scien i ic in e es has eme ged (Shaw e al., 2017; Van Cauwenbe gh e al., 2020). S ill, only a limi ed
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numbe o in e and ansgene a ional s udies a e a ailable in he li e a u e, ocusing on a ew
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endoc ine dis up ing chemicals (EDCs; e.g. Bisphenol A, some pes icides and ph hala es), known o
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hei ubiqui y in he en i onmen and associa ed wi h ep oduc i e endoc ine diso de s (B e ik e al.,
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2018; Li e al., 2020; San angeli e al., 2019). Howe e , many o he na u al and syn he ic subs ances,
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including pha maceu icals, a e suspec ed o hijack endoc ine unc ions and dis up ep oduc ion,
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de elopmen , neu onal p ocesses and/o o he impo an physiological esponses; no only in exposed
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indi iduals, bu also in subsequen non-exposed gene a ions (Gille e e al., 2018; Tusche and Day,
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2019; Walke and Go e, 2011). One o such pha maceu icals is he hypocholes e olaemic
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pha maceu ical sim as a in (SIM). SIM is among he mos p esc ibed human pha maceu icals.
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Ho e e , only a ew s udies ha e examined i s concen a ions in su ace wa e s. Acco ding wi h he
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a ailable in o ma ion, SIM can each he aqua ic en i onmen s, in he icini y o u ban a eas, wi h
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concen a ions abo e 100 ng/L (No way 108 ng/L - Lang o d and Thomas, 2011; India 414.9 ng/L - Khan
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e al., 2021 and Sou h A ica 1585 ng/L - Te e e al., 2020). SIM mode o ac ion ope a es h ough
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inhibi ion o he enzyme 3-hyd oxy-3-me hylglu a yl-coenzyme A educ ase (HMGR), a a e-limi ing
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s ep o he me alona e pa hway (MP), esponsible o he de no o syn hesis o choles e ol in
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e eb a es and he syn hesis o he me hyl a nesoa e ho mone (MF) in c us aceans, wi h a key ole
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in he egula ion o ep oduc ion and mol ing (Bellés X. e al., 2005; Fen e al., 2006; Li e al., 2010;
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San os e al., 2016). Al hough he inal s eps o he me alona e pa hway di e be ween e eb a es
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and c us aceans, mos o he enzyma ic cascade is highly conse ed among me azoans (San os e al.,
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2016). In ou p e ious esea ch (Neupa h e al., 2020a), G. locus a was exposed o 4 consecu i e
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gene a ions (F0 o F3) o en i onmen ally ele an concen a ions o SIM (64 and 320 ng/);
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simul aneously, he o sp ing o he F0-exposed gene a ion we e aised in SIM- ee wa e du ing h ee
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subsequen gene a ions (non-exposed F1 o F3). Ou indings showed ha ep oduc ion and g ow h
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we e pa icula ly impac ed by en i onmen ally ele an concen a ions o SIM, no only in di ec ly
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exposed animals (exposed F0 o F3), bu also in he gene a ions aised in SIM- ee wa e (non-exposed
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F1 and F2, in e gene a ional, and non-exposed F3, ansgene a ional e ec s). In addi ion, a signi ican
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dec ease in MF le els was eco ded in exposed and non-exposed emales du ing ou gene a ions (F0
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o F3). Al hough he molecula mechanism(s) unde lying he obse ed ansgene a ional e ec s o SIM
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a e no ully de ined, epigene ic emodeling s ands as he mos plausible explana ion o hese
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indings, since mos o en i onmen al chemicals do no ha e he abili y o al e DNA sequences o
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p oduce di ec gene ic mu a ions in such a sho ime ame. In ag eemen , Al es e al. (2021) epo ed
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a signi ican down egula ion o DNA me hyl ans e ase 1 (DNMT1) gene exp ession upon exposu e o
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G. locus a o 320 and 8000 ng/L o SIM, concomi an ly wi h global DNA hypome hyla ion.
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Al hough se e e e ec s on ep oduc ion, g ow h, and de elopmen a e known o occu in a h opods,
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including amphipods, ollowing exposu e o SIM o o he s a ins (Dahl e al., 2006; Liu e al., 2019;
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Neupa h e al., 2014; O iz de Ga cía e al., 2014; Zapa a e al., 2003), he absence o mechanis ic
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associa ion be ween exposu e and obse ed e ec s limi s ou unde s anding, no ably ega ding he
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con ibu ion o endoc ine dis up ion p ocesses.
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C us aceans u ilize a a ie y o neu oendoc ine signaling cascades o egula e mol ing, g ow h, and
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ep oduc ion, wi h mos being unique o c us aceans and a h opods in gene al (Rod íguez e al., 2007;
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Waye and T udeau, 2011). The c us acean X-o gan–sinus gland complex is he majo neu oendoc ine
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s uc u e, esponsible o he elease o an a ay o neu opep ides ho mones: including MIH (mol
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inhibi ing ho mone), GIH (gonad inhibi ing ho mone), MOIH (mandibula o gan inhibi ing ho mone)
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and CHH (c us acean hype glycemic ho mone), ho mones ha ha e been ex ensi ely s udied o e he
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las decades and demons a ed o ha e a cen al ole in homeos asis (Gismondi and Joaquim-Jus o,
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2019; Hyne, 2011). Wi h he ecen ad ances in nex -gene a ion sequencing, as well as he g owing
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a ailabili y o a h opods ansc ip omes and genomes, new neu opep ides genes ha e been
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ca aloged in di e en c us acean species, some o which wi h a o eseen ole in ep oduc ion and
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de elopmen , such as Alla os a ins, SIFamide, P oho mones, among o he s (Nguyen e al., 2016).
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Besides X-o gan–sinus gland neu opep ides ho mones, he sesqui e penoid ho mone MF is
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syn he ized in he mandibula o gan, whe eas a ious ypes o s e oidal ho mones implica ed in
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mol ing, such as ecdys e oids, a e p oduced by he Y-o gan. These ho mones, in ol ed in he
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endoc ine sys em o c us aceans, a e physiologically linked o he X-o gan–sinus gland complex
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(Sub amoniam, 2000). Apa om hese a h opod-speci ic neu opep ides, sesqui e penoid and
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s e oidal ho mones, o he neu o ansmi e s, like biogenic amines (e.g. se o onin, dopamine), a e
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associa ed wi h he endoc ine egula ion o physiological unc ions ha con ol ups eam
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neu osec e o y p ocesses sha ed by bo h a h opods and e eb a es (Campbell e al., 2004; Vaud y
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and Kah, 2017). In c us aceans, he biogenic amines a e linked o he X-o gan–sinus gland complex by
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con olling he elease o he neu o-ho mones MIH and CHH.
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In addi ion o dis up ing he homeos asis o endoc ine ho mones, di e en EDCs we e also epo ed
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o p oduce mul iple ac ions in he neu al sys em, being neu o oxic pa icula ly in ea ly li e s ages (Gu
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e al., 2019; Sun e al., 2016; Xu and Yin, 2019). Se e al s udies in humans and oden s ha e p o ided
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e idence o posi i e associa ions be ween p e- /pos -na al exposu e o ce ain EDCs (e.g., bisphenol
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A, ph hala es and pes icides) and impai ed neu ode elopmen al ou comes in o sp ing (e.g. emo ional,
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cogni i e, mo o and muscula diso de s) leading o li elong o e en ansgene a ional dys unc ions
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(Lupu e al., 2020; Repouskou e al., 2020). In ac , ecen epidemiological and oxicological s udies
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es ablished a obus link be ween human neu ological diseases and exposu e o ce ain EDCs
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(T asande e al., 2015). Gi en he impo ance o neu oendoc ine con ol o he main enance o
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homeos asis, u he e o s should be made o add ess he e ec s o EDCs a his le el. Ye , i is no
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clea whe he he obse ed e ec s occu as di ec neu o oxici y o EDCs o esul om an indi ec
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e ec h ough dis up ion o endoc ine unc ions (Knigge e al., 2021; Waye and T udeau, 2011).
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The eme gence o new genomic ools, which can p o ide a high h oughpu sc eening o gene
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exp ession changes, a e e olu ionizing he way eco oxicologis s add ess he impac o en i onmen al
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s esso s on model o ganisms (Chen and Li, 2016; Me ick, 2019; Wang and Chang, 2018). These
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“omics” echnologies p o ide obus app oaches o u he ad ance he mechanis ic linkage be ween
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exposu e and e ec s o po en ial endoc ine dis up o s like SIM, in c us aceans. The e o e, conside ing
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he indings o Neupa h e al. (2020a), epo ing a ma ked ep oduc i e and g ow h e ec in G. locus a
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a e in e and ansgene a ional exposu e o SIM, he p esen s udy pe o med a comp ehensi e
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in es iga ion ha simul aneously assessed he in e and ansgene a ional (F1 and F3) molecula
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changes o genes/pa hways ela ed wi h he egula ion o he endoc ine sys em, wi h a special ocus
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on he neu oendoc ine sys em and b ain unc ions. In o de o imp o e ou knowledge on he
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molecula mechanisms ela ed wi h he po en ial endoc ine/neu o oxic e ec s o SIM h oughou G.
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locus a gene a ions, we add essed he e ec s pa icula ly in i e key signalling pa hways in ol ed in
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c us aceans´ neu oendoc ine egula ion, i.e., Ecdys e oids, Ca echolamines, NO/cGMP/PKG,
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GABAe gic and Choline gic signalling pa hways.
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2. Ma e ial and Me hods
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2.1. Expe imen al design
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To ho oughly analyze he e ec s o en i onmen ally ele an concen a ions o SIM on endoc ine and
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neu al sys ems o G. locus a in an in e and ansgene a ional con ex (non-exposed F1 and F3), we
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ook ad an age o he expe imen conduc ed by Neupa h e al. (2020a). B ie ly, he expe imen
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s a ed wi h one-week-old G. locus a o sp ing (pa en al gene a ion - F0) con inuously exposed o an
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en i onmen ally ele an concen a ion o SIM (320 ng/L p epa ed in 0.0005% ace one), plus con ol
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(0.0005% ace one in il e ed na u al seawa e ). Fi y o sp ing, om ou labo a o y cul u e, we e
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andomly alloca ed in 7L aqua ia ( ou pe ea men ) and exposed o SIM up o adul hood (55-65
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days). In each aqua ium, wi h a 1cm laye o na u al clean sedimen , he amphipods we e kep in
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il e ed na u al sal wa e (33-35‰ salini y) wi h a empe a u e and pho ope iod se o 20°C and 16:8
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hou s (ligh :da k) and eed ad libi um wi h Ul a sp. Wa e enewal in each aqua ium occu ed e e y
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h ee days and inal SIM concen a ion was e-es ablished a e he SIM solu ion was spiked di ec ly in
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he aqua ia and p ope ly s i ed. The ac ual SIM concen a ion was moni o ed in he cou se o he
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assay (Neupa h e al., 2020b). In o de o e alua e he pu a i e in e and ansgene a ional e ec s o
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SIM, he F0 o sp ing o each ea men we e alloca ed in SIM- ee wa e (na u al il e ed seawa e )
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o h ee consecu i e gene a ions (F1, F2 and F3). Each gene a ion was ini ia ed wi h i y o sp ing o
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he p e ious one and kep o 55 o 65 days. A he end o each gene a ion, Females we e sampled
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immedia ely a e he hi d ep oduc ion a e ma u i y o de e mine g ow h, ep oduc ion and
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me hyl- a nesoa e (MF) le els; he las measu ed acco ding o he me hods de eloped by Mon es e
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al. (2017). Fu he mo e, h ee andomly selec ed emales om con ol g oups (F1 and F3) and om
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320 ng/L SIM in e / ansgene a ional g oups (F1 and F3), we e p ese ed in RNA la e a −80°C un il
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indi idual use in RNA-sequencing analyses. Fu he me hodological de ails a e a ailable in Neupa h
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e al., (2020a,b).
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2.2. Analyses o he dis up ed endoc ine and neu al genes in F1 and F3 G. locus a emales’
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ansc ip ome
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The comp ehensi e ansc ip ome assembly p oduced in Neupa h e al. (2020a,b) was used in he
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cu en s udy o ho oughly e alua e he unc ional anno a ed genes o G. locus a emales om in e
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and ansgene a ional exposu e o SIM (non-exposed F1 and F3 gene a ions, espec i ely). B ie ly, o
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p oduce he G. locus a emales ansc ip ome epo ed in Neupa h e al. (2020a,b), RNA was
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ex ac ed om 3 andomly selec ed G. locus a emales pe condi ion [con ol and SIM
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in e / ansgene a ional g oups (F1 and F3)] and indi idually sequenced using he Illumina
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HiSeq2500pai ed-end (2x150) sys em. No ably, he whole amphipod body was used a he han
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speci ic o gans, because G. locus a emales a e oo small o ully sepa a e he o gans o in e es
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wi hou con amina ion om o he issues. The RNA-Seq da ase s we e de no o assembled using he
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T ini y assemble and unc ionally anno a ed wi h he T ino a e sui e. Las ly, he di e en ial gene
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exp ession (DEGs) analyses (False Disco e y Ra e – co ec ed (FDR) p- alue < 0.05, log2| old change|
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≥ 2 and blas e- alue less han 1×10-29) we e pe o med using he Degus pla o m and KEGG pa hways
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sc u inized wi h he KAAS webse e (de ailed in o ma ion a ailable in Neupa h e al., 2020b, sec ions
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2.3 o 2.7).
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In he p esen s udy, all he F1 and F3 unc ional anno a ed genes om he Neupa h e al. (2020b)
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ansc ip ome we e i s manually analized and a high numbe o genes in ol ed in neu oendoc ine
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con ol we e iden i ied. Based on hese indings, we pe o med a da a-d i en app oach sea ching o
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he main molecula componen s o he neu oendoc ine ne wo k om all unc ional anno aned genes.
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We ocused pa icula y on he me abolic pa hways ela ed wi h mol ing, ep oduc ion and neu al
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egula ion (Ecdys e oid, Ca echolamines, NO/cGMP/PKG, GABAe gic and Choline gic signalling
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pa hways). Conside ing ha c us acean neu oendoc ine da a is limi ed in KEGG da abases, o p ope ly
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s udy hese specialized physiological p ocesses in G. locus a, he unc ional anno a ed genes ela ed
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wi h he neu oendoc ine sys em we e sys ema ically e iewed using a manual app oach, hus a oiding
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mis ep esen a ion o G. locus a ansc ip omic da a due o anno a ion omission.
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Thus, o pe o m a gene unc ional analysis, we i s sea ched known p o ein names wi hin se e al
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pla o ms such as Unip o (h ps://www.unip o .o g/ accessed in Ma ch 2020), Geneca d
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(h ps://www.geneca ds.o g/ accessed in Ma ch 2020), NCBI (h ps.//www.ncbi.nlm.nih.go /
203
accessed in Ma ch 2020) and Nex p o (h ps://www.nex p o .o g/ accessed in Ma ch 2020). When
204
a ailable, he in o ma ion was ob ained om species wi h he closes axonomy o G. locus a. We hen
205
used di e en da abases (NCBI-h ps://www.ncbi.nlm.nih.go /gene accessed in Ap il 2020; Reac ome-
206
h ps:// eac ome.o g/ accessed in Ap il 2020; Biog id 4.4-h ps:// hebiog id.o g/ accessed in Ap il
207
2020; STRING 11.5- h ps://s ing-db.o g/ accessed in Ap il 2020; In Ac -
208
h ps://www.ebi.ac.uk/in ac / accessed in Ap il 2020 and Rhea- h ps://www. hea-db.o g/ accessed
209
in Ap il 2020) o e ie e po en ial di ec and/o indi ec biological in e ac ions be ween p o eins
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encoded by selec ed DEGs ela ed o he neu oendoc ine sys em. All pu a i e in e ac ions eco ded
211
by he da abases abo e we e con i med h ough a de ailed e iew o unc ional s udies in he
212
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li e a u e wi h a special ocus on c us aceans and when he in o ma ion o c us aceans was sca ce,
213
on insec s (e.g. Hyne, 2011; S e kel and Oli ei a, 2017; Co i e al., 2009; Mykles e al., 2010; Zhang e
214
al., 2014b; Nako e al., 2018; Takesian and Hensch, 2013). Addi ionally, we used a h opods´ dedica ed
215
unc ional s udies ha we e a ailable in he li e a u e o ind p o eins encoded by he selec ed DEGs
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o in e es ela ed wi h ecdys e oids and mol ing p ocesses ha we e no ound in he da abases used
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(e.g. Qian e al., 2014; S ee e al., 2019; Webs e , 2015a,b; Zhou e al., 2019; Gui a d e al., 2011).
218
All his da a was used o manually design he neu oendoc ine pa hways p esen ed in he p esen s udy
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(Figu e 1).
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8
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Figu e 1 – S udy wo k low
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3. Resul s and discussion
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Due o he ex ensi e use o indus ial, ag icul u al and pha maceu ical p oduc s, ens o housands o
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chemicals a e eleased annually in o aqua ic en i onmen s (E ans e al., 2019; Rzymski, 2017; Wang
225
and Yang, 2016). Howe e , o da e, only an in ini esimal ac ion o en i onmen al chemicals has been
226
assessed o hei endoc ine-dis up ing po en ial in wildli e (B ack e al., 2018; San os e al., 2018).
227
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(A amo e al., 2013; Chang e al., 2015; Gallo e al., 2016). Ca echolamines, pa icula ly DA, media e
399
he elease o se e al c i ical neu o-ho mones, such as C us acean Hype glycaemic Ho mone (CHH)
400
and al e a ions on CHH le els can lead o dis up ion o impo an biological p ocesses such as suga
401
and lipid me abolism (Bulau e al., 2003; Chang, 2005) and osmo egula ion (Chung and Webs e , 2006).
402
In ou ansc ip ome, besides he down egula ion o TH and VSP men ioned abo e, we also de ec ed,
403
in F1, a down egula ion o Dopamine B- hyd oxylase (DBH) ha ca alyzes he con e sion o DA o
404
NEP/EP, and a down egula ion o CHH.
405
406
Figu e 4 – Ca echolamines pa hway in C us aceans. Genes modula ed by SIM and non- esponsi e genes in F1 and F3
407
gene a ions a e p esen ed in boxes. Red ex inside he boxes indica es gene exp ession down egula ion in F1 and/o F3
408
gene a ions and black ex inside he boxes indica es unchanged gene exp ession in F1 and F3 gene a ions. No gene was
409
ound o be up egula ed in his pa hway. . TH –Ty osine hyd oxylase; DHPR – dihyd op e idine educ ase; BH4 –
410
e ahyd obiop e in; L-DOPA – L-3,4-dihyd oxyphenylalanine; CPs – cu icle p o eins; P oPO – P o phenol oxidase; PO – Phenol
411
oxidase hype glycemic ho mone; VSP – enom se ine p o ease Bi- sp; PPAE – p ophenoloxidase ac i a ing enzyme; CHH –
412
c us acean hype glycemic ho mone; DBH – dopamine be a-hyd oxylase.
413
414
3.3. NO/cGMP/PKG signalling pa hway
415
As a o emen ioned, MIH can nega i ely con ol he syn hesis o ecdys e oids. MIH egula es he
416
NO/cGMP/PKG signalling, c i ical o ecdysis (Lee and Mykles, 2006). The e is consensus ha cyclic
417
guanosine monophospha e (cGMP) ac s as an in acellula signalling messenge o MIH-media ed
418
ac ion (Chen e al., 2018; Co i, 2012; F ancis e al., 2010; Nagai e al., 2009; Xu e al., 2019), leading o
419
an ac i a ion o cGMP-dependen p o ein kinase G (PKG) and subsequen inhibi ion o
420
ecdys e oidogenesis (Co i e al., 2009; Mykles e al., 2010). Co i e al. (2009) ound ha MIH induces
421
a sus ained inc ease in cGMP, by he ac i a ion o memb ane-bound guanylyl cyclases (GYC32E and
422
16
GUCY1B1) ha con e guanosine iphospha e (GTP) o cGMP. In he p esen s udy, we obse ed a
423
down egula ion o GYC32E and GUCY1B1 in F1 and F3, indica i e o a dec ease in cGMP, al houg h he
424
ecep o - ype guanyla e cyclase Gyc76C (GYC76C) and he soluble guanyla e cyclase 89Db (GYC89DB)
425
emained unchanged (Figu e 5). In e es ingly, a down egula ion o cGMP-speci ic 3',5'-cyclic
426
phosphodies e ase (PDE5A), ha p e en s he con e sion o cGMP o i s inac i e o m guanosine
427
monophospha e (GMP), was also down egula ed in F1 and F3, sugges ing a cGMP igge ed ac ion
428
(Figu e 5), e en hough 3',5'-cyclic phosphodies e ase pde-4 (PDE4) was non- esponsi e. Also, he
429
guanyla e kinase-like iso o m X1 (GUK1), in ol ed in cGMP and GMP ecycling, was unchanged.
430
Howe e , a down egula ion o nep ilysin-1 (NEP1) ha p omo es he deg ada ion o a ial na iu e ic
431
pep ide (ANP) was obse ed in F1 and F3 which indica es an ac i a ion o cGMP, since mo e ANP is
432
a ailable o cGMP syn hesis. Howe e , he a ial na iu e ic pep ide ecep o B (ANR) was ound o
433
be down egula ed in F1 and F3, sugges ing a dec ease o cGMP, since he binding o ANP o i s ecep o
434
ac i a es he syn hesis o cGMP (Chen e al., 2008; Piggo e al., 2006) (Figu e 5). Thus, wi h hese
435
indings aken oge he , we hypo hesize ha SIM igge s a eedback mechanism o con ol cGMP
436
le els and subsequen inhibi ion o ecdys e oidogenesis h ough he balances be ween he ac ion o
437
GUCYB1/GYC32E and PDE5A and be ween NEP1 and ANR in F1 and F3.
438
439
Figu e 5 – NO/cGMP/PKG signalling pa hway in C us aceans. Genes modula ed by SIM and non- esponsi e genes in F1 and
440
F3 gene a ions a e p esen ed in boxes. Red ex inside he boxes indica es gene exp ession down egula ion in F1 and/o F3
441
gene a ions and black ex inside he boxes indica es unchanged gene exp ession in F1 and F3 gene a ions. No gene was
442
ound o be up egula ed in his pa hway.. MIH – mol inhibi ing ho mone; GTP – guanosine iphospha e; cGMP – cyclic
443
guanosine monophospha e; PKG – cGMP-dependen p o ein kinase; GYC32E – guanyla e cyclase 32E; GUCY1B1 – guanyla e
444
cyclase soluble subuni be a-1; GYC76C – ecep o - ype guanyla e cyclase Gyc76C; GYC89DB – soluble guanyla e cyclase
445
89Db; PDE5A – cGMP-speci ic 3',5'-cyclic phosphodies e ase; PDE4 – 3',5'-cyclic phosphodies e ase pde-4; GMP – guanosine
446
monophospha e; GUK1 – guanyla e kinase iso o m X1; GDP – guanosine diphospha e; NEP1 – nep ilysin1; ANP – a ial
447
na iu e ic pep ide; ANPR – a ial na iu e ic pep ide ecep o B.
448
449
17
3.4. Choline gic synapse pa hway
450
Gi en he impo ance o neu al egula ion o he homeos a ic con ol (Leg adi e al., 2018), we also
451
assessed whe he addi ional pa hways/genes ela ed wi h neu al/beha io al egula ion we e
452
modula ed by SIM. In e es ingly, he ansc ip omic analysis showed ha se e al genes ela ed wi h
453
he syn hesis, anspo , and elease o he neu o ansmi e ace ylcholine (Ach), which plays a key
454
ole in he egula ion o beha io , cogni ion, and con ol o muscle con ac ion, is down egula ed in F1
455
and/o F3 (Figu e 6). The choline gic synapse pa hway is a highly conse ed e olu iona y pa hway in
456
euka yo es, i al o he no mal unc ion o he senso ial and neu omuscula sys ems (Bossy e al.,
457
1988; Zhang e al., 2014b). E en hough he choline anspo e (CHT2) and some ace ylcholine
458
ecep o s subuni s (ACRb1 and CHRNAa4) we e ound o be non- esponsi e o SIM, we obse ed ha
459
SIM se e ely dis up s he choline gic synapse pa hway, as choline O-ace yl ans e ase (ChAT), choline
460
(Ch) and ace ylcholine anspo e s (SLC5A7 and VAChT, espec i ely), ace ylcholine ecep o s -
461
nico inic α7-like subuni s (CHRNA7 and ACR-16) and Ace ylcholines e ase (AChE) a e all
462
down egula ed in F1 and ChAT, SLC5A7, VAChT, CHRNA7 and AChE a e also down egula ed in F3
463
(Figu e 6). Since his pa hway is c i ical o he no mal unc ion o choline gic ansmission—i.e., he
464
communica ion o neu ons in he ne ous sys em, including mo o neu ons, ha will ac i a e muscle
465
con ac ion—we hypo hesize ha in e and ansgene a ional exposu e o SIM could modula e he
466
muscle sys em o G. locus a. In e es ingly, p e ious s udies epo ed he occu ence o ad e se
467
neu omuscula eac ion upon adminis a ion o SIM, possibly media ed by he nico inic ace ylcholine
468
ecep o (nAChRs) a ec ing ne e ansmission ac oss he synapse and esul ing in muscle weakness
469
and pain by neu omuscula degene a ion (G ajales-Reyes e al., 2013). Ou indings also sugges ha
470
he obse ed SIM modula ion o he choline gic synapse pa hway could po en ially dis u b he mol ing
471
cycle o G. locus a in F1 and F3 gene a ions. In ac , simul aneous con ac ions o specialized muscle in
472
a h opods a e equi ed o spli he old cu icle du ing mol ing and gene a e p essu e pulses o body
473
expansion (Nako e al., 2018). Mol ing, as desc ibed abo e, is a key p ocess o c us aceans´ g ow h
474
and ep oduc ion. The e o e, he in e and ansgene a ional dis up ion o he choline gic
475
synapse pa hway could be po en ially linked wi h he obse ed e ec s o SIM on G. locus a g ow h
476
and ep oduc ion p e iously epo ed (Figu e 2) (Neupa h e al., 2020a; Neupa h e al., 2014) and be
477
po en ially ela ed wi h he ecdys e oid cascade dis u bance desc ibed abo e, also in ol ed in he
478
egula ion o mol ing, ep oduc ion and g ow h.
479
18
480
Figu e 6 – Choline gic synapse pa hway in C us aceans. Genes modula ed by SIM and non- esponsi e genes in F1 and F3
481
gene a ions a e p esen ed in boxes. Red ex inside he boxes indica es gene exp ession down egula ion in F1 and/o F3
482
gene a ions and black ex inside he boxes indica es unchanged gene exp ession in F1 and F3 gene a ions. No gene was
483
ound o be up egula ed in his pa hway. . Ch – Choline; Ach – Ace ylcholine; ChAT – choline O-ace l ans e ase; ACR16 –
484
ace ylcholine ecep o subuni alpha- ype ACR-16-like; ACR b1 – ace ylcholine ecep o subuni be a; CHRNA7 – ace ylcholine
485
ecep o subuni alpha-7-like; CHRNA α4 – ace ylcholine ecep o subuni alpha-4; SLC5A7 – high a ini y choline anspo e
486
1; CHT2 – choline anspo e p o ein 2; VAChT – esicula ace ylcholine anspo e 1; AChE – ace ylcholines e ase.
487
488
489
3.5. GABAe gic signalling pa hway
490
A down egula ion o se e al p o eins in ol ed in he neu o ansmi e γ-aminobu y ic acid (GABA)
491
cycle was also obse ed. GABA is p ima y in ol ed in hype pola ising inhibi o y synap ic ansmission
492
by coun e balancing he depola iza ion o neu onal memb anes (C owley e al., 2016) and is
493
syn hesized om L-glu ama e, he p incipal exci a o y neu o ansmi e in ol ed in p ocesses such as
494
synap ic memo y and plas ici y (Bena och, 2010; Fagg and Fos e , 1983; Fonnum, 1984). I s ac ion
495
educes he excessi e glu ama e gic signalling and subsequen exci o oxici y, ac ing as a eedback
496
con ol sys em (Gonzalez-Bu gos and Lewis, 2008; Lau and Tymianski, 2010), egula ing neu al ac i i y
497
and ene gy me abolism and hus, modula ing b ain homeos asis (Takesian and Hensch, 2013; Xu e al.,
498
2011). Figu e 7 displays he ansc ip omic esul s o GABA signalling pa hway whe e down egula ion
499
o genes coding o key p o eins in F1 was obse ed: i.e., glu aminase li e , mi ochond ial-like (GLS),
500
4-aminobu y a e amino ans e ase (ABAT), GABA ecep o del a (GABA-R) and glu aminase, sodium
501
and chlo ide dependen GABA anspo e 1-like (GAT1) ha a e in ol ed in he ac ion, anspo and
502
deg ada ion o GABA. On he o he hand, i was also obse ed ha some genes ela ed wi h he
503
in e ac ion, ancho ing and o ganiza ion o GABA ecep o s wi h he cy oskele on, such as gephy in
504
19
(GPHN) and gamma-aminobu y ic acid ecep o -associa ed p o ein (GABARAP), and he o ma ion o
505
ke oglu a a e om glu ama e, i.e., mi ochond ial glu ama e dehyd ogenase 1 (GAD), we e non-
506
esponsi e o SIM.
507
508
Figu e 7 – GABAe gic signalling pa hway in C us aceans. Genes modula ed by SIM and non- esponsi e genes in F1 and F3
509
gene a ions a e p esen ed in boxes. Red ex inside he boxes indica es gene exp ession down egula ion in F1 and/o F3
510
gene a ions and black ex inside he boxes indica es unchanged gene exp ession in F1 and F3 gene a ions. No gene was
511
ound o be up egula ed in his pa hway.. GLS – glu aminase li e mi ochond ial-like; GAD – mi ochond ial glu ama e
512
dehyd ogenase 1; ABAT – 4-aminobu y a e amino ans e ase; GPHN – gephy in; GABA-R – GABA ecep o del a; GABARAP –
513
gamma-aminobu y ic acid ecep o -associa ed p o ein; GAT1 – sodium and chlo ide dependen GABA anspo e -1-like.
514
515
Besides he neu oendoc ine pa hways discussed abo e, able 2 displays addi ional genes in ol ed in
516
he egula ion o ne ous and neu omuscula unc ions ha showed a shi in gene exp ession p o ile
517
a e in e and ansgene a ional SIM exposu e.
518
519
Table 2 –Genes ela ed wi h neu o oxic e ec s and hei espec i e unc ion
520
Gene
F1
s a us
F3
s a us
E alue/
Bi sco e
Func ion
Species
p oho mone-4-like iso o m
X1
-
1.3e-86
329.3
Clea ed in o he ollowing six chains: B ain pep ides IDLSRFYGHFNT, IDLSRFYGHFN,
IDLSRFYGHF, DLSRFYGHFNT, DLSRFYGHFN o B ain pep ide DLSRFYGHF
Ame
pu a i e lachesin-like
2.9e-94
356.7
Func ions as a homophilic cell-adhesion molecule. May play a ole in ea ly neu onal
di e en ia ion and axon ou g ow h
Dme
p oho mone-3-like
-
1.6e-34
156.0
Clea ed in o B ain pep ide ITGQGNRIF
Ame
p oho mone-1-like
3.1e-31
144.8
Clea ed in o h ee chains: B ain pep ide LRNQLDIGDLQ, B ain pep ide LRNQLDIGDL
and B ain pep ide SYWKQCAFNAVSCF-amide
Ame
synap ob e in-1-like
iso o m X4
-
1.1e-48
202.6
Ac s in neu onal exocy osis o synap ic ansmission. Po en ial ole in choline gic
ansmission
Cb
p o ein unc-79 homolog
8.5e-299
1036.9
Componen o he NALCN channel is esponsible o Na(+) leak cu en s and
ac i a ed by neu opep ides
Hsa
neu opilin-1-like iso o m
X1
2.1e-99
372.5
Recep o in ol ed in he o ma ion o ce ain neu onal ci cui s and in o ganogenesis
ou side he ne ous sys em.
Hsa
nep ilysin-1-like
5.9e-109
405.2
Me alloendop o ease which unc ions in e ili y and memo y o ma ion.
Dme
20
zwei Ig domain p o ein zig-
8-like
3.2e-98
368.6
Requi ed pos emb yonically o main ain he posi ion o se e al neu on cell bodies
and en al ne e co d axons o speci ic neu ons
Cel
neu o ophin 1-like
1.3e-90
342.4
Ligand o he Toll- ela ed ecep o s, p omo ing mo o axon a ge ing and neu onal
su i al
Dme
pu a i e neu al-cadhe in 2
2.9e-261
912.5
Cell adhesion p o eins
in ol ed in he ansmission o de elopmen al in o ma ion
Dme
con ac in-6-like
0.0e+00
1128.6
Media e cell su ace in e ac ions. Pa icipa es in oligodend ocy es gene a ion by
ac ing as a ligand o NOTCH1.
Hsa
po assium ol age-ga ed
channel sub amily B
membe 1-like iso o m X1
1.3e-54
223.4
Media es ansmemb ane anspo in exci able memb anes
Regula ion o ac ion po en ial in neu ons, muscle and endoc ine cells
Hsa
i egula chiasm C-
oughes p o ein-like
2.5e-308
1067.8
Requi ed o co ec axonal pa hway o ma ion in he op ic lobe and o
p og ammed cell dea h in he de eloping e ina.
Dme
pu a i e neu o imin-like
9.2e-69
271.2
Neu al cell adhesion molecule
Hsa
p o ein RUFY3-like iso o m
X1
-
1.0e-176
630.2
Plays a ole in he gene a ion o neu onal pola i y o ma ion and axon g ow h
Hsa
Down synd ome cell
adhesion molecule-like
p o ein Dscam2
0.0e+00
1729.9
Cell adhesion molecule. In ol ed in axon guidance.
Dme
ninju in-2-like iso o m X2
-
6.0e-39
170.6
Homophilic cell adhesion molecule ha p omo es axonal g ow h. May play a ole in
ne e egene a ion and in he o ma ion and unc ion o o he issues
Hsa
p o ein u le homolog B-
like iso o m X7
-
1.3e-53
220.7
Abundan ly exp essed in in e neu ons, whe e i may egula e inhibi o y synapse
de elopmen
Hsa
Down synd ome cell
adhesion molecule-like
p o ein 1 homolog, pa ial
1.4e-196
696.8
Cell adhesion molecule ha plays a ole in neu onal sel -a oidance.
Hsa
sodium-coupled
monoca boxyla e
anspo e 1-like
-
2.8e-210
741.5
May play a c i ical ole in an elec ochemical Na+ g adien on neu ons. Main enance
o he ene gy s a us and unc ion o neu ons.
Hsa
pu a i e neu o ypsin-like
-
3.0e-274
954.5
Plays a ole in neu onal plas ici y and is associa ed wi h lea ning and memo y
ope a ions
Hsa
neu oligin-4, Y-linked-like
-
0.0e+00
1387.1
Neu onal cell su ace p o ein in ol ed in cell-cell-in e ac ions
Hsa
neu oligin-4, X-linked like
1.8e-295
1025.4
Neu onal cell su ace p o ein in ol ed in cell-cell-in e ac ion
Hsa
neu oligin-2-like, pa ial
1.4e-219
772.3
In ol ed in cell-cell in e ac ions ia i s in e ac ions wi h neu exin amily membe s.
Media es cell-cell in e ac ions bo h in neu ons and in o he ypes o cells
Hsa
synap o agmin-11-like
iso o m X2
5.7e-179
637.1
Plays an impo an ole in dopamine ansmission by egula ing endocy osis and he
esicle- ecycling p ocess
Hsa
a ylsul a ase B-like
2.8e-78
302.0
Regula o o neu i e ou g ow h and neu onal plas ici y
Hsa
RNA-binding p o ein
Musashi homolog Rbp6-
like
4.3e-66
261.2
May play a ole in he p oli e a ion and main enance o s em cells in he cen al
ne ous sys em
Dme
iono opic ecep o 25a-
like
-
0.0e+00
1389.4
In eg al pa o a ious neu al senso y sys ems in he an enna ha p o ide he
neu al basis o he esponse o en i onmen al changes
Dme
se ine/ h eonine-p o ein
kinase BRSK2-like iso o m
X2
-
3.4e-296
1027.7
Se ine/ h eonine-p o ein kinase ha plays a key ole in pola iza ion o neu ons and
axonogenesis,
Hsa
innexin inx1-like iso o m
X2
-
1.2e-31
145.6
S uc u al componen o he gap junc ions
Dme
myelin P2 p o ein-like
-
4.5e-49
203.8
May play a ole in lipid anspo p o ein in Schwann cells
Hsa
calcium-ac i a ed
po assium channel
slowpoke-like
1.9e-252
881.3
Po assium channel ac i a ed by bo h memb ane depola iza ion o inc ease in
cy osolic Ca2+ ha media es expo o K+. I s ac i a ion dampens he exci a o y
e en s ha ele a e he cy osolic Ca2+ concen a ion and/o depola ize he cell
memb ane. I he e o e con ibu es o epola iza ion o he memb ane po en ial.
Dme
pep idyl-glycine alpha-
amida ing monooxygenase
B-like
-
4.9e-32
147.5
Bi unc ional enzyme ha ca alyzes he pos - ansla ional modi ica ion o inac i e
pep idylglycine p ecu so s o he co esponding bioac i e alpha-amida ed pep ides,
a e minal modi ica ion in biosyn hesis o many neu al and endoc ine pep ides
Hsa
pep idyl-alpha-
hyd oxyglycine-alpha-
amida ing lyase I
-
1.8e-76
296.2
P obable lyase ha ca alyzes an essen ial eac ion in C- e minal alpha-amida ion o
neu opep ides
Dme
pep idyl-alpha-
hyd oxyglycine alpha-
amida ing lyase 2-like
-
2.8e-88
335.5
P obable lyase ha ca alyzes an essen ial eac ion in C- e minal alpha-amida ion o
neu opep ides
Dme
neu opep ide SIFamide
ecep o -like
-
2.8e-159
573.5
Con ols di e en ep oduc i e beha iou s, e.g., sexual ecep i i y in emales,
dominance and agg ession beha iou s
Dme
Red indica es down egula ion, g een indica es up egula ion; (-) indica es no changes in gene exp ession; Hsa – Homo sapiens;
521
Ame – Apis melli e a, Dme – D osophila melanogas e , Cb - Caeno habdi is b iggsae, Cel - Caeno habdi is elegans
522
523
21
4. Conclusions
524
O e all, we demons a e he e ha in e and ansgene a ional exposu e o SIM, a en i onmen ally
525
ele an le els, has signi ican e ec s in he egula ion o key signaling pa hways in ol ed in c us acean
526
neu oendoc ine egula ion, concomi an ly o changes in apical endpoin s, such as dep essed
527
ep oduc ion and g ow h. The neu oendoc ine sys em plays a majo ole in me azoan homeos a ic
528
con ol, pa icula ly in axa displaying a cen al neu oendoc ine sys em: egula ing beha io ,
529
ep oduc ion, mol ing, among o he majo biological p ocesses. The neu oendoc ine me abolic
530
pa hways he e designed p o ide addi ional genomic in o ma ion in his poo ly anno a ed species and
531
will ep esen a e e ence sou ce o u he and mo e ocused neu oendoc ine molecula analysis on
532
his and o he amphipod species.
533
Ye , despi e he biological impo ance o neu oendoc ine egula ion, he impac o en i onmen al
534
chemicals a his le el has been poo ly s udied. The e o e, he indings epo ed he e suppo he
535
impo ance o add essing he e ec s o en i onmen al chemicals in neu oendoc ine egula ion, as a
536
s ep owa ds imp o ing haza d and isk assessmen o biological ac i e compounds. Impo an ly, he
537
dis up ion in signaling pa hways epo ed he e was no only obse ed in F1 bu also in he F3
538
gene a ion, which implies ansgene a ional e ec s. Neupa h e al. (2020a,b) epo ed he
539
modula ion o se e al genes in ol ed in he egula ion o he epigenome in F3, which may explain he
540
obse ed ansgene a ional e ec s. A de ailed e alua ion o he epigene ic machine y in ol ed in his
541
dis up ion should be disclosed in he u u e, gi en ha en i onmen al chemicals inducing
542
ansgene a ional e ec s o e se e al non-exposed gene a ions a e o majo conce n.
543
544
5. Au ho ship con ibu ion s a emen
545
T. Neupa h: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis, In es iga ion, Resou ces,
546
Da a cu a ion, W i ing – o iginal d a , W i ing - e iew & edi ing, Supe ision, P ojec adminis a ion,
547
Funding acquisi ion. N. Al es: Concep ualiza ion, Me hodology Fo mal analysis, In es iga ion, Da a
548
cu a ion, Da a cu a ion, W i ing – o iginal d a , W i ing - e iew & edi ing. A.M. Machado: Fo mal
549
analysis, In es iga ion, Da a cu a ion. M. Pinhei o: Fo mal analysis, In es iga ion, Da a cu a ion,
550
W i ing - e iew & edi ing. R. Mon es: In es iga ion, W i ing - e iew & edi ing. R. Rodil: In es iga ion,
551
W i ing - e iew & edi ing. S. Ba os: Fo mal analysis, In es iga ion, Da a cu a ion, W i ing - e iew &
552
edi ing. R. Rui o: Fo mal analysis, In es iga ion, Da a cu a ion, W i ing - e iew & edi ing. L. Filipe C.
553
Cas o: Valida ion, W i ing - e iew & edi ing. J.B. Quin ana: In es iga ion, W i ing - e iew & edi ing,
554
Funding acquisi ion. M.M. San os: Concep ualiza ion, Me hodology, Valida ion, Fo mal analysis,
555
In es iga ion, Resou ces, Da a cu a ion, W i ing - o iginal d a , W i ing - e iew & edi ing, Supe ision,
556
P ojec adminis a ion, Funding acquisi ion.
557
558
6. Decla a ion o Compe ing In e es
559
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships
560
ha could ha e appea ed o in luence he wo k epo ed in his pape .
561
562
563
22
7. Acknowledgmen s
564
This wo k was inancially suppo ed by: i) T ansobesogen P ojec – “T ans-phyle ic obesogenic
565
esponses: om epigene ic modules o ansgene a ional en i onmen al impac s” e e ence:
566
PTDC/CTA-AMB/31544/2017 – NORTE-01-0145-FEDER-031544, co- inanced by he Po uguese
567
Founda ion o Science and Technology (FCT), No h Regional Ope a ional P og am (NORTE 2020) and
568
he Eu opean Regional De elopmen Fund (FEDER). ii) No -Wa e P ojec – “Poluen es eme gen es nas
569
águas da Galiza-No e de Po ugal: no as e amen as pa a ges ão de isco” e e ence:
570
0725_NOR_WATER_1_P inanced by INTERREG VA Spain-Po ugal coope a ion p og amme, C oss-
571
Bo de No h Po ugal/Galizia Spain Coope a ion P og am (POCTEP) 2014–2020. iii) he Na ional Funds
572
h ough FCT unde he p ojec s (UIDB/04423/2020;
573
UIDP/04423/2020). Nélson Al es acknowledges FCT o his Ph.D. g an DFA/BD/6218/2020. And e M
574
Machado acknowledges FCT o his Ph.D. g an DFA/BD/8069/2020. Ma lene Pinhei o acknowledges
575
FCT o he Ph.D. g an SFRH/BD/147834/2019. Susana Ba os acknowledges FCT o he Ph.D. g an
576
PD/BD/143090/2018. Financial suppo by Xun a de Galicia (ED431C2021/06) and he Spanish Agencia
577
Es a al de In es igación - AEI (PID2020-117686RB-C32) is also g a e ully acknowledged.
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Au ho s a emen
Concep ualiza ion, Te esa Neupa h and Miguel M. San os; Me hodology, Te esa Neupa h,
And é M. Machado, Rosa Mon es and Miguel M. San os; Valida ion, Te esa Neupa h, And é M.
Machado, L. Filipe C. Cas o and Miguel M. San os; Fo mal analysis, Te esa Neupa h, And é M.
Machado, Rosa Mon es, Rosa io Rodil, Susana Ba os, Nelson Al es., Raquel Rui o, L. Filipe C.
Cas o, José B.Quin ana and Miguel M. San os; In es iga ion, Te esa Neupa h, And é M.
Machado, Rosa Mon es, Rosa io Rodil, Susana Ba os, Nelson Al es., Raquel Rui o, L. Filipe C.
Cas o, José B.Quin ana and Miguel M. San os; So wa e, Te esa Neupa h, And é M. Machado;
Resou ces, Te esa Neupa h, L. Filipe C. Cas o and Miguel M. San os; Da a cu a ion, Te esa
Neupa h, And é M. Machado, Rosa Mon es, Rosa io Rodil, Susana Ba os, Nelson Al es., Raquel
Rui o, L. Filipe C. Cas o, José B.Quin ana and Miguel M. San os; W i ing-o iginal d a
p epa a ion, Te esa Neupa h and Miguel M. San os; W i ing- e iew and edi ing Te esa
Neupa h, And é M. Machado, Raquel Rui o, L.Filipe C. Cas o, José B.Quin ana and Miguel M.
San os; Supe ision, Te esa Neupa h and Miguel M. San os; P ojec adminis a ion, Te esa
Neupa h and Miguel M. San os Funding acquisi ion, José B.Quin ana and Miguel M. San os
