T ansc ip ion o ibogenesis genes in ish gonads: Applica ions in he iden i ica ion o s ages o
oogenesis and in en i onmen al moni o ing o in e sex condi ion
I a xe Rojo-Ba olomé, Aina a Valencia, Ibon Cancio*
CBET Resea ch G oup, Dep . o Zoology and Animal Cell Biology, Resea ch Cen e o Expe imen al Ma ine
Biology and Bio echnology o Plen zia (PiE-UPV/EHU), Uni e si y o he Basque Coun y (UPV/EHU), A ea za,
z/g, E-48620 Plen zia, Basque Coun y, Spain
Co esponding au ho : [email p o ec ed]
ABSTRACT
One o he bes desc ibed e ec s o en i onmen al xenoes ogens in ish is he gene a ion o
in e sex gonads in males. Conside ing 5S RNA a ma ke o he p esence o oocy es, a 5S/18S RNA
index was calcula ed in 296 hicklip g ey mulle s (Chelon lab osus) om pollu ed en i onmen s. In
addi ion, qPCR analysis o ansc ip ion ac o s g 3a and ub 1, ela ed espec i ely o 5S and 18S
RNA syn hesis, was conduc ed along emale-oogenesis. 5S/18S RNA index iden i ied sex wi h a
h eshold alue o 0.4521 sepa a ing males om emales. His ological analysis iden i ied 38
in e sex indi iduals. In e sex se e i y and 5S/18S RNA indexes we e co ela ed. 5S/18S RNA
index iden i ied o a ian de elopmen al s age wi h high 5S RNA le els du ing ea ly oogenesis and
18S RNA ela i e alues inc easing owa ds ma u a ion. g 3a and ub 1 ansc ip ion le els
ollowed he pa e n o 5S RNA accumula ion. Thus, ibogenesis genes p o ide easy/quan i a i e
me hods o molecula ly iden i y he sex, emale game ogenic s age and in e sex se e i y
in mulle s.
INTRODUCTION
O ganisms inhabi ing es ua ine wa e s a e no mally exposed o complex con aminan cock ails
wi h an h opic o igin (Boehm and Bischel, 2011). Among o he con aminan s de ec ed in he
aqua ic en i onmen s, endoc ine dis up ing chemicals (EDCs) ha e ecei ed special a en ion a e
hey we e i s highligh ed by he Eu opean En i onmen al Agency in 1997 (EEA, 1997). EDCs we e
hen de ined as “chemical pollu an s able o in e e e wi h he no mal unc ioning o ho mones”
(EEA, 2012) and hey can cause al e a ions a di e en biological o ganisa ion le els, om he
molecula one o he indi idual o he (sub)popula ion one (B ande , 2013, WHO/UNEP, 2013).
They include a complex a ay o subs ances, wi h di e en chemical s uc u es and sou ces (Tyle
e al., 1998, López de Alda and Ba celó, 2001, Po e e al., 2006, Casals-Casas and Des e gne,
2011, Khe an, 2014) which in indus ialized coun ies mainly a i e o he aqua ic en i onmen
h ough he municipal, indus ial and hospi al was ewa e ea men plan s e luen s (Campbell e
al., 2006). Such compounds in complex mix u es, can in e ac enhancing hei po ency and
biological ac i i y, and in some ci cums ances, ac ing in an addi i e manne (Tho pe e al., 2003).
Xenoes ogens a e conside ed EDCs wi h he abili y o mimic es ogens o o cause es ogen-like
esponses in exposed o ganisms (Campbell e al., 2006). They al e ho monal homeos asis
in e e ing wi h no mal sexual di e en ia ion and game ogenesis, which in consequence a ec s
he de elopmen and ep oduc ion o exposed indi iduals/popula ions (Tyle e al., 1998, Goksøy
e al., 2003, Mills and Chiches e , 2005). The e ec s o xenoes ogenic compounds in some aqua ic
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Ma ine Pollu ion Bulle in 121(1/2) : 292-301 (2017),
which has been published in inal o m a h ps://doi.o g/10.1016/j.ma polbul.2017.06.027. ©2017 Else ie unde CC BY-NC-ND
license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
o ganisms a e well known, one o he bes desc ibed esponses being he eminiza ion o ju enile
and male ish (WHO/UNEP, 2013, Tyle and Jobling, 2008, Goksøy e al., 2003, Goksøy , 2006,
Biza o e al., 2014). Some imes his leads o he gene a ion o in e sex gonads, when oocy es
di e en ia e wi hin he no mal es icula issue in gonocho is ic ish species (Ma hiessen, 2003,
Bahamonde e al., 2013, Biza o e al., 2014, O iz-Za agoi ia e al., 2014). In e sex condi ion has
been epo ed in bo h eshwa e and ma ine ish ela ed o chemical exposu e in highly o
mode a ely con amina ed a eas (Bahamonde e al., 2013, O iz-Za agoi ia e al., 2014). These
in e sex males display lowe ep oduc ion capaci y han no mal males, wi h logical consequences
in popula ion iabili y (Jobling e al., 2002b, Ha is e al., 2011).
Xenoes ogenic e ec s ha e been epo ed also in mugilid ish popula ions om con amina ed
es ua ies (Fe ei a e al., 2004) and in e sex gonads ha e been desc ibed in hicklip g ey mulle s
(Chelon lab osus) used as pollu ion sen inel o ganisms in es ua ies in he Sou he n Bay o Biscay
(Diaz de Ce io e al., 2012, Puy-Azu mendi e al., 2013, Biza o e al., 2014, Valencia e al., 2017).
Oocy es ha e been ound in es es o mulle s om Bilbao, Pasaia and Onda oa ha bou s, and in
es ua ies in Deba and in he Biosphe e Rese e o U daibai in Ge nika (Puy-Azu mendi e al., 2013,
Biza o e al., 2014, Valencia e al., 2017). These si es ha e been associa ed o was e wa e
ea men plan e luen s and/o o indus ial ac i i ies in ha bou a eas. In e sex mulle s ha e
been desc ibed all along he ep oduc i e cycle, wi h pe cen ages anging om 3% o 60% o
analyzed males depending on he si e and mon h o cap u e (O iz-Za agoi ia e al., 2014). Mos
in e sex mulle s p esen low o mode a e in e sex se e i y indexes ollowing he sco e de eloped
by Jobling e al. (2006) ha anks he se e i y acco ding o he numbe and dis ibu ion o oocy es
p esen ed in he es is. Fu he mo e, ele a ed i ellogenin ansc ip and p o ein le els ha e been
measu ed in hese males, accompanied by an up- egula ion o he a oma ase coding
gene cyp19a1b in b ain (Biza o e al., 2014). Thus, hicklip g ey mulle is conside ed an impo an
sen inel species o he biomoni o ing o exposu e o ep oduc ion EDCs in he Sou he n Bay o
Biscay (O iz-Za agoi ia e al., 2014).
In spi e o he inc easing numbe o s udies desc ibing he in e sex condi ion in ish in he las
decades, he physiological and molecula mechanisms go e ning he p ocess emain unknown
(Abdel-Moneim e al., 2015). In addi ion, and al hough i is known ha he ansc ip ion le els o
se e al genes ela ed o sex di e en ia ion a e al e ed a e exposu e o xenoes ogens
(Bahamonde e al., 2015b), no di ec ela ionship wi h he in e sex condi ion has been es ablished
ye and mo e esea ch is needed in o de o ind speci ic molecula ma ke s o his condi ion
(Bahamonde e al., 2013, O iz-Za agoi ia e al., 2014, Abdel-Moneim e al., 2015).
In his espec , ecen ly, accumula ion o 5S RNA and o ansc ip s coding o 5S RNA
accompanying p o eins ha e been s udied in mulle s om pollu ed a eas (Diaz de Ce io e al.,
2012, Valencia e al., 2017). The ansc ip ion le els o ibogenesis genes enabled he
iden i ica ion o sex o each indi idual in a compa a i e manne , when a leas one indi idual o
each sex is p esen in he s udy, i espec i e o hei si e o collec ion and hei s age wi hin he
ep oduc i e cycle. In addi ion, such genes we e up- egula ed in in e sex es is in compa ison o
no mal es is. S ong 5S RNA ansc ip ion, speci ic o o a ies, can be easily iden i ied by a simple
elec opho esis o o al RNA ex ac ed om he gonads (Diaz de Ce io e al., 2012). The ela i e
amoun o 5S o 18S RNA calcula ed a e elec opho e ic analysis (5S/18S RNA index) iden i ies
he p esence o oocy es in gonads and allows dis inguishing no only he sex bu also he oogenic
s age in emales, as demons a ed in di e en comme cial ish species o he Bay o Biscay (Rojo-
Ba olomé e al., 2016). This is so because 5S RNA le els ela i e o 18S RNA a e highe in
p e i ellogenic oocy es han in ma u e ones (Rojo-Ba olomé e al., 2016). In his con ex , he
5S/18S RNA index was applied in he p esen s udy o he unambiguous molecula iden i ica ion
o sex, emale ep oduc i e s age and in e sex se e i y in mulle s collec ed om pollu ed si es o
he Sou he n Bay o Biscay du ing a comple e annual ep oduc i e cycle. In addi ion, ansc ip ion
le els o he gene al ansc ip ion ac o IIIA (g 3a) and ups eam binding ansc ip ion ac o 1
(ub 1), genes ela ed o ibogenesis h ough egula ion o 5S and 18S RNA syn hesis, we e
s udied in o a ies o emales a di e en de elopmen al s ages. Such analysis could elucida e he
possibili y o using ansc ip ion le els o ibogenesis genes in an en i onmen al moni o ing
con ex o assess he impac o xenoes ogenic compounds and he incidence o in e sex condi ion
in pollu ion sen inel ish species.
MATERIAL AND METHODS
S udy a ea and biological samples
F om Sep embe 2010 o Sep embe 2011 wel e o hi y adul (> 20 cm leng h) hicklip g ey
mulle s (Chelon lab osus) we e mon hly collec ed by ishing- od in he ha bou o Pasaia
(43°19′35″N, 1°55′9″W), loca ed on he Basque coas (SE Bay o Biscay). Mulle s we e also sampled
in June 2013 and in Feb ua y 2014 in he es ua ies o Ge nika (43°19′26″N, 2°40′26″W) and
Galindo (43°18′11″N, 2°59′55″W) close he poin s o discha ge o he was e wa e ea men
plan s o Ge nika and o he Bilbao me opoli an a ea. The o al amoun o indi iduals collec ed
o his s udy was o 296.
All animal manipula ions conduc ed we e au ho ised by compe en egional au ho i ies a e he
e alua ion and app o al o all p o ocols by he E hics and Animal Wel a e Commission o he
Uni e si y o he Basque Coun y (CEBA/152/2010). A e cap u e, indi iduals we e immedia ely
anaes he ized in a sa u a ed e hyl 4-aminobenzoa e, sac i iced by decapi a ion and gonads we e
emo ed. A po ion o each gonad was embedded in RNA la e (Ambion, Li e Technologies,
Ca lsba d, USA), ozen in liquid ni ogen and hen s o ed in he labo a o y a − 80 °C un il u he
used.
Fo his ological analysis, a po ion was aken om he middle pa o he gonad om each ish
(a ound 1 cm in leng h ac oss he whole gonad). This was hen ixed in 4% neu al bu e ed
o malin and anspo ed a 4 °C o he labo a o y o u he p ocessing.
His ological analysis o he gonads
A e 24 h in he ixa i e, gonads we e dehyd a ed in a g aded se ies o e hanol (70%, 90% and
96%) and embedded in me hac yla e esin acco ding o he manu ac u e 's ins uc ions (Techno i
7100; He aeus Kulze GmbH & Co. KG, We heim, Ge many). 6 o 9 esin sec ions (5 μm) we e cu
in a 2065 Supe cu mic o ome (Leica Ins umen s GmbH, We zla , Ge many) and s ained wi h
hema oxylin/eosin. Sex and he de elopmen al s age o he oocy es in o a ies we e de e mined
mic oscopically, ollowing he game ogenic s age g ading desc ibed by McDonough e al.
(2005) o Mugil cephalus. The s ages we e as ollows. Res ing (R), wi h he p esence o a e ic
oocy es (> 20%) in an o he wise emp y o a y wi h sca ce oogonia and lamellae p esen ing some
muscle and connec i e issue bundles while o a ian wall look hickened. Pe inucleola (Pn), wi h
inac i e o a y con aining pe inucleola oocy es and a e y hin o a ian wall. Co ical al eoli (Ca),
wi h oocy es a co ical al eoli s age some o hem s a ing i ellogenesis (< 50%). Vi ellogenesis
(V), wi h oocy es ull o yolk globules and enla ged plasma memb ane. Ma u e/spawning (M), wi h
hyd a ed oocy es showing coalescence o lipid d ople s and e y hick oocy e en elope, and
p esence o some a e ic oocy es (< 20%). No ull ma u e o spawning indi iduals could be
iden i ied as spawning occu s in he open sea (O iz-Za agoi ia e al., 2014) and samplings we e
always ca ied ou in es ua ine a eas.
In he case o iden i ied in e sex indi iduals, he in e sex se e i y was es ablished mic oscopically.
Fo ha , up o 9 non-consecu i e 5 μm his ological sec ions we e comple ely examined wi h a 20X
objec i e, di iding each sec ion in se e al ields o iew. To de e mine he numbe o oocy es in
each his ological sec ion, he ield o iew wi h he maximum oocy e amoun eco ded was
conside ed, ollowing he me hodology desc ibed by Blaze e al. (2007). Then, he in e sex
se e i y o each ish was es ablished depending on he mean numbe o oocy es wi hin all
his ological sec ions analyzed pe indi idual and ollowing he index de eloped by Jobling e al.
(2006).
Ex ac ion o o al RNA, capilla y elec opho esis and 5S/18S RNA index
To al RNA was ex ac ed om 50 o 100 mg o issue using TRIzol® (In i ogen, Li e Technologies)
and ollowing he manu ac u e 's ins uc ions. Ob ained RNA was pu i ied using Qiagen RNeasy ki
(Qiagen, Cali o nia, USA) a e a DNase diges ion s ep (RNase-F ee DNase Se , Qiagen). A e
pu i ica ion, he same amoun o RNA (250–500 ng), as es ima ed h ough abso bance a 260 nm
(good quali y RNA es ablished a abso bance a ios o 260/280 and 260/230 a ound 2), was loaded
in an Agilen RNA 6000 Nano Ki Bioanalyze (Agilen Technologies, San a Cla a, Cali o nia, USA).
Elec ophe og ams p o ided by he Bioanalyze we e used o quan i y he concen a ion o he
bands co esponding o 5S RNA and 18S RNA in each sample using he “Time Co ec ed A ea” o
each peak o calcula e he 5S/18S RNA a io (Rojo-Ba olomé e al., 2016). When he p esence o
one o he RNAs was below he le els o de ec ion o he machine a 0.1 alue was gi en o each
sample ins ead o 0 ( he lowes eco dable alue was 0.2). The bina y loga i hm o he a io was
calcula ed in o de o de elop a 5S/18S RNA index ha allowed clea isualiza ion o he
di e ences be ween es es and o a ies.
cDNA syn hesis and PCR analysis in o a ies
2 μg o o al RNA om each indi idual gonad we e used o cDNA syn hesis wi h he A ini ySc ip
Mul iple Tempe a u e cDNA Syn hesis Ki (Agilen Technologies) using andom p ime s, acco ding
o manu ac u e 's ins uc ions. 5S RNA is a e y small ansc ip and andom p ime s may no
adequa ely e o ansc ibe such small nucleic acids. Fo ha eason, and o compa a i e
pu poses, an addi ional e o ansc ip ion assay was pe o med wi h RNA om o a ies wi h
pe inucleola (Pn) and i ellogenic (V) oocy es using a e e se p ime speci ic o mulle 5S RNA
(5′-AAGCTTACAGCACCTG-3′).
P ime s o ob ain mulle 45S p e- RNA sequence agmen s we e designed h ough alignmen
(Clus al Omega, h p://www.ebi.ac.uk/Tools/msa/clus alo/) o piscine 45S p e- RNA sequences
a ailable in NCBI (h p://www.ncbi.nlm.nih.go /) and sea ching o highly conse ed nucleo ide
egions. P ope ies o designed p ime s we e checked employing he IDT OligoAnalyze Tool
(h ps://eu.id dna.com/calc/analyze ). Con en ional PCRs we e un wi h 0.8 nM o o wa d 5′-
GAGGCCCTGTAATTGGAATGAG-3′, e e se1 5′-CAAAGTGCGTTCGAAGTGTCGA-3′ o e e se2 5′-
AGAGAAGGCGCGAGGACAC-3′ p ime s. The ampli ica ion was un wi h comme cial Taq DNA
Polyme ase, ecombinan Ki and 100 mM dNTP Mix (In i ogen) o 35 cycles in a 2720 The mal
Cycle (Applied Biosys ems, Ca lsba d, Cali o nia, USA). PCR p ocedu e was as ollows: 94 °C o
2 min, and 35 cycles wi h a dena u a ion s ep a 94 °C o 30 s, annealing a 61 °C o 30 s and
elonga ion a 72 °C o 30 s. A inal s ep a 72 °C was added o 8 min. Ob ained PCR p oduc s we e
sequenced in he SGIke Sequencing Se ice o he Uni e si y o Basque Coun y. Once sequenced,
agmen s we e aligned o ob ain a consensus sequence and analyzed using CAP3
(h p://pbil.uni lyon1. /cap3.php) and Clus alW2 ools. Sequences ob ained ha e been published
in GenBank (KX358060, KX358061).
Speci ic p ime s we e designed o ampli y he agmen anging om he 18S RNA 3′- egion in o
he in e nal ansc ibed space 1 and eaching he 5.8S RNA 5′- egion in cDNA gene a ed om
RNA ex ac ed om Pn and V o a ies. This egion su e s 45S p e- RNA in e nal splicing leading o
he gene a ion o he ma u e 18S RNA. Thus, he ampli ica ion o he sequence in his case is only
possible when 18S RNA and 5.8S RNA emain oge he . The agmen was ampli ied using
con en ional PCR and employing 0.8 mM o o wa d 5′-AACCTCAGTGCGTGGCGGA-3′ and e e se
5′-TCCACCGCTAAGAGTAGTCATG-3′ p ime s. Ampli ica ion was pe o med as desc ibed abo e
(annealing empe a u e 61 °C). Finally, PCR p oduc s we e analyzed by elec opho esis in e hidium
b omide s ained aga ose gels (1.5%).
qPCR analyses in o a ies
cDNA ob ained by andom and speci ic e o ansc ip ion om o a ies a di e en de elopmen al
s ages was quan i ied in he Syne gy HT Mul i-Made Mic opla e Reade (BioTek, Winooski, USA) by
Quan -i TM OliG een® s ain (In i ogen). The quan i ica ion was pe o med in a eac ion olume
o 100 μl wi h a heo e ical cDNA concen a ion ange o 0.02–0.2 ng/μl, a 485/20 nm exci a ion
and 528/20 nm emission wa eleng hs. Real PCR inpu cDNA concen a ion was calcula ed using
he high- ange s anda d cu e acco ding o manu ac u e 's ins uc ions.
5S and 18S RNA, g 3a and ub 1 ansc ip le els we e quan i ied (only in o a ies) by SYBR
G een® qPCR (Roche, Basel, Swi ze land). qPCR was conduc ed in iplica es using a 7300 Applied
Biosys ems The mocycle . The PCR eac ion mix u es (20 μl) consis ed o 10 μl o 2 × SYBR® G een
PCR mas e mix, app op ia e concen a ion o p ime s dilu ed in RNase- ee wa e (see Table 1)
and 2 μl cDNA empla e. qPCR p ocedu e was as ollows: 50 °C o 2 min, 95 °C o 10 min, hen
40 cycles consis ing o a dena u a ion s ep a 95°C o 15 s and an annealing s ep o 30 s
( empe a u e o each p ime se in Table 1). A dissocia ion s ep was added a he end consis ing
o 15 s a 95 °C, 1 min a annealing empe a u e and a inal s ep a 95 °C o 15 s.
Table 1. P ime sequences used o he qPCR analysis o he ansc ip ion le els o 5S RNA, 18S
RNA, g 3a and ub 1 in o a ies o hicklip g ey mulle s.
Gene
Fo wa d sequence (5′3′)
Re e se sequence (5′3′)
P ime
(nM)
Sample dilu ion
Annealing T
(°C)
5S RNAa
Fw-CTTACGGCCATACCACCCTG
R -GTATTCCCAGGCGGTCTCCC
6.25
1/200 &
1/12800b
60
18S RNA
Fw-GAGGCCCTGTAATTGGAATGAG
R -TAAGATACGCTATTGGAGCTGGAA
6.25
1/12800
60
g 3a
Fw-CCAGGAGAAGCGATATAAATGTGA
R -TCGTGATGCTTCAGTTTTCCATG
12.5
1/400
59
ub 1
Fw-CTCTAAAGCAAAGGTCAGTCCAGA
R -AATATGAACATGGCTGAGATGGGC
12.5
1/400
60
A P o ec ed unde Spanish pa en P201130778.
B Dilu ion o cDNA ob ained om 5S RNA speci ic e o ansc ip ion.
All ampli ica ion esul s we e no malized wi h he amoun o cDNA cha ged in he qPCR acco ding
o Rojo-Ba olomé e al. (2016) using an adap ed ΔCT o mula (RQ) wi h e iciency co ec ion (E):
m being he slope o he s anda d cu e o he qPCR eac ion.
whe e ∆ CT = CT sample − CT pla e in e nal con ol.
5S s. 18S RNA ansc ip ion le el indexes we e calcula ed using qPCR esul s ob ained o 5S
RNA om cDNA p oduced using bo h andom o 5S RNA speci ic p ime s.
S a is ical analysis
To assess whe he emale and male indi iduals could be seg ega ed in o wo s a is ically di e en
g oups acco ding o hei 5S/18S RNA index he R 3.3.1 (2016) so wa e e sion was used. To
gene a e he clus e s a logis ic eg ession was ca ied ou using he balance be ween speci ici y
and sensi i i y. Then, a maximum A ea Unde he Cu e (AUC) was calcula ed using he “pROC”
package e sion 1.8 a ailable o R (AUC = 0.996). Finally, he 5S/18S index cu poin alue
di e en ia ing males and emales was ob ained. The s a is ical analyses o he es o he esul s
we e unde aken using SPSS (SPSS Inc., Chicago, Illinois). Da a ailed in no mali y and a iance
equali y a e applying he Shapi o-Wilk (n < 30) es and Le ene's es , bo h a a 0.05 signi icance
le el (p < 0.05). Signi ican di e ences be ween g oups we e hen e alua ed using he non-
pa ame ic K uskal-Wallis es . When only wo g oups we e compa ed (Pn s. V), da a analysis was
pe o med wi h he non-pa ame ic Mann-Whi ney es o wo independen samples. In all he
cases, signi ican di e ences we e es ablished a p < 0.05. In addi ion, Spea man unila e al
co ela ion analysis was pe o med o compa e he his ological in e sex se e i y index and he
5S/18S RNA index. Spea man signi ican di e ences we e es ablished a p < 0.01.
RESULTS
5S/18S RNA index: iden i ica ion o sex
5S/18S RNA index was used o iden i y he sex o hicklip g ey mulle indi iduals s udied du ing a
whole annual cycle (Fig. 1). O all analyzed indi iduals (296) his ological analysis was only possible
in 215 (me hac yla e blocks o 81 samples we e los ) which we e his ologically sexed (colo ed do s
in Fig. 1), iden i ying 91 emales, 86 males and 38 in e sex indi iduals. Then wo popula ion
clus e s we e dis inguished based on hei 5S/18S RNA index alues wi h a cu poin alue o
0.4251 (Fig. S1). When he 177 his ologically sexed males and emales we e classi ied using his
clus e dis ibu ion 95% o emales and 100% o males we e co ec ly iden i ied. No male
indi idual showed a 5S/18S RNA index alue highe han he cu poin and only wo males ou o
86 did no show nega i e alues (one in Feb ua y and ano he one in June). The majo i y o he
emales (86 om 91) showed an index alue highe han he cu poin , wi h he excep ion o only
one emale in No embe and ou in Janua y. Sample dis ibu ion e ealed ha while all male
indi iduals displayed a na ow no mal dis ibu ion wi h an index peak a ound − 3.0, wo
popula ions could be dis inguished wi hin emales wi h peaks a ound 5.0 and 12.0 (Supplemen a y
Fig. S1).
Fig. 1. 5S/18S RNA in gonads o hicklip g ey mulle s collec ed du ing a comple e annual cycle. Each ed do
co esponds o one his ologically iden i ied emale indi idual (n = 91) and each blue do o a male (n = 86).
Each black line co esponds o an indi idual whose sex was no iden i ied his ologically (n = 81). Box in pink
g oups mos o emale indi iduals, and he blue box encompasses 100% o he males. (Fo in e p e a ion o
he e e ences o colo in his igu e legend, he eade is e e ed o he web e sion o his a icle.)
This sex dis ibu ion was used o classi y a o al o 81 indi iduals no sexed his ologically (black lines in Fig. 1,
Supplemen a y Fig. S2). 35 indi iduals displayed a 5S/18S RNA index alue abo e 0.4251 and hus hei
p obabili y o be emales was 100%. Indi iduals (46 ou o 81) wi h an index alue lowe han 0.4251 we e
males wi h a 94.5% p obabili y. This applies in he case ha none o he 81 indi iduals we e in e sex.
5S/18S RNA index and in e sex se e i y
A o al o 38 in e sex indi iduals, his ologically iden i ied du ing ou samplings in Pasaia, Galindo
and Ge nika, we e analyzed in he p esen s udy. They all displayed low o mode a e in e sex
se e i y (se e i y indexes 1 o 3) acco ding o he anking me hodology used (Jobling e al.,
2006, Blaze e al., 2007).
When compa ing 5S/18S RNA index om hese in e sex indi iduals and hei his ologically anked
in e sex se e i y index, a posi i e co ela ion (Spea man p < 0.01) wi h a magni ude o 0.638 was
obse ed be ween bo h indexes. As he amoun o oocy es in he es es inc eased, he 5S/18S
RNA index inc eased (Fig. 2). The lowes 5S/18S RNA index alues we e eco ded wi h in e sex
index alues o 1, whe e es is p esen ed om 1 o 5 oocy es sca e ed wi hin each es is sec ion,
and he highes a in e sex index 3, wi h clus e s o 21 o 50 oocy es in each es is sec ion. When
compa ing in e sex indi iduals wi h males and emales, in e sex indi iduals wi h lowe se e i y
indexes did no show signi ican di e ences in 5S/18S RNA index alues wi h males, while
indi iduals wi h se e i y index 3 showed 5S/18S RNA index alues simila o emales and di e en
om males (Fig. 2, Supplemen a y Fig. S2).
Fig. 2. Compa ison be ween he 5S/18S RNA index as calcula ed om he elec ophe og ams and he
his ologically calcula ed in e sex se e i y index in hicklip g ey mulle s. Box plo s ep esen he da a wi hin
he 25 h and 75 h pe cen iles, wi h he median indica ed by a line, and op and bo om whiske s indica ing
he minimum and maximum alues. Di e en le e s indica e signi ican di e ences be ween g oups
(K uskal-Wallis, p < 0.05). M: male, I.I.: in e sex se e i y index, F: emale. I.I. anked om 1 o 3 ollowing he
desc ip ion p oposed by Jobling e al. in 2006. The o al amoun o indi iduals in each g oup we e; 86 in M,
22 in I.I.1, 7 in I.I.2, 9 in I.I.3 and 91 in F ( o al n = 215).
5S/18S RNA index: iden i ica ion o emale ep oduc i e s age
5S and 18S RNA ela i e luc ua ions allowed dis inguishing o a ies a di e en de elopmen al
s ages (Fig. 3). A ea ly s ages du ing oogenesis (Pn and Ca) 5S RNA le els we e e y high in
compa ison wi h 18S RNA, bu as oogenesis ad anced ( om Pn o V), he ela i e amoun o 5S
RNA dec eased and he 18S RNA ela i e amoun inc eased in elec ophe og ams. In
consequence, he 5S/18S RNA index ha was high a he beginning o he oogenesis (Pn = 6.11
and Ca = 7.69), dec eased as oogenesis ad anced; he lowes index alues being eco ded a
i ellogenesis (V = 0.90). Values inc eased again a eg essing s age (R = 4.59).
Fig. 3. Va ia ions in he ela i e amoun o 5S RNA, 18S RNA and 5S/18S index du ing hicklip g ey
mulle oogenesis. Rep esen a i e mic og aphs o o a ies wi hin each o he de elopmen al s ages analyzed
a e shown (scale ba = 200 nm): R: es ing, Pn: pe inucleola , Ca: co ical al eoli, EV: ea ly i ellogenesis and
V: i ellogenesis. No indi iduals wi h ma u e hyd a ed o a ies (M) we e a ailable as mulle s we e ne e
sampled a open sea. The alue abo e each mic og aph depic s he mean 5S/18S RNA index alue a each
o he analyzed s ages. In he o al RNA elec ophe og ams ( ep esen a i e o indi iduals in each s age) he
peak a 25 s co esponds o 5S RNA and he one a 40–45 s co esponds o 18S RNA. The las peak
co esponds o 28S RNA. [FU]: luo escence, [s]: ime in seconds.
To p o e ha we we e eally measu ing 5S and 18S RNA le els in he elec ophe og ams and no
any hing else as i could be a gued by somebody, speci ic RNA ansc ip ion le els we e analyzed
h ough qPCR (Fig. 4). Compa ison o he 5S/18S RNA indexes ob ained om Bioanalyze
elec ophe og ams (Fig. 4A) and a e qPCR analysis (Fig. 4B) showed iden ical di e ences
be ween g oups. Anyhow, i was no iceable ha 5S/18S RNA index alues calcula ed wi h qPCR
analyses we e always nega i e, his meaning ha qPCR always iden i ied highe le els o 18S RNA
han o 5S RNA, also in Pn o a ies. In o de o cla i y his inconsis ency wi h he Bioanalyze
esul s, 5 Pn o a ies and 5 V o a ies we e andomly chosen and e o ansc ip ion was ca ied ou
using 5S RNA speci ic p ime s ollowed by a qPCR analysis o 5S RNA ansc ip ion le els. 5S/18S
RNA index a e speci ic 5S RNA e o ansc ip ion (Fig. 5C) showed he same ela i e p o ile
ob ained wi h he o al cDNA (Pn > V) (Fig. 5B), bu displaying posi i e alues, as i happened wi h
he index ob ained om he elec ophe og ams (Fig. 5A).
CONTRIBUTIONS OF AUTHORS
IR conduc ed he labo a o y measu emen s and d a ed he manusc ip . AV conduc ed mos o he
samplings and pa icipa ed in his ological analysis o he samples. IC designed and supe ised he
s udy di ec ing analysis o esul s and he w i ing p ocess.
ACKNOWLEDGEMENTS
Funded h ough p ojec s o he Uni e si y o he Basque Coun y (UFI 11/3), Basque
Go e nmen (S-PEN13UN101 and IT810-13) and he Spanish MINECO + Eu opean ERDF
Funds (AGL2012-33477 and AGL2015-63936-R). IR and AV a e ecipien o a g an o p edoc o al
s udies o he Basque Go e nmen . We hank he echnical suppo o he SGike Se ice
(UPV/EHU) and P o . Inma A os egui and D . I an zu Ba io o he Depa men o Applied
Ma hema ics and S a is ics and Ope a ional Resea ch o UPV/EHU o hei help wi h he R
so wa e s a is ical analysis.
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