Gill ansc ip omic analysis in as - and slow-g owing 1
indi iduals o My ilus gallop o incialis2
Daniel P ie o*[1], Pablo Ma kaide[1], Iñaki U u xu u[1], En ique Na a o[1], Sebas ien 3
A igaud[2], Elodie Fleu y[3], I in zi Iba ola[1] & Mi en Bego U u ia[1] 4
[1] GIU 17/061, GI 544, Depa amen o de Gené ica, An opología Física y Fisiología Animal, Facul ad5
de Ciencia y Tecnología, Uni e sidad del País Vasco/Euskal He iko Unibe si a ea, UPV/EHU, Apa ado 6
644, 48080 Bilbao, Spain. *Co esponding au ho , e-mail: Daniel.p ie [email protected] 7
[2]LEMAR UMR 6539 CNRS/UBO/IRD/I eme , IUEM, ue Dumon d’U ille, 29280 Plouzané, F ance8
[3]LEMAR, UMR 6539 UBO-CNRS-I eme -IRD, Technopole B es I oise 29280 Plouzané, F ance9
Abs ac 10
The molecula basis unde lying he mechanisms a he o igin o g ow h a ia ion 11
in bi al es is s ill poo ly unde s ood, al hough se e al genes ha e been desc ibed as 12
up egula ed in as -g owing indi iduals. In he p esen s udy, we ea ed mussel spa o 13
he species My ilus gallop o incialis unde die s below he pseudo aeces h eshold (BP) 14
and abo e he pseudo aeces h eshold (AP). A e 3 mon hs, F and S mussels om each 15
condi ion we e selec ed o ob ain 4 expe imen al g oups: FBP, SBP, FAP and SAP. We 16
hypo hesized ha he nu u ing condi ions du ing he g owing pe iod would modi y he 17
molecula basis o hei g ow h a e di e ences. 18
To deciphe he molecula mechanisms unde lying he g ow h a ia ion, he gill 19
ansc ip omes o he ou mussel g oups we e analysed. Gene exp ession analysis 20
e ealed i) a low numbe (12) o genes di e en ially exp essed in associa ion wi h die 21
and ii) 117 genes di e en ially exp essed by he as - and slow-g owing mussels. 22
Acco ding o Biological P ocess GO e m analysis ansc ip omic di e ences be ween 23
he F and S mussels we e mainly based on he up egula ion o : esponse o he s imulus, 24
g ow h and cell ac i i y. Rega ding he KEGG e ms, ca bohyd a e me abolism and he 25
K ebs cycle we e up egula ed in F mussels, whe eas biosyn he ic p ocesses we e 26
up egula ed in S mussels. In acco dance wi h hei la ge gill su ace a ea and highe 27
a es o eeding and g ow h, he F indi iduals o e exp essed genes in hei gill issues, 28
and hese we e in ol ed in i) g ow h (insulin-like g ow h ac o s and myos a in); ii) 29
main enance o he s uc u e and unc ioning o ex acellula ma ix (collagen, laminin, 30
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Aquacul u e 511 : (2019) // A icle ID
734242, which has been published in inal o m a h ps://doi.o g/10.1016/j.aquacul u e.2019.734242. © 2019 Else ie
unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
ibulin and deco in); iii) il a ion and cilia y ac i i y (mucin, ib ocys in, dynein and 31
ilB homologue p o ein genes); i ) ae obic me abolism (ci a e syn hase and ca bonic 32
anhyd ase); and ) he immune-sys em, p obably in associa ion wi h haemocy es. In 33
con as , S indi iduals o e exp essed a di e en se ies o genes pe aining o immune 34
sys em (leucine- ich epea p o ein and galec in), along wi h genes in ol ed in he 35
esponse o cellula s ess (Hea shock p o ein (HSP24) and me alloendopep idase) as 36
well as anae obic me abolism (C4-dica boxyla e anspo e ). These esul s migh 37
sugges ha S indi iduals would ha e a g ea e p e alence o pa hogens/diseases o a 38
highe suscep ibili y o he pa hogens. 39
40
Keywo ds: Fas -g owing, mussel, gill, ansc ip ome 41
42
1. In oduc ion 43
Bo h endogenous ac o s and en i onmen al condi ions in luence he g ow h a e 44
in bi al es (B own, 1988; Dickie e al. 1984; Malle and Haley, 1983; Pace e al. 2006; 45
Tamayo e al. 2011). S udies compa ing he physiological beha iou be ween as - and 46
slow-g owing indi iduals ha e g ea ly con ibu ed, in he las wo decades, o he 47
unde s anding o he physiological basis o di e en ial g ow h. The main conclusions 48
we e ha di e ences in g ow h a es esul ed om di e ences in i) he capaci y o 49
acqui e and abso b ood, ii) he e iciency o ene gy con e sion p ocesses and/o iii) he 50
alloca ion o ene gy o g ow h and main enance (Koehn and Shumway, 1982; To o and 51
Ve ga a, 1998; Bayne e al. 1999a, 1999b; Tamayo e al. 2014; Fe nández-Rei iz e al. 52
2016). 53
The mul ilocus he e ozygosi y hypo hesis o mula ed by Sighn and Zou os 54
(1978) es ablished he exis ence o a posi i e co ela ion be ween he deg ees o 55
he e ozygosi y and g ow h a e. Aneuploidy was also demons a ed o play a ole in 56
in e indi idual di e ences in g ow h a e in bi al es: signi ican ly highe alues o 57
aneuploidy we e obse ed in slow-g owing specimens o oys e s (C assos ea gigas) 58
and mo e ecen ly in he clam Rudi apes decussa us, wi h a high nega i e co ela ion 59
obse ed be ween g ow h a e and aneuploidy pe cen age (Lei ao e al. 2001; Teixei a 60
De Sousa e al. 2011). 61
In ecen yea s, high h oughpu gene exp ession analyses ha e used nex -62
gene a ion sequencing (NGS) o mic oa ays, wi h he aim o deciphe ing he 63
unde lying mechanisms, and hese echniques ha e allowed he iden i ica ion o genes 64
in ol ed in g ow h p ocesses (G acey e al. 2008; Lockwood e al. 2010; Sussa ellu e 65
al. 2010; De os e al. 2015; Sua ez-Ulloa e al. 2015; Xu e al. 2016). Fo ins ance, 66
Zhang e al. (2012) epo ed ha collagen and laminins, (ex acellula ma ix p o eins 67
om connec i e issue) and ib onec ins a e in ol ed in he o ma ion o he shell in he 68
oys e C assos ea gigas. Bassim e al. (2014) analysed he gene exp ession o he 69
mussel My ilus edulis du ing ea ly de elopmen ( om egg o pos -la ae), iden i ied a 70
se o genes ela ed o g ow h p ocesses in ea ly de elopmen (e.g., GATAD1, 71
PIP5K1A and ATRX) and highligh ed (Bassim e al. 2015) 29 gene ma ke s ela ed o 72
g ow h and mo ali y o bi al e la ae. 73
Ve y ew s udies ha e a emp ed o speci ically analyse he di e en ial gene 74
exp ession be ween as - and slow-g owing specimens o bi al es. Using di e en 75
c osses be ween inb ed lines o C assos ea gigas, Meye and Manahan (2010) ound 76
signi ican di e ences be ween as - and slow-g owing la al amilies in he ansc ip 77
abundance o ibosomal p o eins as well as in he a es o exp ession o genes encoding 78
o he small ca dioac i e pep ide p ecu so (ScPB), which is in ol ed in eeding 79
egula ion and in se e al p o eins in ol ed in he ene gy me abolism. Some o hem 80
we e elec on anspo componen s encoding genes (ND4L and ND1), ATP-syn hase ȣ, 81
and wo coiled-coil-helix-coiled-coil-helix domains (CHCHD2 and CHCHD). Mo e 82
ecen ly, De la Peña e al. (2016) epo ed he exis ence o signi ican di e ences in he 83
a e o exp ession o e i ins (Ap e 1) be ween as - and slow-g owing indi iduals o 84
A gopec en pu pu a us a di e en de elopmen al s ages (5 s ages om emb yos o 85
ju eniles). Wilson e al. (2016) p oduced an inb ed as g ow h line (F) o Mya a ena ia 86
clams and analysed he gene exp ession o es he hypo hesis ha speci ic g ow h-87
ela ed genes will be up egula ed in F indi iduals. These au ho s es ablished a posi i e 88
co ela ion be ween some me abolic genes ( a y acid syn hase and ATPase) wi h as 89
g ow h. These au ho s also ound some up egula ed genes in ol ed in s uc u al 90
emodelling in a as -g owing pheno ype in ag eemen wi h p e ious s udies indica ing 91
p o ein u no e as he main de e minan p ocesses o g ow h he e osis. Finally, 92
Saa ed a e al. (2017) concluded ha a se o genes con olling issue and o gan g ow h 93
p ocesses in model o ganisms (named ‘GCGC’) displayed a mino ole in de e mining F 94
and S in Rudi apes decussa us s ocks. Howe e , hey ound ha he insulin-media ed 95
p ocesses had an essen ial ole in in e indi idual di e ences in g ow h a e. 96
Al hough he a ailable gene ic in o ma ion is inc easing (Saa ed a and Bache e, 97
2006; Tanguy e al. 2008; As o ga e al. 2014)—e.g., he genome o he oys e 98
C assos ea gigas was published in 2012 (Zhang e al. 2012)—knowledge ega ding 99
molecula and gene ic in e indi idual di e ences in he g ow h po en ial o bi al es 100
emains a low s anda ds. La ge-scale sequencing p ojec s (e.g., NGS) ha e p oduced 101
la ge amoun s o sequences in da abases, bu a signi ican pa o hese sequences lack 102
an assigned unc ion o simila i y. The e o e, a combina ion o analyses o he 103
ansc ip ome and o he o ganiza ional le el esponses is necessa y o unde s and he 104
oles o speci ic genes in he unc ional esponses a he le el o he whole o ganism 105
(Bassim e al. 2014). 106
In he p esen s udy, we ha e analysed he gene exp ession in gill issue o 107
mussel (My ilus gallop o incialis) specimens ha we e selec ed as as (F) and slow (S) 108
g owe s a e ea ing hem o h ee mon hs in he labo a o y unde wo di e en 109
nu i ional en i onmen s. A e he ea ing pe iod, he physiological componen s o he 110
Scope o G ow h o he selec ed F and S mussels we e eco ded unde di e en 111
expe imen al die s o assess he in luence o ea ing condi ions on he pa ame e s o he 112
physiological beha iou s esponsible o as e g ow h (P ie o e al., in p epa a ion). 113
I espec i e o eeding condi ions du ing ea ing, as e g owe s exhibi ed highe Scope 114
o G ow h alues ha mainly esul ed om hei inc eased capaci y o acqui e ood. 115
Indeed, as g owe s displayed highe clea ance a es, and hey consis en ly we e ound 116
o ha e signi ican ly highe gill-su ace a ea pe mass uni han hei slow-g owing 117
coun e pa s. The combina ion o highe gill-su ace a ea wi h highe clea ance a e in 118
as -g owing indi iduals is a pheno ypical ea u e ha we ha e also obse ed in 119
p e ious s udies pe o med wi h mussels (P ie o e al. 2018) and clams (Tamayo e al. 120
2011). 121
Thus, he gill is one o he o gans likely playing a majo ole in de e mining he 122
in e indi idual g ow h a e di e ences in he mussel My ilus gallop o incialis. 123
Acco dingly, in he p esen s udy, we ha e selec ed he gill issue as he a ge o gan o 124
compa e gene exp ession in hese g oups o as and slow-g owing mussels. The aims o 125
his s udy we e o sea ch o candida e genes o eco ded di e ences in physiological 126
beha iou and, ul ima ely, in g ow h, o asce ain biological p ocesses accoun ing o 127
such di e ences a he molecula le el. Addi ionally, he e ec o he ea ing nu i ional 128
condi ion was also conside ed as a possible modula o o molecula p ocesses 129
unde lying he in e indi idual di e ences in g ow h a e. Speci ically, emphasis was 130
placed on linking physiological (P ie o e al., in p epa a ion) and ansc ip omic esul s 131
(p esen s udy) o achie e a mo e holis ic unde s anding o he o ganism beha iou in 132
di e en g ow h scena ios. 133
134
135
2. Ma e ial and Me hods 136
137
2.1. Selec ion o mussels
138
Some 400 mussels (My ilus gallop o incialis) o app oxima ely 10 mm shell 139
leng h (~150 mg li e weigh ) we e collec ed in a ocky sho e in An zo as (Bizcay, 140
No h Spain) in Feb ua y 2014. Once a he lab, we ea ed each hal o he mussels a 141
one o he wo “main enance condi ions” (named AP and BP) designed o o ce 142
di e en eeding s a egies in bo h g oups: a g oup o 200 mussels was ed a high-143
quali y die (o ganic con en = 80%) dosed a a pa icle olume concen a ion o 1.0-1.5 144
mm3/L (below he pseudo aeces h eshold; main enance condi ion BP), and he o he 145
g oup o 200 mussels was ed a low-quali y die (o ganic con en = 30%) dosed a 146
pa icle olume concen a ion o 3.0-3.5 mm3/L (abo e he pseudo aeces h eshold: 147
main enance condi ion AP). Die s we e a mix u e o cul u ed Isoch ysis galbana (T-148
iso), lyophilized Phaeodac ylum ico nu um and eshly collec ed and sie ed pa icles 149
o na u al sedimen . 150
Shell leng h was measu ed wi h a 0.05 accu acy callipe , and li e-weigh was 151
de e mined using a 0.01 mg accu acy balance. A e h ee mon hs, he la ges and 152
smalles 24 indi iduals, ep esen ing he pe cen iles P12.5 and P87.5 in size dis ibu ion o 153
each g oup, we e selec ed as as (F) and slow (S) g owe s, espec i ely. Acco dingly, 154
ou expe imen al g oups o mussels we e ob ained combining main enance (BP and 155
AP) and g ow h (F o S) condi ions: i) as -g owing mussels ed below he pseudo aeces 156
h eshold (FBP), ii) slow-g owing mussels ed below he pseudo aeces h eshold (SBP), 157
iii) as -g owing mussels ed abo e he pseudo aeces h eshold (FAP), and i ) slow-158
g owing mussels ed abo e he pseudo aeces h eshold (SAP). The g ow h a es o he 159
mussels we e calcula ed as GR = he inc ease in he shell-leng h o li e-weigh /elapsed 160
ime (days). A e he physiological expe imen s had been comple ed, he gills o he 161
mussels we e dissec ed and p ocessed o gill su ace a ea de e mina ion and RNA 162
ex ac ion. 163
2.2. RNA ex ac ion 164
Gill samples we e s o ed imme sed in RNAla e a -80 ºC un il he RNA was 165
indi idually ex ac ed wi h a ‘RiboPu e RNA Pu i ica ion Ki ’ (Ambion ki ). The 166
analysis o he quali y and in eg i y o he RNA was checked wi h F agmen Analyze TM 167
Au oma ed CE Sys em equipmen om Ad anced Analy ical wi h ‘DNF-471 S anda d 168
Sensi i i y RNA Analysis ki ’, (15 n ) and F agmen Analyze TM 1.1.0.11 so wa e. The 169
RNA quali y was checked using PROSize 2.0. The RNA concen a ion was measu ed in 170
he spec opho ome e UV/VIS Nanod op 1000 (The mo Fishe ). 171
We used 20 indi idual mussels pe expe imen al condi ion (20 om FBP, 20SBP, 172
20FAP and 20SAP). The gill RNA was ex ac ed indi idually. Once ex ac ed, he RNA 173
was quan i ied acco ding o he me hod desc ibed abo e. Each indi idual RNA sample 174
was hen dilu ed o a common concen a ion o 100 ng/ul. A e ha , he 20 indi idual 175
RNA samples pe expe imen al g oup we e andomly combined o c ea e 4 di e en 176
pools composed o 5 di e en indi idual . To c ea e he pools, he same RNA quan i y 177
(500 ng) om each o he 5 indi iduals was added. Once c ea ed, he concen a ion o 178
he pools was quan i ied in he Nanod op 1000. Thus, we ob ained 16 di e en pools (4 179
pools om each expe imen al g oup x 4 expe imen al g oups), each one con aining 180
RNA om 5 di e en indi iduals. A e ha , he 16 pools we e ma ked as desc ibed in 181
he sec ion 2.3.1. 182
2.3. Mic oa ay design and hyb idiza ion 183
We used a Su eP in G3 Cus om mic oa ay (8x60 k) om Agilen o analyse he 184
ansc ip ome o he gill samples. Mic oa ay p obes we e designed using Agilen 185
eA ay pla o m, using My ilus gallop o incialis sequences downloaded om NCBI in 186
Feb ua y o 2015. Sequences wi h he bes Blas x hi (e- alue <10e-10) o unique 187
p o eins agains non edundan da abase we e selec ed. Th ee noniden ical p obes we e 188
designed o each sequence. Housekeeping genes ( hose usually used in My ilus qPCR 189
analysis) we e added as posi i e con ols, alongside de aul Agilen nega i e con ols. 190
The emaining spo s in he a ay we e illed wi h sequences o he genus My ilus 191
ep esen ing unique p o eins (we e My ilus gallop o incialis o hologue was missing). 192
Two p obes o he unanno a ed sequence (one in each eading di ec ion) we e included 193
in he a ay. Thus, he a ay was based on 17,491 unanno a ed and 7,806 anno a ed 194
sequences. The pla o m is a ailable in gene exp ession omnibus (GEO) eposi o y wi h 195
he accession numbe GPL25650. Hyb idiza ion was pe o med in 16 pools (4 di e en 196
pools o di e en 5 indi iduals pe expe imen al g oup). Pools we e andomly 197
hyb idized in he a ays, including a leas one pool pe expe imen al g oup in each 198
a ay. 199
2.3.1. Ma king p o ocol 200
We used he ‘One-Colo Mic oa ay-Based Exon Analysis’ ma king p o ocol 201
om Agilen . Samples we e ma ked using ‘Low Inpu Quick Amp WT Labeling ki , 202
One-Colo ’ (p/n 5190-2943) ki . In o al, 100 ng o RNA was used o he ma king 203
eac ion. Ma ked samples we e quan i ied wi h a Nanod op ND-1000 o de e mine he 204
e iciency o he speci ic ac i i y o he luo och omes. 205
2.3.2. Hyb idiza ion 206
Samples we e manually hyb idized wi h Su eHyb Hyb idiza ion Chambe s 207
(Agilen echnologies). Hyb idiza ion was conduc ed in he o en o Agilen 208
Technologies acco ding o he Agilen p o ocol. The cha ac e is ics we e as ollows: 600 209
ng o ma ked cRNA, 40 µl olume, 65 ºC empe a u e, and 20 hou s du a ion a 10 pm 210
in he hyb idiza ion. 211
2.3.3. Scanning 212
The scanning was ca ied ou on he DNA Mic oa ays G2565CA scanne wi h 213
ozone–ba ie slide co e s wi h he Scan Con ol So wa e e sion 8.5.1., using he 214
de aul p o ocol Agilen G3_GX_1Colo . The Scanning esolu ion was 3 µm, he g een 215
channel was used, and he size o he esul ing Ti image was 20 bi s. 216
2.3.4. Fea u e ex ac ion 217
We used Agilen Fea u e Ex ac ion So wa e ( e . 10.7.3.1) (Agilen 218
Technologies) o p ocess he mic oa ay images and o quan i y he luo escence o he 219
p obes. The quali y o all a ays was e alua ed using he 9 QC-me ic pa ame e s 220
gene a ed in he ea u e ex ac ion. Following his p ocedu e, he p ocessed luo escence 221
signal (gene a ed by he ea u e ex ac ion) was ob ained. 222
223
2.4. Da a ea men 224
Da a ea men was ca ied ou in R ( . 3.3.2.) using he limma package ( . 225
3.30.13) om Bioconduc o (Ri chie e al. 2015). P obes we e p e il e ed using 226
gIsPosAndSigni ag; a Boolean alue indica ing i he signal o he p obe exceeds he 227
backg ound signal. P obes wi h a nonsigni ican signal in all he samples o a leas one 228
expe imen al g oup (n=4) we e emo ed. Backg ound was co ec ed using no mexp 229
me hod, and no maliza ion be ween he a ays was pe o med using he quan ile 230
me hod, as desc ibed in Smy h e al. (2002). Fold-change and s anda d e o we e 231
es ima ed by i ing he da a o a linea model and an empi ical Bayes (eBayes) 232
smoo hing was applied o he s anda d e o s. The inal gene exp ession alue was he 233
a e age o he noniden ical p obes co esponding o each sequence. Di e en ial 234
exp ession quan i ica ion was based on a loga i hmic scale (logFC), he adjus ed p-235
alue o False Disco e y a e (Benjamin–Hochbe g me hod) ep esen ing he s a is ical 236
signi icance o he obse ed changes. P obes wi h FDR<0.05 we e conside ed 237
di e en ially exp essed, as sugges ed in Cheng and Pounds (2007). Hie a chical 238
clus e ing (HCL) analysis was pe o med using dendex end package ( .1.5.2.) o 239
analyse simila i y be ween samples. 240
No malized hyb idiza ion alues, as well as he aw da a, we e deposi ed in he 241
gene exp ession omnibus (GEO) eposi o y wi h he accession numbe GSE120975. 242
243
244
2.5. Anno a ion and gene on ology 245
246
Mic oa ay sequences we e anno a ed using Annoc ip 1.3. agains Swiss-P o 247
and UniRe da abases ( . ma ch-2017). Gene On ology (GO) o h ee domains 248
(Cellula Componen , Molecula Func ion and Biological P ocess) was analysed o 249
ansc ip ome da a in e p e a ion, al hough we ocused ou analysis mainly on he 250
Biological P ocess (Sua ez-Ulloa e al. 2015). The GO e ms lis was summa ized using 251
REVIGO (Supek e al. 2011). Di e en ially exp essed genes we e also mapped o he 252
Kyo o Encyclopaedia o Genes and Genomes (KEGG) da abase o pa hway analysis 253
(Kanehisa, 2002). Conse ed p o ein domains we e iden i ied using PROSITE (Sig is 254
e al. 2009) and NCBI conse ed p o ein domain inde ools. 255
256
257
258
3. Resul s 259
260
261
3.1. G ow h a es o he expe imen al mussel g oups 262
263
A e 3 mon hs o main enance o he mussels unde BP o AP condi ions, he 264
li e weigh o F indi iduals was 2.5- old highe han ha o S indi iduals, and he shell 265
leng h was 45% longe . Acco dingly, li e-weigh and shell-leng h g ow h a es o F 266
indi iduals was ound o be app oxima ely 3 imes g ea e han ha o S indi iduals in 267
bo h main enance condi ions (Table 1). 268
269
Table 1. Shell-leng h (mm), li e-weigh (g), shell-leng h g ow h a e (mm/day) and li e-weigh g ow h 270
a e (g/day) (mean alues ± SD) o FBP, SBP, FAP and SAP mussel g oups. Numbe o indi iduals pe 271
mussel g oup = 24 272
273
Mussel
g oup Leng h (mm) Li e weigh (g) G ow h a e
(mm/day)
G ow h a e
(g/day)
FBP
21.2 ± 0.7
0.9 ± 0.1
0.146 ± 0.009
0.012 ± 0.002
SBP
13.9 ± 1.2
0.3 ± 0.1
0.055 ± 0.015
0.004 ± 0.001
FAP
21.9 ± 0.6
1.0 ± 0.1
0.144 ± 0.007
0.011 ± 0.001
SAP
15.4 ± 1.0
0.5 ± 0.1
0.060 ± 0.013
0.004 ± 0.001
274
3.2. Quali y and ep oducibili y o he DNA mic oa ay da a 275
The ma ked RNA quali y was good in all samples. The yield and he Cyanine 3 276
speci ic ac i i y we e highe han 0.825 µg/ eac ion and 15 pmol/µg, espec i ely, in all 277
ma ked samples. In all cases, he hyb idiza ion wi h he a ay sui ed (o passed) he 278
quali y s anda ds, e alua ed wi h 9 QC me ics pa ame e s. Only in 0.95% o he p obes 279
(568 p obes ou o 59,539) did he signal ha e a lowe exp ession alue han he 280
backg ound on all samples. Fo ou analysis, we used he p obes ha had a posi i e 281
signal on all he samples in a leas one expe imen al g oup. Mean exp ession alues 282
and s anda d de ia ions o he housekeeping genes o he a ay a e shown in he 283
addi ional ile 1. The a iabili y among samples was lowe han 3% in 19 o 20 284
housekeeping genes. 285
3.3. T ansc ip anno a ion 286
The unc ional anno a ion o he genes on he a ay ca ied ou by Blas x agains 287
Swiss-P o and UniRe da abases had 38.8% signi ican ma ches (E- alue 10-5): 10,001 288
ou o 25,781 genes. In o al, 27.7% o he anno a ed genes we e ma ched on 289
4. Discussion 367
In he p esen s udy, we analysed he gene exp ession di e ences in he gill 368
issue be ween as -g owing (F) and slow-g owing (S) mussels ha we e main ained o 369
he long e m in he labo a o y, while being ed expe imen al die s o phy oplank on and 370
sil dosed ei he below (BP) o abo e (AP) he pseudo aeces h eshold. In acco dance o 371
wha we epo ed p e iously om a simila expe imen (P ie o e al., 2018), he as e 372
g ow h o he F mussels (bo h FBP and FAP) was based on hei capaci y o display highe 373
clea ance a es and highe p e-inges i e selec ion e iciencies (physiological esul s will 374
be published elsewhe e). I espec i e o he die ed (BP o AP), inc eased capaci y o 375
wa e il a ion and pa icle acquisi ion in F mussels ha e been ound o be coupled wi h 376
he possession o signi ican ly highe gill-su ace a eas, a ea u e o he as -g owing 377
pheno ype ha we ha e also ound in ou p e ious s udies on mussels (P ie o e al. 378
2018) and clams (Tamayo e al. 2011). 379
The expe imen s o physiological ene ge ics (P ie o e al., in p ep) e ealed only 380
mino di e ences in he physiological pa ame e s o mussels ed BP and AP die s. In 381
good ag eemen wi h he physiological esul s, he ansc ip omic p o iles (HCL esul s) 382
we e e y simila be ween hem and only a educed numbe o genes we e di e en ially 383
exp essed. Th ee o he anno a ed DEGs (glu a hione S- ans e ase, headcase p o ein 384
and p o ocadhe in β) p e iously ha e been epo ed o be up egula ed in esponse o 385
en i onmen al s ess and/o bac e ial in ec ion in bi al es (Manduzio e al. 2004; Pa k 386
e al. 2009; Kim e al. 2009; De Zoysa e al. 2011; Li e al. 2018; Rey-Campos e al. 387
2019). Howe e , any in e p e a ion ega ding possible di e ences in he s imula ion o 388
immune esponse in mussels eed below o abo e he pseudo aeces le el is complica ed 389
because BP mussels o e exp essed glu a hione S- ans e ase, whe eas he o he wo 390
genes we e di e en ially exp essed in AP mussels. The emaining anno a ed DEGs 391
(No ch- egula ed anky in epea -con aining p o ein (NRARP), TNFAIP3-in e ac ing 392
p o ein 2 and FAM60A p o ein and RNA 2'-O-me hyl ans e ase ib illa in) ac in 393
se e al pa hways in ol ed in cell di e en ia ion, p oli e a ion, apop osis and RNA and 394
p o ein me hyla ion in bi al es such as no ch pa hway (Bassim e al. 2014), TFG-be a 395
signalling pa hway (Wei e al. 2017) and MAP/ERK pa hway. Li e al. (2016) epo ed 396
ha TNFAIP3-in e ac ing p o ein was down egula ed in indi iduals o Chlamys a e i 397
exposed o Benzopy ene and sugges ed ha he educ ion in TNFAIP3 was indica i e o 398
dep essed me abolic a e and hampe ed p og ession o mi osis. In he p esen 399
expe imen , o e exp ession o 4 genes in ol ed in cell p oli e a ion pa hways in he 400
mussels ha we e ed abo e pseudo aeces le el could sugges he exis ence o an 401
inc eased gill cell enewal equi emen in AP mussels. Howe e , mo e analysis should 402
be pe o med o con i m such a hypo hesis. 403
The low impac ha nu i ional condi ion and eeding mode (below o abo e he 404
pseudo aeces h eshold) exe on he gill ansc ip ome con as s wi h he b oad 405
di e ences associa ed wi h he di e ences in g ow h a e be ween F and S specimens: 406
117 di e en ially exp essed genes in gill issues. The classi ica ion o hese genes 407
acco ding o Biological P ocess GO e ms indica ed ha he di e ences mainly a ec 408
esponses o s imulus, g ow h and cellula ac i i y p ocesses. Thus, he GO e m 409
indings suppo ed he highe g ow h a es and ac i i y le els o F indi iduals in 410
compa ison wi h S mussels. No su p isingly, p e ious wo ks on in e indi idual g ow h 411
a e di e ences in bi al es ha e also desc ibed simila GO e ms as he main p ocesses 412
unde lying g ow h di e ences; o ins ance, Wilson e al. (2016) epo ed ha 19% o 413
GO e ms o di e en ially exp essed genes in as -g owing Mya a ena ia a e associa ed 414
wi h cell s uc u e, whe eas 17% e e o signalling and g ow h, 12% o ene gy and 415
nu ien me abolism and 10% o DNA/RNA and p o ein syn hesis. Rega ding he 416
KEGG e ms, ene ge ic me abolism e ms we e e e ed o F indi iduals in good 417
co espondence o hei highe ac i i y le els. Up egula ion o Co ac o and P5P 418
biosyn hesis pa hway in S indi iduals seems o in ol e di e ences in p o ein 419
me abolism ha could unde lie di e ences in he p o ein u no e be ween g ow h 420
g oups, as desc ibed in p e ious s udies (Hawkins e al. 1986, 1996). The P5P 421
biosyn hesis pa hway could ei he indica e a highe a e o anae obic me abolism, which 422
in bi al es is based on he u iliza ion o amino acids ia opine dehyd ogenases, o 423
aspa a e-succina e pa hway (Hochachka and Some o. 2002) 424
Mos di e en ially exp essed (DE) genes be ween he p esen F and S 425
indi iduals lack a clea associa ion o GO e ms because he s udied model p esen s 426
only ew sequences anno a ed in he ools allowing pe o mance o he GO analysis. 427
Thus, emphasis has been placed on he indi idual ( a he han he g oup) analysis o DE 428
genes and hei unc ions o deciphe he ansc ip omic basis o g ow h a e di e ences. 429
430
431
4.1. Up egula ed genes in F mussels. 432
Up egula ion o g ow h di e en ial ac o -8, also known as myos a in, and 433
insulin-like g ow h ac o in he gill o F mussels would appea meaning ully associa ed 434
wi h he highe gill su ace a ea exhibi ed by as g owe s. Myos a in is a nega i e 435
egula o o muscle g ow h in e eb a es, and Wang e al. (2010) ound ha 436
polymo phism o he myos a in gene was co ela ed wi h di e en ial g ow h ai s in 437
mammals. In bi al es, myos a in ha e been sugges ed o ha e al e na i e unc ions ha 438
a e ela ed wi h cell de elopmen (Saina and Technau, 2009; Núñez-Acuña and 439
Galla do-Ésca a e, 2014; Mo elos e al. 2015; Niu e al. 2015). Insulin-like pep ides 440
ha e been epo ed o ac as g ow h egula o s o so issues and shell in bi al es 441
(Taylo e al. 1996; G icou e al. 2003), and hei oles in de e mining in e indi idual 442
g ow h a e di e ences in bi al es ha e been ecen ly sugges ed by Saa ed a e al. 443
(2017), who ound a signi ican o e exp ession o NOV-like p o ein in he gills o as -444
g owing Rudi apes decussa us. Using he PROSITE ool on he highly di e en ially 445
exp essed genes (FC>8, FDR<0.01), we ha e ound ha , in addi ion o myos a in and 446
insulin-like pep ides, F indi iduals up egula ed an epide mal g ow h ac o -like (EGF). 447
Valenzuela-Mi anda e al. (2015) also epo ed he o e exp ession o EGFs in he 448
muscle o F specimens in he abalone Halio is u escens. EGF is exp essed in a ious 449
issues o oys e s (Sun e al. 2014) and has been sugges ed o induce cell p oli e a ion 450
and mig a ion du ing wound healing and o s imula e glycoly ic enzymes such as 451
phospho uc okinase and py u a e kinase (Canesi e al. 2000). 452
In addi ion o o e exp essing g ow h- egula o s, he gills o F mussels 453
o e exp essed genes in ol ed in he s uc u e and unc ionali y o he ex acellula 454
ma ix (ECM), such as collagen, laminin, ibulin and deco in. Some o hese genes ha e 455
been p e iously epo ed o be di e en ially exp essed be ween as - and slow-g owing 456
indi iduals o di e en in e eb a es: Fo ins ance, collagen, has been ound o be 457
up egula ed in F indi iduals in abalones (Valenzuela-Mi anda e al. 2015) and clams 458
(Saa ed a e al. 2017). Genomic (Zhang e al. 2012) and ansc ip omic (Zhao e al. 459
2012) analyses ha e sugges ed ha collagen migh play an impo an ole in shell 460
o ma ion and so issue g ow h and epai in bi al es. In addi ion, collagen also 461
appea s o play a ele an ole in he adhesion and mig a ion o haemocy es o he ECM 462
(Kou sogiannaki and Kaloyianni, 2011) and likely plays a c ucial ole in he p ocess o 463
cell immuni y du ing in lamma o y esponse (Adams, 2018). Fibulin ha e been epo ed 464
o ac in associa ion wi h laminin and collagen in de elopmen and biomine aliza ion 465
p ocesses (Timpl e al. 2003; Sleigh e al. 2015). Deco in in e ac s wi h some g ow h 466
ac o s such as EGF, and i s binding wi h myos a in has been desc ibed o cause 467
hype ophy in human muscle cells (Kanzlei e e al. 2014). 468
The F mussels in he p esen expe imen we e ound o o e exp ess mucin, he 469
backbone glycop o ein ha o ms he ma ix o he mucus (Espinosa e al. 2016). In 470
bi al es, he il e ed pa icles a e e ained in he mucus s ings ci cula ing h ough he 471
cilia ed g o es and anspo ed o he labial palps o be ei he inges ed o ejec ed as 472
pseudo aeces (Beninge and S -Jean, 1997; U u ia e al. 2001). A highe pu a i e 473
mucus p oduc ion in F indi iduals would be in conco dance wi h hei highe clea ance 474
a es and highe p e-inges i e selec ion e iciencies, wi h bo h physiological pa ame e s 475
g ea ly con ibu ing o in e indi idual di e ences in he g ow h a e o mussels (P ie o 476
e al. 2018). Consis en wi h he highe clea ance a es and highe mucin exp ession, he 477
gills o F mussels also o e exp essed ib ocys in, which is in ol ed in ubulogenesis 478
and cilia y ac i i y (Wa d e al. 2003), as well as he dynein and ilB homologue p o ein 479
genes ha a e in ol ed in he con e sion o ATP hyd olysis in o mechanical wo k 480
(Gibbons and Rowe. 1965; Ka lie e al. 2010; Ho ani e al. 2013). Dynein 481
o e exp ession in F specimens has also been epo ed in Halio is u escens (Valenzuela-482
Mi anda e al., 2015). O e exp ession o hese genes in F mussels seems o co ela e 483
well wi h he highe il e ing ac i i y o he mussels. Recen ly, La on e al. (2019) 484
epo ed ha ib ocys in was one o he up egula ed genes in oys e la ae wi h highe 485
a es o su i al o he pes i us (OsHV-1) in ec ion in an expe imen ha showed 486
ansgene a ional immune p iming in C assos ea gigas. 487
P ocesses in ol ed in he me abolic ene gy supply and ATP u no e a e 488
especially ele an o he g ow h a e o bi al es, and hus, he inding ha 2 genes 489
ela ed o ene gy me abolism we e up egula ed in F indi iduals is highly meaning ul. 490
P e ious app oaches o he cha ac e iza ion o gene ic di e ences be ween as - and 491
slow-g owing indi iduals o di e en species ha e emphasized he impo ance o 492
di e en ial aspec s o he ene ge ic me abolism be ween g ow h lines. Fo ins ance, 493
Meye and Manahan (2010) ound ATP-syn hase and wo di e en NADH 494
dehyd ogenase subuni s up egula ed in F indi iduals o he oys e C. gigas, Wilson e 495
al. (2016) ound a y acid syn hase like-1 and a y acid syn hase like-2 genes 496
up egula ed in as -g owing indi iduals o M. a ena ia, and Saa ed a e al. (2017) 497
ound he NADH subuni up egula ed in F indi iduals o he clam Rudi apes 498
decussa us. In he p esen s udy, we ound ci a e syn hase (CS) and ca bonic anhyd ase 499
up egula ed in he gill o F indi iduals. Ci a e syn hase is a speci ic ma ke o ae obic 500
me abolism conside ed an indica o o he gene al physiological s a us o he o ganism 501
(Ga cia-Esqui el e al. 2001, 2002; Pe ne e al. 2012; Gué élou e al. 2013) and has 502
been shown o co ela e wi h espi a ion a es in acul a i e anae obes such as in e idal 503
in e eb a es (Dahlho e al. 2002). Highe ci a e syn hase exp ession in ou F mussels 504
migh hus be indica i e o inc eased ene gy equi emen s o gill issue o sus ain highe 505
il e ing ac i i y. The ca bonic anhyd ase enzyme amily main ains he pH/salini y 506
balance and a ou s he exchange o espi a o y gases (B e on, 2001) and has been 507
epo ed o play a ole in he p ocess o biomine aliza ion in he man le issue (Zhang e 508
al. 2012; Hüning e al. 2016). Finally, DENN domain-con aining p o ein 3, also ound 509
o be up egula ed in F indi iduals, is in ol ed in he egula ion con e sion o inac i e 510
GDP-bound o GTP o m and esicle-media ed anspo pa hways (Ma a e al. 2011). 511
We ha e no ound e idence o a DENN domain-con aining p o ein unc ion in 512
bi al es. 513
The gill o F indi iduals up egula ed wo genes di ec ly ela ed wi h he immune 514
sys em, p obably loca ed in he haemocy es: ni ic oxide syn hase (NOS) and he 515
sca enge ecep o MARCO (mac ophage ecep o wi h collagenous s uc u e). NOS 516
has been de ec ed in haemocy es o se e al bi al es (Liu e al. 2018) and p oduces 517
ni ic oxide, a pa hogen-killing molecule wi h b oad an i i al and an ipa asi ic e ec s 518
(Pau z e al. 2010). In mammals, MARCO is a ecep o o bac e ia exp essed mainly in 519
mac ophages; in bi al es, i has been p e iously epo ed in My ilus gallop o incialis 520
(Mo ei a e al. 2015). 521
4.2. Up egula ed genes in S mussels. 522
The gills o he slow-g owing mussels o e exp ess many genes in ol ed in 523
immune, de ence and cell s ess esponses, such as HSP24, leucine- ich epea p o eins, 524
me alloendopep idase and galec in. The o e exp ession o hea shock p o eins has been 525
commonly ound in o ganisms main ained unde empe a u e s ess (Ho man and 526
Some o, 1996; Some o 2012; Lockwood e al. 2013), salini y s ess (Zhao e al. 2012) 527
me al exposu e (Zhang e al. 2012) and/o bac e ial exposu e (Gena d e al. 2013). In 528
addi ion, Zhang e al. 2012 ound an o e exp ession o HSP genes in he oys e 529
C assos ea gigas unde a ious s ess condi ions (ai exposu e, he mal s ess, salini y 530
s ess and me al exposu e) and concluded ha HSP induc ion could be a common 531
de ence agains all s esses in C. gigas. Leucine- ich epea p o eins ha e been 532
desc ibed o be in ol ed in he immuni y o in e eb a es (Wang e al. 2016) and 533
me alloendopep idase, seems o be key componen o he esponse agains bac e ial 534
in ec ions (Miyoshi and Shinoda, 2000). Galec in is p obably associa ed wi h 535
haemocy es (Espinosa e al. 2016; Vas a e al. 2015) and pa icipa es in he ecogni ion 536
o glycans o he su ace o i us and bac e ia (Nikapi iya e al. 2014). In good 537
co espondence wi h he p esen s udy, Saa ed a e al. (2017) also ound di e ences 538
be ween as - and slow-g owing indi iduals o he clam Rudi apes decussa us in he 539
immune and de ence p ocesses o diges i e gland and gills. S indi iduals o e exp essed 540
genes in ol ed in immune and de ence p ocesses such as de ensin and umou nec osis 541
ac o membe 11, whe eas F indi iduals we e ound o o e exp ess di e en genes, 542
such as sialic acid-binding lec in and hyd amacin-1. They conclude ha he obse ed 543
high di e ences in he exp ession o immune and de ence genes could e lec a 544
di e en ial i ness among indi iduals, p omo ing as e g ow h a es in hose indi iduals 545
able o igh mo e e icien ly agains diseases. In he p esen s udy, mos o he 546
o e exp essed genes in S mussels we e ound o belong o he immune and de ence 547
sys em and cellula s ess, which s ongly sugges s a g ea e p e alence o 548
pa hogens/diseases o a highe suscep ibili y o he pa hogens. As sugges ed by Gena d 549
e al. (2013), when analysing he physiological esponse o C. gigas la ae submi ed o 550
bac e ial in ec ion, ex a in es men s in suppo ing de ence mechanisms migh d ain 551
ene gy esou ces om no mal p ocesses in heal hy o ganisms, esul ing in educed 552
eeding and g ow h pe o mances. 553
In addi ion, he s ong up egula ion o coun in-1 (FC≈4), a cell-coun ing ac o 554
ha limi s he maximum size o he mul icellula s uc u e by he down egula ion o he 555
cell adhesion media o gp24, seems o indica e de elopmen al p ocess inhibi ion in S 556
indi iduals. Symp oms o impai men in he espi a o y unc ion o he gill a ec ing 557
ae obic ATP p oduc ion a e also e iden in S mussels: E idence o inc eased use o 558
anae obic me abolic pa hways includes s ong up egula ion o anae obic C4-559
dica boxyla e anspo e (FC ≈ 8), as well as he inc eased biosyn hesis o py idoxal-5 560
–phospha e. Simila ly, Saa ed a e al. (2017) ha e epo ed up egula ion in he 561
diges i e gland o S clams o enzymes e y likely in ol ed in anae obic me abolism 562
(e.g., mala e dehyd ogenase and glyce ol-3-phospha e dehyd ogenase). 563
564
4.3. Conclusions and p ospec s 565
The p esen esul s show he exis ence o subs an ial di e ences in he 566
ansc ip ome o he gills o F and S indi iduals. The gills o he as -g owing mussels 567
o e exp essed g ow h ac o s and genes ha a e in ol ed in he main enance o ele an 568
cellula unc ions, such as he main enance o he cilia y ac i i y, he de elopmen o a 569
obus ex acellula ma ix con ibu ing o an ibac e ial de ence and he main enance o 570
ae obic me abolic pa hways. This ansc ip omic p o ile in he F mussels sugges s ha 571
he gills a e well equipped o main ain highe il e ing ac i i ies ha enable as -572
g owing mussels o maximize ood acquisi ion and sus ain as g ow h a es. In con as , 573
slow-g owing mussels o e exp ess genes in ol ed in he immune sys em and genes ha 574
pa icipa e in cellula -s ess esponses and anae obic me abolic pa hways. These esul s 575
could sugges ha S indi iduals would ha e a g ea e p e alence o pa hogens/diseases 576
o a highe suscep ibili y o he pa hogens. Fu he analysis wi h di e en o gans (e.g., 577
diges i e gland) a e needed o ob ain a holis ic iew o he ansc ip omic basis o as -578
g ow h in bi al es; howe e , he p esen s udy sugges s ha he immune esponse migh 579
be a c ucial componen o he in e indi idual di e ences in g ow h a e in My ilus 580
gallop o incialis mussel spa s. 581
582
5. Acknowledgemen s 583
584
This s udy was unded h ough he p ojec AGL2013-49144-C3-1-R o he Spanish 585
Minis y o Economy and Compe i i eness. D. P ie o was unded by an FPI g an om 586
he Basque Go e nmen . The au ho s a e hank ul o he echnical and human suppo 587
p o ided by SGIke o UPV/EHU (Eu opean unding: ERDF and ESF). Finally, D. 588
P ie o especially wan s o hank D . A. Hu e o all he help p o ided. 589
590
6. Re e ences 591
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