Accep ed Manusc ip
Tau ine me abolism and e ec s o inclusion le els in o i e
(B achionus o undi o mis, Tschuguno , 1921) on A lan ic
blue in una (Thunnus hynnus, L.) la ae
Mónica B. Be anco , Guillaume R. Lau en , Au elio O ega,
Fe nando de la Gánda a, Douglas R. Toche , Gab iel Mou en e
PII: S0044-8486(19)30320-5
DOI: h ps://doi.o g/10.1016/j.aquacul u e.2019.05.040
Re e ence: AQUA 634155
To appea in: aquacul u e
Recei ed da e: 7 Feb ua y 2019
Re ised da e: 19 Ap il 2019
Accep ed da e: 18 May 2019
Please ci e his a icle as: M.B. Be anco , G.R. Lau en , A. O ega, e al., Tau ine
me abolism and e ec s o inclusion le els in o i e (B achionus o undi o mis,
Tschuguno , 1921) on A lan ic blue in una (Thunnus hynnus, L.) la ae, aquacul u e,
h ps://doi.o g/10.1016/j.aquacul u e.2019.05.040
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Tau ine me abolism and e ec s o inclusion le els in o i e
(B achionus o undi o mis, Tschuguno , 1921) on A lan ic blue in
una (Thunnus hynnus, L.) la ae
Mónica B. Be anco a*, Guillaume R. Lau en a, Au elio O egab, Fe nando de la
Gánda ab, Douglas R. Toche a and Gab iel Mou en ec
a Ins i u e o Aquacul u e, Facul y o Na u al Sciences, Uni e si y o S i ling, FK9 4LA S i ling,
Sco land, UK
b Plan a Expe imen al de Cul i os Ma inos, Ins i u o Español de Oceanog a ía (IEO), 30860
Pue o de Maza ón (Mu cia), Spain
c Depa amen o de Biología, Facul ad de Ciencias del Ma y Ambien ales, Uni e sidad de Cádiz,
11510 Pue o Real, Cádiz, Spain
* Co esponding au ho :
Tel.: +44-1786-467892
E-mail add ess: m.b.be anco @s i .ac.uk
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Abs ac
Tau ine appea s o be a c ucial nu ien o eleos s, especially op p eda o species such as A lan ic
blue in una (Thunnus hynnus, L.; ABT). While die a y au ine supplemen a ion has been highly
ecommended, he e is a lack o s udies on au ine assimila ion and biosyn hesis o his iconic
species. The p esen s udy aims o p o ide insigh in o he molecula mechanisms in ol ed in au ine
biosyn hesis and anspo in ABT by s udying issue dis ibu ion and on ogene ic de elopmen o
exp ession o cys eine dioxygenase (cdo), cys eine sul inic acid deca boxylase (csad), 2-
aminoe hane hiol dioxygenase (ado) and au ine anspo e ( auT) in esponse o g aded le els o
die a y au ine supplemen a ion. The ull open eading ame (ORF) o cdo and pa ial sequences o
csad, ado and auT we e ob ained, wi h he ansla ed polypep ides being 202, 176, 166 and 324
amino acids, espec i ely. All h ee showed cha ac e is ics such as cupin mo i s in Cdo and p edic ed
N-glycosyla ion si es in Tau ha a e common o hese genes in o he species. Phylogene ic analysis
showed ha he ABT sequences clus e ed wi h sequences o o he eleos s, and sepa a ely om
mammals and molluscs. Tissue dis ibu ion a ied, wi h adipose issue, kidney, whi e muscle and
es is/b ain showing highes exp ession o cdo, csad, ado and auT, espec i ely. Whole la ae
exp ession o csad peaked a 15 dah, whe eas he o he genes gene ally inc eased h oughou
de elopmen o show highes exp ession a 25 dah. The nu i ional ial was ca ied ou by eeding
ABT la ae om mou h opening o 14 days a e ha ching (dah) wi h o i e s (B achionus
o undi o mis) en iched wi h 4 di e en le els o au ine: 0.0 ( au0), 0.5 ( au0.5), 1.0 ( au1), and 2.0
g au ine pe 106 o i e s ( au2). Ro i e s e ec i ely accumula ed au ine wi h ABT la ae ed on
ea men au2 a aining he highes concen a ion o au ine. Howe e , ABT la ae ed au1 displayed
highe g ow h and su i al, and lexion index a 14 dah, han la ae ed he o he au ine le els.
La ae ed au1 also showed gene ally highe exp ession o auT and cdo and diges i e and
an ioxidan enzyme genes. While his s udy showed ha la al ABT exp ess au ine me abolism
genes, sugges ing possible syn hesis ha could con ibu e o he au ine pool in he ish, la al
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pe o mance was enhanced by a le el o die a y au ine (3.7 mg au ine g-1 o i e ) supplied by
en ichmen o o i e s a 1 g au ine pe 106 o i e s.
Keywo ds: blue in una, la ae, au ine, gene exp ession, o i e en ichmen , cDNA
Abb e ia ions: aa, amino acids; ABT, A lan ic blue in una (Thunnus hynnus); alp, alkaline
phospha ase; amy, amylase; anpep, amino pep idase; bac in, be a ac in; bal1, bile sal ac i a ed lipase
1; bal2, bile sal ac i a ed lipase 2; ca , ca alase; cdo, cys eine dioxygenase; csad, cys eine sul inic
acid deca boxylase; dah, days a e ha ch; e 1α, elonga ion ac o 1 alpha; FC, old change; gpx1,
glu a hione pe oxidase 1; gpx4, glu a hione pe oxidase 4; myhc, myosin hea y chain; ORF, open
eading ame; pl, panc ea ic lipase; pla2, phospholipase A2; qPCR, quan i a i e eal ime PCR; sod,
supe oxide dismu ase; auT, au ine anspo e ; opo, opomyosin; yp, ypsin; ubiq, ubiqui in;
UTR, un ansla ed egion.
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In oduc ion
A lan ic blue in una (ABT, Thunnus hynnus, L.) is a species wi h high ma ke alue al hough
i s closed aquacul u e is cu en ly ine icien and a om la ge-scale comme cial p oduc ion wi h
low su i al o la al s ages, (De la Ganda a e al., 2016; Van Beijnen, 2017). In o de o op imize
he ABT p oduc ion cycle, u he knowledge o he nu i ional equi emen s o he species is pi o al,
and unde s anding biological mechanisms o nu ien assimila ion in la ae is a key a ea. Al hough
some s udies ha e been pe o med on di e en aspec s o ABT nu i ion (Mo ais e al., 2011;
Be anco e al., 2017a,b; Ko en e al., 2018) he e is limi ed in o ma ion ega ding equi emen s o
many nu ien s ha can be c i ical o la al and ju enile s ages o his species.
Tau ine is he common name o 2-aminoe hanesul onic acid, an amino sul onic acid which
is no inco po a ed in o p o eins bu , a he , esides in he ee amino acid pool (Ham e e al., 2013).
Despi e his, au ine is no conside ed an amino acid since i con ains a sulphonyl acid g oup a he
han a ca boxyl acid g oup (Pin o e al., 2012). Howe e , au ine plays a c i ical ole in many majo
biological unc ions and, in eleos s, is in ol ed in bile sal conjuga ion, osmo egula ion, memb ane
s abiliza ion, modula ion o neu o ansmi e s, an ioxidan unc ion and ea ly de elopmen o isual,
neu al and muscula sys ems (Hux able, 1992; Salze and Da is, 2015). In e eb a es, he e a e wo
main pa hways o biosyn hesizing au ine om cys eine wi h he inal s ep in bo h pa hways being
he oxida ion o hypo au ine o au ine, wi h he p oduc ion o hypo au ine a ying (Salze and Da is,
2015). One pa hway in ol es he pa icipa ion o wo enzymes, cys eine dioxygenase (Cdo; EC
1.13.11.20) and cys eine sul ina e deca boxylase (Csad; EC 4.1.1.29), which p oduce hypo au ine
om cys eine. A second ou e o hypo au ine p oduc ion is h ough he ac ion o he enzyme 2-
aminoe hane hiol dioxygenase (Ado; EC 1.13.11.19), which con e s cys eamine, de i ed om
coenzyme A deg ada ion, o hypo au ine. In addi ion o hese enzymes, au ine anspo e (Tau ), a
highly conse ed memb ane anspo e is c i ical o he anspo and ecycling o au ine and plays
c ucial oles in in es inal unc ions (O’Flahe y e al., 1997; Shimizu and Sa su, 2000). Fish ha e
a ied au ine biosyn hesis capabili y, possibly eflec ing di e ences in he exp ession
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le els/ac i i ies o he key biosyn he ic enzymes and he au ine anspo e (Liu e al., 2017). Fo
ins ance, Csad ac i i y has been epo ed o di e among di e en eleos species (El-Sayed, 2014;
Salze and Da is, 2015) and an appa en lack o Csad ac i i y has been epo ed in ish amilies such
as he Lab idae, Scomb idae and Soleidae (Salze and Da is, 2015) and ABT (Yokoyama e al., 2001).
So a , i is unknown i he me abolic pa hway o biosyn he izing au ine using enzymes o
ans o m me hionine-de i ed cys eine is ac i e in ABT. The e o e, i ABT is unable o syn hesize
au ine by endogenous me abolism, die a y inpu would be essen ial especially o la al s ages whe e
biosyn he ic unc ions in gene al a e s ill de eloping and incomple e (De la Rosa and S ipanuk, 1985).
In he wild, ABT la ae can assimila e au ine om na u al ood, mainly copepods (Uo ani e al.,
1990; Ca alan e al., 2011) ha con ain high le els o au ine (Van de Mee en e al., 2008; Ka lsen
e al., 2015). In a ming, au ine would ha e o be supplied by eed and, gi en he p esen end in
aqua eed p oduc ion, wi h ish meal and oil being eplaced by e es ial plan sou ces ha a e de oid
o au ine, i is c ucial o de e mine he au ine biosyn he ic capaci y o ABT, as a de iciency in his
nu ien could appea (Ga lin e al., 2007; Ba ows e al., 2008; Takagi e al., 2008). This is
pa icula ly impo an in ABT, a op p eda o in he ophic chain, sugges ing ha au ine en ichmen
o eed migh be essen ial. Some p e ious s udies ha e indica ed he posi i e e ec ha die a y au ine
can ha e on eleos la ae, such as enhancemen on g ow h (Ma suna i e al., 2005a,b, 2008, 2013;
Ka lsen e al., 2015; Kim e al., 2016), eed con e sa ion a io and lipid me abolism (Cha zi o is e
al., 2007), diges i e enzyme ac i i ies (Salze e al., 2012), and me amo phosis (Pin o e al., 2010).
Indeed, a ecen s udy in Paci ic blue in (Thunnus o ien alis) and yellow in una (T. albaca es) la ae
demons a ed ha eeding o i e s en iched wi h 800 mg au ine L-1 p omo ed la al g ow h and o al
p o ein con en (Ka agi i e al., 2017), sugges ing ha au ine is an impo an nu ien o he ea ly
s ages o apidly g owing eleos species.
The aim o he p esen s udy was o p o ide insigh in o he molecula mechanisms in ol ed
in au ine biosyn hesis and anspo in ABT by s udying he issue dis ibu ion, on ogene ic
de elopmen and esponse o g aded die a y au ine supplemen a ion o cdo, csad, ado and auT genes
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Fo his pu pose, he open eading ames (ORF) o he genes we e sequenced and hei exp ession
de e mined by eal ime quan i a i e PCR (qPCR) in issues and du ing de elopmen . Addi ionally, a
dose- esponse nu i ional ial was pe o med by eeding ABT la ae om mou h opening o 14 days
a e ha ching (dah) wi h o i e s en iched wi h ou inc easing le els o au ine (0.0 g au ine pe 106
o i e s, au0; 0.5 g au ine pe 106 o i e s, au05; 1.0 g au ine pe 106 o i e s, au1 o 2.0 g au ine
pe 106 o i e s, au2). Mo eo e , he e ec s o g aded au ine inclusion in o i e s on he exp ession
o la al ABT genes ela ed o an ioxidan and diges i e enzymes was also in es iga ed.
2. Ma e ials and Me hods
2.1. Isola ion o genes o au ine me abolism
Sequences o genes encoding o au ine me abolism ( auT, cdo, ado and csad) we e ob ained
by iden i ying he sequences om Sequence Read A chi es (SRA) SRX2255758, ERX555873 and
ERX555874. The se o con iguous sequences we e assembled using CAP3 (Huang and Madan,
1999) and iden i y o he deduced amino acid (aa) sequences con i med using he BLASTp sequence
analysis se ice o he Na ional Cen e o Bio echnology In o ma ion (NCBI)
(h p://www.ncbi.nlm.nih.go ). P ime s we e designed in o de o sequence he open eading ames
(ORF) o each gene (Supplemen a y Table) using cDNA om whole ABT la ae (see below) as
empla e. PCR p oduc s ob ained we e pu i ied using he Illus a GFX PCR DNA and Gel Band
Pu i ica ion ki (GE Heal hca e, Li le Chal on , UK) and sequenced o con i m iden i y (Sange
ABI3730xl, Eu o ins Genomics, Kons anz, Ge many). Subsequen ly, p ime s o qPCR we e
designed on hese PCR agmen s using he online so wa e P ime 3 (Un e gasse e al., 2012;
Supplemen a y Table).
The deduced aa sequences o he newly sequenced ABT auT, cdo, ado and csad and
sequences o hese genes o a a ie y o species ac oss e eb a e and in e eb a e lineages we e
aligned wi h he Clus alW ool (BioEdi 7.0.9, Tom Hall, Depa men o Mic obiology, No h
Ca olina S a e Uni e si y, USA). Phylogene ic analysis was pe o med using he neighbou -joining
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me hod wi h MEGA 5.1 (h p://www.megaso wa e.ne /) (Sai ou and Nei, 1987). Con idence in he
esul ing ee b anch opology was measu ed using boo s apping h ough 1,000 eplica ions.
2.2. Tissue RNA ex ac ion and cDNA syn hesis
Samples o 100 mg o la ae o issue we e homogenized in 1 mL o TRI Reagen (Sigma-
Ald ich, Do se , UK) using a bead issue dis up o (BioSpec, Ba les ille, OK, USA) be o e being
mixed wi h 100 µL BCP (Phase sepa a ion eagen , 1–b omo–3–chlo op opane, Sigma-Ald ich). The
uppe aqueous phase was ans e ed o a esh ube and mixed wi h RNA p ecipi a ion solu ion
(sodium chlo ide + sodium ci a e sesquihyd a e, Sigma-Ald ich) and isop opanol. A e
cen i uga ion, he RNA pelle was washed wice wi h e hanol and esuspended in molecula biology
g ade wa e . Quan i y and quali y o he RNA we e de e mined by spec opho ome y using a
NanoD op ND-1000 (Lab ech In ., Eas Sussex, UK), and in eg i y de e mined by elec opho esis
using 200 ng o o al RNA in 1 % aga ose gel. cDNA was syn hesized using 2 μg o o al RNA and
andom p ime s in 20 μL eac ions and he high capaci y e e se ansc ip ion ki wi hou RNase
inhibi o acco ding o he manu ac u e ’s p o ocol (Applied Biosys ems, Wa ing on, UK).
2.3. Quan i a i e PCR (qPCR) analysis o gene exp ession
P ime s o qPCR we e designed on he abo e PCR agmen s o au ine me abolism genes
using he online so wa e P ime 3 (Un e gasse e al., 2012), and we e a ailable o ABT genes
ela ed o an ioxidan enzymes, diges i e enzymes and housekeeping om p e ious s udies (Be anco
e al., 2017a,b) (see Supplemen a y Table). Th ee housekeeping genes we e es ed (elonga ion ac o -
1α, el 1α, ubiqui in, ubiq and β-ac in, bac in), wi h el 1α and ubiq selec ed as being mo e s able
acco ding o geNo m (Vandesompele e al., 2002; M s abili y alue = 0.165 o bo h genes). The
e iciency o p ime s o each gene was e alua ed by se ial dilu ions o cDNA pooled om he
samples o con i m i was > 85 % o all p ime pai s. qPCR was pe o med using a Biome a TOp ical
The mocycle (Analy ik Jena, Goe ingen, Ge many) in 96-well pla es in duplica e 20 μL eac ion
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olumes con aining 10 μL o Lumina is Colo HiG een qPCR Mas e Mix (The mo Scien i ic, Hemel
Hemps ead, UK), 1 μL o he p ime co esponding o he analyzed gene (10 pmol concen a ion), 3
μL o molecula biology g ade wa e , and 5 μL o cDNA (1/20 dilu ed). In he case o housekeeping
genes only 2 μL o cDNA we e used inc easing he molecula biology g ade wa e o 6 μL. In
addi ion, ampli ica ions we e ca ied ou wi h a sys ema ic nega i e con ol (NTC, no empla e
con ol) con aining no cDNA. S anda d ampli ica ion pa ame e s con ained a UDG p e- ea men a
50 °C o 2 min and an ini ial dena u a ion s ep a 95 °C o 10 min, ollowed by 35 cycles: 15 s a
95 °C, 30 s a he annealing empe a u e (Supplemen a y Table 1) and 30 s a 72 °C. A he end o
he qPCR un, a mel cu e o 0.5 °C inc emen s om 75 °C o 90 °C was pe o med, enabling
con i ma ion o he ampli ica ion o a single p oduc in each eac ion. Fo gene exp essions in
on ogenesis and he die a y ial, he exp ession le els (gene exp ession old change) o he a ge
genes we e calcula ed ollowing he me hod desc ibed by P a l (P a l, 2001). The ela i e
exp ession o each gene among he issues was calcula ed as he loga i hm o a bi a y uni s a e
no maliza ion agains he exp ession le el o he housekeeping gene el 1α. One a bi a y uni was
equal o he lowes exp ession le el o he gene in each da ase .
2.4. Tissue dis ibu ion o au ine me abolism genes
Samples o issues including b ain, gills, hea , kidney, spleen, li e , in es ine, whi e muscle,
ed muscle, adipose issue, o a y and es is we e ob ained om b oods ock una (n = 4; 2 males and
2 emales; be ween 200 - 250 kg o al weigh and 10 o 15 yea s old) ha we e being sac i iced as
pa o he no mal ope a ing p ocedu es o check o ma u a ion s age and gonadal de elopmen .
Addi ionally, o a ies and es is om a u he wo emales and males we e collec ed in o de o ha e
an adequa e sample size (n = 4). All issue samples (~ 100 mg) we e placed in RNALa e ® (Sigma-
Ald ich, Do se , UK), le o e nigh a 4 °C and subsequen ly s o ed a -70 °C p io o RNA
ex ac ion.
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The au ine me abolism genes showed a ied issue dis ibu ions (Fig. 2). The highes numbe
o ansc ip s o cdo was ound in adipose issue, ollowed by li e and in es ine. In con as , he
exp ession le el o csad was highes in kidney ollowed by in es ine wi h li e showing he lowes
alue. The highes numbe o mRNA copies o auT we e ound in ed muscle, ollowed by whi e
muscle ≥ spleen, wi h only a low le el ound in li e . Wi h ado, es is and b ain we e he issues wi h
he highe numbe s o ansc ip s whe eas exp ession was much lowe in all he o he issues.
3.4. Die a y ial
3.4.1. Tau ine con en in ABT la ae
ABT la ae e ec i ely accumula ed au ine in hei bodies as a s ong and posi i e co ela ion
was ound be ween die a y au ine and la al au ine le els (Tables 1 and 2). This ela ionship was
ound o be linea wi h an R2 alue o 0.95 (y = 5.3x – 4.3) (Table 2).
3.4.2. G ow h, de elopmen and su i al o ABT la ae
G ow h pe o mance o ABT la ae 14 dah and ed on o i e s B. o undi o mis en iched wi h
Algamac 3050 Bio Ma ine® and di e en doses o au ine (0.0, 0.5, 1.0 and 2.0 g au ine.10-6 o i e )
is shown in Table 3. To al leng h and weigh s we e signi ican ly highes when ABT la ae we e ed
die au1 ( o i e s en iched wi h 0.5 g au ine pe 106 o i e s), which co esponded o 3.7 mg au ine
g-1 o i e d y mass based on he measu ed au ine con en o he o i e s (Table 1), and nume ically
lowes in hose ed au0. Flexion index was signi ican ly highe in ABT la ae ed au1 compa ed o
la ae ed au0 and au0.5, wi h la ae ed au2 showing an in e media e alue. While ABT la ae
ed he au1 die showed he nume ically highes a e age su i al, he e we e no s a is ically
signi ican di e ences in su i al among ABT la ae ed he di e en au ine doses la gely due o
a ia ions wi hin ea men s.
3.4.3. Gene exp ession in ABT la ae
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The exp ession le els o bo h cdo and csad we e bo h signi ican ly highe in la ae ed die
au1 compa ed o la ae ed au0 and he o he le els o die a y au ine (Fig. 3). In con as , he
exp ession o ado showed he opposi e pa e n o his wi h exp ession being lowe in la ae ed au1
compa ed o la ae ed he o he die s. The auT exp ession le els showed a dec easing end as
die a y au ine inc eased wi h exp ession in la ae ed au0 being signi ican ly highe han in la ae
ed he die s supplemen ed wi h au ine (Fig. 3).
The exp ession o all he diges i e genes measu ed showed a simila pa e n wi h highes
exp ession in ABT la ae ed au1 (Fig. 4). The exp ession o bo h bile sal -ac i a ed lipase 1 (bal1)
and phospholipase A2 (pla2) was signi ican ly highe in ABT la ae ed au1 compa ed o la ae ed
au0. While a simila pa e n in exp ession was obse ed wi h bile sal -ac i a ed lipase 2 (bal2) he
di e ences did no each s a is ical signi icance.
All he genes o he an ioxidan sys em ha we e measu ed showed a simila pa e n wi h he
highes exp ession in ABT la ae ed he au1 die (Fig.5). While his was signi ican o supe oxide
dismu ase (sod), glu a hione pe oxidase 1 (gpx1) and glu a hione pe oxidase 4 (gpx4), he di e ences
in exp ession o ca alase (ca ) we e no s a is ically signi ican .
4. Discussion
The p esen s udy aimed o in es iga e he impac s o die a y au ine le el ia en ichmen o
o i e on g ow h and me abolism o i s eeding ABT la ae. Fi s ly, key genes o au ine
me abolism we e cloned, wi h he ull ORF sequence ob ained o cdo, and pa ial sequences achie ed
o auT, csad and ado. Fo auT he pa ial ORF (324 aa) con ained po en ial N-glycosyla ion si es
and six ansmemb ane domains, which was in ag eemen wi h auT o o he species (Wang e al.,
2017). Phylogene ic analyses showed a clea dis inc ion be ween eleos and mammal clus e s wi h
simila i y sco es o mo e han 90 % and 81 %, espec i ely. Fu he mo e, molluscs we e clea ly
sepa a ed om bo h mammals and eleos s, which may indica e ha au ine anspo e de eloped
ea lie in e olu ion as p e iously sugges ed (Hui e al., 2012). In ag eemen he phylogene ic ees
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o he h ee genes g ouped ABT oge he wi h o he eleos species indica ing high e olu iona y
conse a ion.
The ull mRNA sequence o Cdo was ob ained wi h an ORF coding o a p o ein o 202 aa,
whe eas a pa ial ORF sequence o 166 aa was acqui ed o Ado. Alignmen o aa om bo h genes
e ealed cupin mo i s 1 and 2 sepa a ed by an in e mo i egion, which a e common cha ac e is ics
o cupin p o eins (Dunwell e al., 2001; S ipanuk e al., 2011; Wang e al., 2016). The pa ial ORF
sequence coding o 176 aa ound o csad con ained he impo an py idoxal-dependen
deca boxylase conse ed domain, an enzyme g oup which is also p esen in csad o Pag us majo ,
Se iola quinque adia a, O eoch omis nilo icus, and O yzias la ipes (Haga e al., 2015). The
phylogene ic analyses also e ealed high simila i y sco es o he ABT genes wi h genes o o he
eleos s o he han salmonids in he case o Csad, and Salmo sala and Anguilla japonica o Cdo.
This highligh s in e es ing di e en ia ion in au ine me abolism genes, on one hand, be ween
eshwa e and ma inewa e species and, on he o he hand, be ween anad omous and ca ad omous
ish. Thus, e olu iona y adap a ions o di e en li es yles, including mig a ions and ans e be ween
eshwa e and ma ine en i onmen s wi h associa ed di e en equi emen s o osmo egula ion may
ha e gene a ed di e en ia ion in genes o au ine assimila ion and/o biosyn hesis.
The exp ession le els o he ou ABT au ine me abolism genes was e alua ed du ing ea ly
on ogenesis om 1 dah o 25 dah. Resul s showed ha , du ing ea ly la al de elopmen , he
exp ession le el o he csad gene peaked ea lie han he exp ession le els o cdo, ado and auT. In
gene al, exp ession o he genes was low 1 dah and inc eased du ing de elopmen sugges ing
inc easing biosyn hesis o au ine, which may e lec ha au ine is necessa y o la al de elopmen
o ABT. As he ansc ip copies could be de ec ed a 1 dah, i is possible ha ma e nal mRNA is
p esen in he egg, as has been obse ed in zeb a ish emb yos (Chang e al., 2013). The peak o auT
ansc ip copy numbe a 25 dah was simila o esul s ound in Senegalese sole a 30 dah by Pin o
e al. (2010), which may indica e ha du ing he in e media e la al s age (18-25 dah), ma ine ish
la ae including ABT ha e inc eased capaci y o anspo au ine. Al hough he on ogenic analysis
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o gene exp ession was ca ied ou on whole la ae, muscle is he main issue and, gi en ha auT
exp ession was g ea es in ABT muscle issues, i is likely ha he peak in auT exp ession e lec s
he enhanced anspo o au ine in muscle, whe e g ow h po en ial is e y high a his s age o
de elopmen . In ag eemen wi h his, Ado, an enzyme ha p oduces hypo au ine by he oxida ion o
cys eamine h ough a pa hway di e en o ha o Csad and Cdo (Salze and Da is, 2015), also peaked
a 25 dah. Howe e , he highes old change (FC) o hese genes is ela i ely low (1.8 o auT and
2.4 o ado), whe eas a FC o 18.3 and 33.3 was obse ed o csad and cdo, espec i ely, bo h
enzymes pa icipa ing in he same biosyn he ic pa hway. These high FC indica e ha he Csad/Cdo
combina ion is he main pa hway o au ine biosyn hesis and ha csad is he a e limi ing enzyme
o au ine biosyn hesis in bo h mammals (De La Rosa and S ipanuk, 1985) and ish (Chang e al.,
2013).
The ou au ine me abolism genes we e exp essed o some ex en in all issues o ABT
examined, in ag eemen wi h o he ish species (Pin o e al., 2012; Haga e al., 2015; Plasus e al.,
2019). Howe e , in he p esen s udy, auT was p edominan ly exp essed in muscle issue (whi e >
ed), which is consis en wi h ish muscle con aining ela i ely high le els o au ine (Hux able,
1992). The e o e, he high exp ession le els obse ed in his issue migh e lec he physiological
unc ion o auT, inducing he up ake o au ine in o skele al muscle cells. Adipose issue displayed
he highes cdo ansc ip copy numbe , indica ing a high po en ial o au ine biosyn hesis in his
issue, as ound p e iously in mice (Ueki and S ipanuk, 2008). Howe e , au ine also plays an
impo an ole in osmo egula ion and his may be e lec ed in he high mRNA copy numbe s o csad
in kidney, which has also been obse ed in o he eleos species (Haga e al., 2015). In he p esen
s udy, he highes exp ession le els o ado in ABT we e obse ed in es is and b ain. The high le el
o exp ession o hese genes in gonads is ela ed o he high concen a ion o au ine in hese issues
(Plan e e al., 2008). Li le in o ma ion is cu en ly a ailable ega ding he cys eamine pa hway
in ol ing ado, al hough a ecen s udy in ca p (Cyp inus ca pio) epo ed b ain o be he main issue
exp essing he enzyme, al hough es is was no included in ha s udy (Plasus e al., 2019). S udies in
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di e en animal species ha e also shown ha ac i i ies o he au ine me abolism enzymes a y
among issues (Kuo and S ipanuk, 1984; S ipanuk and Ueki, 2011). The e o e, i seems ha he
pa e n o issue exp ession o he au ine me abolism genes in ABT is ela ed o he biochemical
unc ions each enzyme and he ole o di e en issues. On he o he hand, i should no ed ha high
mRNA le els o hese genes ha e no been co ela ed o highe enzyme ac i i y (Higuchi e al., 2012).
This could explain why, o ins ance, he exp ession le els o csad in kidney we e ele a ed whe eas
cdo le els we e qui e low, sugges ing ha egula ion migh be a he p o ein le el as opposed o he
ansc ip ional le el. O e all hough, he p esence and exp ession o hese genes indica es ha ,
despi e being a op p eda o , ABT has some capaci y o biosyn hesize au ine, and does no ely
en i ely upon die a y in ake. Howe e , no au ine was de ec ed in la ae ed au0, which indica es
ha al hough hey con ain he enzyma ic machine y, i is no e icien . In con as , nei he mRNA no
enzyme ac i i y o some o he au ine me abolism enzymes ha e been iden i ied in some ish species
such as cobia (Rachycen on canadum; Go o e al., 2001a; Wa son e al., 2014).
In o de o con i m an ac i e ole o au ine me abolism including biosyn hesis in ABT, a
ial was ca ied ou by eeding la ae om mou h opening o 14 dah wi h di e en le els o au ine
supplied ia o i e s en iched wi h inc easing le els o au ine. Tau ine concen a ion in la ae was
s ongly co ela ed o he le el o au ine en ichmen in o i e in ag eemen wi h p e ious ials
(Ma suna i e al., 2007; Ka agi i e al., 2017; Ko en e al., 2018). This con i ms ha ABT la ae a e
able o assimila e die a y au ine in o hei issues and may e lec a au ine equi emen . The lack o
au ine in he en ichmen media ( au0) led o poo g ow h in e ms o o al leng h and o al d y mass
and impai ed de elopmen indica ed by educed lexion index. In con as , he highes g ow h and
mos apid de elopmen was ob ained in la ae ed au1 ha co esponded o 3.7 mg au ine pe g
o i e d y mass. These esul s a e consis en o wha has been obse ed in la ae o o he una
(Ka agi i e al., 2017) and eleos species (Ma suna i e al., 2005a.b, 2013; Pin o e al., 2010;
Hawkya d e al., 2015; Kim e al., 2016), whe e en ichmen o o i e s wi h au ine p omo ed la al
g ow h. None heless, he inc ease o die a y au ine om 3.7 o 9.0 mg g-1 o i e s did no u he
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p omo e la ae g ow h, simila ly o a s udy in humpback g oupe (C omilep es al i elis), whe e
inc easing he le els om 2.7 o 8.5 mg au ine g-1 o i e did no lead o inc eased la al o al leng h
(Ridwan and Ha ya i, 2017). These esul s indica e ha le els o au ine o a ound 3.8 mg g-1 may
sa is y he equi emen s o ABT la ae o his nu ien . In con as , su i al o la al ABT was no
signi ican ly a ec ed by die a y au ine in he p esen s udy in con as o se e al p e ious s udies in
Pag us majo and Pa alich hys oli aceus (Chen e al., 2004a,b), Se iola dume ili (Ma suna i e al.,
2013), Nibea albiflo a (Xie e al., 2015) o Se iola lalandi (Ro man e al., 2017). This is likely due
o he la ge in e - ank a iabili y obse ed in he p esen ial, al hough a lack o e ec o die a y
au ine has also been epo ed in o he species such as A ac oscion nobilis (Ro man e al., 2017) and
Solea Senegalensis (Pin o e al., 2010).
While he abo e con i med a ole o die a y au ine in la al ABT, he p esen ial also
demons a ed a ole o endogenous au ine me abolism. The mRNA copy numbe o auT was
egula ed by die a y au ine in a dose dependen manne , wi h he gene being down- egula ed as
die a y le els o au ine inc eased. This indica es ha when subs a e ( au ine) le els a e low, auT
exp ession is up- egula ed o p omo e and enhance he abso p ion and anspo o au ine. Simila
esul s we e obse ed in u bo (Scoph almus maximus) bo h in i o (Wang e al., 2017) and in i o
(Wei e al., 2018) as well as in A lan ic salmon smol s (Za a e and B adley, 2007). Aside om auT,
o he genes in eleos s ha e been specula ed o ake pa in au ine homeos asis, pa icipa ing in he
biosyn hesis o his amino acid. In his espec , he egula ion o au ine biosyn hesis is complica ed,
as i is no only egula ed by he p oduc au ine bu also he le els o subs a e sul u amino acids,
wi h di e en ial egula ion o csad and cdo (Wang e al., 2016). I would be expec ed ha bo h
enzymes would be up- egula ed when au ine le els we e low/de icien , bu his was no he case as
peak mRNA copy numbe s we e obse ed in la ae ed au1 wi h 3.7 mg au ine pe g o i e s. Se e al
s udies in eleos s ha e epo ed he lack o egula ion by au ine o cdo exp ession/ac i i y, which
was mainly egula ed by cys eine and me hionine (Gaylo d e al., 2006; Wang e al., 2015, 2016).
The e o e, he consis en pa e n o exp ession o bo h cdo and csad in ABT could be in luenced by
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he combina ion/ a io o sulphu amino acids a he han solely by he le els o die a y au ine.
Addi ionally, he lack o egula ion by die a y au ine could indica e a low capaci y o biosyn hesize
au ine in ABT, gi en ha in he wild hese ish usually consume au ine- ich p ey, such as smalle
ish. Consis en wi h his, no Csad ac i i y was ound in Paci ic blue in una (Yokoyama e al., 2001).
The e is ano he pa hway o p oduce au ine in eleos s using cys eamine, p oduced om he
b eakdown o coenzyme A, which is hen he subs a e o cys eamine dioxygenase (Ado). Mos o
he s udies in eleos s ha e ocussed on he cys eine sul inic acid pa hway, and paid li le a en ion o
he exp ession and/o ac i i y o ado. In he p esen s udy, a pa ial ado mRNA was epo ed o he
i s ime in una, and i was shown ha i s ansc ip copy numbe was modula ed by die a y au ine
le el. A die a y au ine le el o 3.7 mg g-1 o i e ( au1) lead o down- egula ion o ado exp ession
al hough he le els we e no s a is ically di e en o hose in ish ed au0 o au2. P e ious s udies
showed no egula ion o ado exp ession by au ine in a zeb a ish cell line, which could indica e ha ,
simila o csad and cdo, ado could be egula ed pos - ansc ip ionally (Liu e al., 2017). These esul s
sugges ha he cys eamine pa hway is no e y ac i e in ABT, as has been shown o o he
ca ni o ous ma ine eleos s (Go o e al., 2001b).
In addi ion o p omo ing g ow h, au ine has also been shown o enhance diges ibili y in ish
(Lunge e al., 2007). The diges i e enzymes, bile sal -dependan lipases 1 and 2 (bal1 and bal2),
ha e been epo ed o be he main enzymes in ol ed in lipid diges ion in Paci ic blue in una
(Mu ashi a e al., 2014). In he p esen ial, bo h bal1 and bal2 showed a simila pa e n o
exp ession, wi h highes exp ession le els in la ae ed au1 (3.7 mg g-1 o i e s). Fu he mo e, pla2,
an enzyme in ol ed in in es inal phospholipid diges ion (Toche , 2003), showed he same pa e n as
bal1, again wi h highes exp ession le el in la ae ed au1. Taken oge he hese esul s indica e a
diges i e p omo ing e ec o au ine a an en ichmen le el o 3.7 mg au ine g-1 o i e , which was
en i ely consis en wi h he impac o die a y au ine on ABT la al g ow h. Howe e , i is wo h
no ing ha he exp ession le els o he diges i e genes could be in luenced by g ow h a he han
die a y au ine le els, as p e iously sugges ed (Be anco e al., 2017b). Indeed, simila esul s we e
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ound by Sæle e al. (2010), whe e a ela ionship be ween bal genes and cod (Gadus mo hua) la ae
body size was shown.
Tau ine is known o ha e an ioxidan p ope ies, and can se e as a sca enge o some eac i e
oxygen species (Me aye e al., 2008). Indeed, au ine de iciency can ha e an impac on ed-ox
balance ha can, consequen ly, esul in mi ochond ial oxida i e s ess in i o (Jong e al., 2012). A
p e ious s udy ound ha Ca , Sod and Gpx ac i i ies inc eased wi h die a y au ine le el in se e al
ish species (Li e al., 2016). In ag eemen , he exp ession le els o sod, gpx1 and gpx4 in ABT in he
p esen s udy we e highes in la ae ed au1, hese la ae also showing he highes g ow h and a e
o de elopmen . Indeed, a s ong co ela ion was ound be ween la al o al leng h, d y weigh and
gpx1 exp ession le els ( = 0.6 and 0.5, espec i ely), which co obo a es he ole o au ine as an
an ioxidan . In con as , ano he s udy showed dec eased exp ession o an ioxidan enzymes when
sea b eam la ae we e ed inc eased die a y au ine le els (Izquie do e al., 2019).
In summa y, he p esen s udy indica ed ha ABT la ae possess enzymes necessa y o
biosyn hesize au ine h ough he wo main pa hways. The h ee enzymes and he au ine anspo e
showed di e en ial issue exp ession and could be de ec ed be o e he onse o ex e nal eeding.
Exp ession o he biosyn hesis enzymes was no ob iously egula ed by die a y au ine le el, possibly
indica ing a nu i ional equi emen o his nu ien . In con as , auT exp ession was up egula ed
when die a y le els o au ine we e low, indica ing a ole o his gene in main aining au ine le els
in muscle and au ine homeos asis in ABT. Ro i e s supplemen ed wi h au ine a 1 g pe 106 o i e s
imp o ed he g ow h o ABT la ae, wi hou a ec ing inal su i al. In conclusion, despi e he
p esence o au ine biosyn hesis genes, ABT la ae equi ed a supply o die a y au ine a a ound 3.7
mg g-1 eed ( o i e ) in o de o ensu e adequa e g ow h and de elopmen .
Acknowledgemen s
We wish o hank he echnical s a a Labo a o y o Ma ine Aquacul u e (IEO), Pue o de Maza ón
(Mu cia), Spain and Nu i ional Analy ical Se ices (NAS), Ins i u e o Aquacul u e, Uni e si y o
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S i ling, UK ha con ibu ed o his wo k. This wo k was suppo ed by he Conseje ía de Inno ación,
Ciencia y Emp esa de la Jun a de Andalucía, P oyec o de Excelencia de P omoción Gene al del
Conocimien o [Re . RNM 733, 2012), and P og ama Es a al de In es igación del Minis e io de
Economía y Compe i i idad [Re . AGL2014-52003-C2-1-R, 2014].
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Figu e Legends
Figu e 1. Exp ession o cys eine dioxygenase (cdo), cys eine sul inic acid deca boxylase (csad),
au ine anspo e ( auT) and 2-aminoe hane hiol dioxygenase (ado) du ing de elopmen o A lan ic
blue in una (Thunnus hynnus) la ae (1 dah-25 dah) ea ed unde s anda d p ocedu es. Resul s
ep esen means ± s anda d e o (n = 4) o ela i e exp ession no malized wi h wo housekeeping
genes (ubiqui in and elonga ion ac o 1 alpha). Di e en le e s show signi ican di e ences o he
exp ession o each gene du ing de elopmen .
Figu e 2. Tissue dis ibu ion o cdo, csad, auT and ado ansc ip s in A lan ic Blue in una
b oods ock. T ansc ip exp ession le el was de e mined by qPCR in 12 issues wi h alues deno ing
he log-no malized (e 1

) ela i e exp ession o he a ge genes in each issue. Da a ep esen he
a e age o ou indi iduals (n = 4) wi h s anda d e o s (SEM). B, b ain; G, gills; H, hea ; K, kidney;
S, spleen; L, li e ; I, in es ine; R, ed muscle; W, whi e muscle; A, adipose issue; O, o a y; T, es is.
Figu e 3. Nu i ional egula ion o au ine me abolism genes, cys eine dioxygenase (cdo), cys eine
sul inic acid deca boxylase (csad), au ine anspo e ( auT) and cys eamine dioxygenase (ado) in
la ae o A lan ic blue in una (T. hynnus). La ae we e ed o i e s (B achionus o undi o mis)
en iched wi h 4 le els o au ine: 0.0 ( au0); 0.5 ( au0.5); 1.0 ( au1); 2.0 ( au2) g au ine.10-6 o i e s.
Values a e no malized exp ession a ios, co esponding o an a e age o 6 pools o la ae (n = 6) wi h
s anda d e o s (SEM). Le e s deno e signi ica e di e ences as de e mined by one-way ANOVA (p
< 0.05).
Figu e 4. Nu i ional egula ion o diges i e enzymes, bile sal -ac i a ed lipase 1 (bal1), bile sal -
ac i a ed lipase 2 (bal2) and phospholipase A2 (pla2) in la ae o A lan ic blue in una (T. hynnus).
La ae we e ed o i e s (B achionus o undi o mis) en iched wi h 4 le els o au ine: 0.0 ( au0); 0.5
( au0.5); 1.0 ( au1); 2.0 ( au2) g au ine.10-6 o i e s. Values a e no malized exp ession a ios,
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co esponding o an a e age o 6 pools o la ae (n = 6) wi h s anda d e o s (SEM). Le e s deno e
signi ica e di e ences as de e mined by one-way ANOVA (p < 0.05).
Figu e 5. Nu i ional egula ion o an ioxidan enzymes, glu a hione pe oxidase 1 (gpx1), glu a hione
pe oxidase 4 (gpx4), ca alase (ca ), supe oxide dismu ase (sod) in la ae o A lan ic blue in una (T.
hynnus). La ae we e ed o i e s (B achionus o undi o mis) en iched wi h 4 le els o au ine: 0.0
( au0); 0.5 ( au0.5); 1.0 ( au1); 2.0 ( au2) g au ine.10-6 o i e s. Values a e no malized exp ession
a ios, co esponding o an a e age o 6 pools o la ae (n = 6) wi h s anda d e o s (SEM). Le e s
deno e signi ica e di e ences as de e mined by one-way ANOVA (p < 0.05).
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Table 1. To al amino acid con en including au ine (mg/g d y mass) o o i e s B. o undi o mis
en iched wi h Algamac 3050® and inc easing doses o au ine (0.0 g/106 o i e s ( au0), 0.5 g/106
o i e s ( au0.5), 1.0 g/106 o i e s ( au1) and 2.0 g/106 o i e s ( au2).
au0
au0.5
au1
au2
Tau ine
0.0
±
0.0e
2.5
±
0.2d
3.7
±
0.1c
9.0
±
0.1a
EAA
Valine
18.5
±
3.4
22.8
±
0.8
20.4
±
1.2
22.2
±
3.0
Isoleucine
1.7
±
0.3
2.1
±
0.1
1.9
±
0.1
2.0
±
0.3
Leucine
26.8
±
1.8b
30.3
±
0.6a
26.4
±
1.6b
31.3
±
0.1a
Phenylalanine
17.3
±
1.2b
19.5
±
0.5a
16.9
±
1.0b
20.2
±
0.3a
His idine
6.1
±
0.8b
7.1
±
0.2a
6.0
±
0.4b
7.4
±
0.6a
Lysine
24.0
±
1.8b
28.0
±
0.6a
23.0
±
2.1b
30.1
±
0.1a
A ginine
17.5
±
3.2b
22.1
±
0.4a
18.6
±
1.7ab
23.0
±
0.3a
Th eonine
11.3
±
1.6b
14.7
±
0.6a
11.5
±
0.6b
14.3
±
0.3a
Me hionine
7.2
±
0.1b
8.4
±
0.1a
7.1
±
0.6b
8.4
±
0.1a
NEAA
Aspa ic acid
33.9
±
2.1b
38.1
±
0.8a
32.5
±
1.9b
38.2
±
0.2a
Glu amic acid
42.4
±
2.8b
49.0
±
1.4a
42.3
±
2.4b
49.5
±
0.3a
Se ine
12.1
±
0.4bc
16.1
±
0.4a
10.4
±
0.6c
13.3
±
0.7b
P oline
17.9
±
1.2ab
19.7
±
0.7a
16.7
±
1.0b
19.8
±
0.3a
Glycine
15.7
±
1.5b
17.2
±
0.4ab
16.2
±
1.1b
18.9
±
0.3a
Alanine
15.4
±
1.0bc
17.1
±
0.5ab
15.5
±
0.7bc
18.1
±
0.2a
Ty osine
13.4
±
0.8b
15.6
±
0.7a
12.5
±
0.8bc
15.3
±
0.2a
Cys eine
3.4
±
0.1ab
4.0
±
0.1a
3.4
±
0.3ab
3.0
±
0.2b
Da a a e means ± SD (n = 3). Means wi hin a ow bea ing di e en supe sc ip le e s a e signi ican ly
di e en as de e mined by one-way analysis o a iance (ANOVA), and Tukey’s mul iple
compa ison es (P < 0.05). EAA, essen ial amino acids; NEAA, non-essen ial amino acids.
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Table 2. To al amino acid con en including au ine (mg/g d y mass) o A lan ic blue in una (T.
hynnus L.) la ae 14 days a e ha ch ed on o i e s B. o undi o mis en iched wi h Algamac 3050
® and inc easing doses o au ine; 0.0 g/106 o i e s ( au0), 0.5 g/106 o i e s ( au05), 1.0 g/106 o i e s
( au1) and 2.0 g/106 o i e s ( au2).
au0
au0.5
au1
au2
Tau ine
0.0
±
0.0d
1.8
±
0.1c
3.8
±
0.1b
6.4
±
0.2a
EAA
Valine
35.6
±
0.6
35.9
±
0.1
32.5
±
4.6
36.8
±
0.6
Isoleucine
25.8
±
0.4a
26.1
±
0.2a
25.7
±
0.4a
26.3
±
0.5a
Leucine
42.4
±
0.3bc
43.1
±
0.2ab
42.8
±
0.5b
44.4
±
0.4a
Phenylalanine
23.9
±
0.7
24.2
±
0.7
24.1
±
0.4
25.1
±
0.8
His idine
3.2
±
0.4
3.3
±
0.2
3.2
±
0.2
3.1
±
0.6
Lysine
45.6
±
0.5b
46.5
±
0.2b
46.6
±
0.6b
48.5
±
0.7a
A ginine
8.5
±
0.5
9.1
±
0.2
8.9
±
0.2
9.1
±
0.3
Th eonine
10.2
±
0.4c
11.7
±
0.4ab
11.5
±
0.3b
12.7
±
0.5a
Me hionine
22.0
±
0.8ab
22.4
±
0.3ab
20.9
±
1.1b
23.8
±
1.3a
NEAA
Aspa ic acid
9.4
±
0.8
9.7
±
0.2
9.2
±
0.5
9.7
±
0.2
Glu amic acid
18.2
±
0.6
19.4
±
0.9
19.6
±
0.6
20.1
±
1.7
Se ine
3.3
±
0.2b
3.9
±
0.5ab
4.7
±
0.6a
4.6
±
0.3a
P oline
15.1
±
0.6ab
15.8
±
0.5ab
14.8
±
0.3b
16.0
±
0.3a
Glycine
10.2
±
0.5
10.4
±
0.4
9.5
±
0.8
9.2
±
1.0
Alanine
13.7
±
0.6
14.8
±
0.5
14.0
±
0.5
14.5
±
0.2
Ty osine
17.4
±
0.5
17.9
±
0.8
17.2
±
0.6
18.3
±
0.8
Cys eine
4.1
±
0.4
3.6
±
0.4
3.2
±
0.8
4.1
±
0.8
Da a a e means ± SD (n = 3). Means wi hin a ow bea ing di e en supe sc ip le e s a e signi ican ly
di e en as de e mined by one-way analysis o a iance (ANOVA), and Tukey’s mul iple
compa ison es (P < 0.05). EAA, essen ial amino acids; NEAA, non-essen ial amino acids.
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Table 3. G ow h pe o mance o 14 days a e ha ch ABT la ae ed on o i e s B achionus
o undi o mis en iched wi h Algamac 3050 Bio Ma ine® and di e en doses o au ine (0.0, 0.5, 1.0
and 2.0 g o au ine pe 106 o i e s).
Resul s o g ow h pe o mance a e p esen ed as means ± SD (n = 25 pe eplica e o o al leng h,
o al weigh and lexion index, and n = 3 o su i al a es. An SD o 0.0 implies an SD o < 0.05.
Means wi hin a ow bea ing di e en supe sc ip le e s a e signi ican ly di e en as de e mined by
one-way analysis o a iance (ANOVA), and Tukey’s mul iple compa ison es (P < 0.05).
au0
au0.5
au1
au2
To al leng h (mm)
6.6
±
0.4c
6.7
±
0.1bc
6.9
±
0.3a
6.8
±
0.3b
D y weigh (mg)
0.41
±
0.04c
0.45
±
0.01bc
0.55
±
0.06a
0.46
±
0.08bc
Flexion index
38.7
±
16.2b
40.0
±
7.2b
51.0
±
10.4a
45.7
±
9.7ab
Su i al (%)
12.4
±
1.8
9.6
±
2.8
14.7
±
7.8
10.5
±
8.5
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Highligh s
- Tissue dis ibu ion, on ogene ic de elopmen and exp ession in esponse o g aded le els o die a y
au ine supplemen a ion o genes in ol ed in au ine me abolism we e e alua ed in A lan ic Blue in
una.
- Tissue dis ibu ion a ied, wi h adipose issue, kidney, whi e muscle and es is/b ain showing
highes exp ession o cys eine dioxygenase (cdo), cys eine sul inic acid deca boxylase (csad), 2-
aminoe hane hiol dioxygenase (ado) and au ine anspo e ( auT), espec i ely.
- Whole la ae exp ession o csad peaked a 15 dah, whe eas he o he genes gene ally inc eased
h oughou de elopmen o show highes exp ession a 25 dah.
- A lan ic Blue in una la ae ed 1 g au ine pe 106 o i e s (3.7 mg 3 au ine g-1 o i e ) displayed
he bes g ow h and exp ession le els o auT, cdo, diges i e and an ioxidan enzymes.
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Figu e 1
Figu e 2
Figu e 3