Dicentrarchus Labrax): Molecular Characterization, Quantification in Plasma, Liver and Ovary, and Maturational Proteolysis

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Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis. Ozlem Yilmaz, Francisco Prat, A. Jose Ibáñez, Sadi Köksoy, Haruna Amano, Craig V. Sullivan

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Ozlem Yilmaz, Francisco Prat, A. Jose Ibáñez, Sadi Köksoy, Haruna Amano, et al.. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.. Comparative Biochemistry and Physiology - Part B: Biochemistry and Molecular Biology, Elsevier, 2016, 194-195, pp.71-86. 10.1016/j.cbpb.2015.11.010. hal-01303975

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Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint ÅÒ Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. mn,Hrn,Slia,CagV,Mlil ielgnn n rdc okproteins yolk product and vitellogenins ( bass Multiple sea V., European Craig in Sullivan, Haruna, Amano, eoei spbihdi t nlfr.Pes oeta uigtepouto process production the during that th pertain. note manuscript. affect journal Please the the could of to form. which proof apply version publication. discovered final resulting for its early be the in may accepted this of errors published providing review been is and are has it typesetting, we that copyediting, before customers manuscript undergo will unedited our manuscript an to The of service file a PDF As a is This proteolysis, maturational and ovary, B Part Physiology, and liver plasma, in Yilm as: article this cite Please 2015 November 26 2015 November 3 2015 May date: 7 Accepted date: Revised doi: date: Received S1096-4959(15)00210-9 9960 CBB in: appear To Reference: DOI: PII: Sullivan V. Craig Amano, ze imz rnic rt .Js Ib« Jose A. Prat, Francisco Yilmaz, bass Ozlem sea European in proteolysis maturational and proteins ovary, and liver yolk product ( and vitellogenins Multiple ÔØ ietacu labrax Dicentrarchus 21) doi: (2015), DOI :10.1016/j.cbpb.2015.11.010 oprtv iceityadPyilg,Pr B Part Physiology, and Biochemistry Comparative Comment citer cedocument: :Mlclrcaatrzto,qatfiaini plasma, in quantification characterization, Molecular ): 10.1016/j.cbpb.2015.11.010 ietacu labrax Dicentrarchus z ze,Pa,Facso Ib´ Francisco, Prat, Ozlem, az, 10.1016/j.cbpb.2015.11.010

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Akdeniz University,Akdeniz Fisheries07070, TURKEY Faculty, Antalya, (

Instituto de Ciencias MarinasdeAndalucía Department of Biology, College of Agriculture and LifeDepartmentCollegeBiology, AgricultureCarolina and of ofSciences,State North School ofMarineBiosciences, 1 Kitasato University, CentralImmunology Research University, Akdeniz Laboratories, Faculty and ofMedi Instituto de Acuicultura de Torre laSal de Dicentrarchus labrax * Institut* Present NationalINRA address: dela Recherche Agronomique,

Dr Moliner, 50 Present and Electron address: Confocal MicroscopyValência Service, University of Campus de 35000Rennes Beaulieu, FRANCE Cedex, University, Raleigh, NC Kanagawa 252 Antalya, 07070,TURKEY Torre 12595,Ribera Sal, s/n de de la Cabanes, Castell Avda. Real, República2,11510Puerto Saharaui NC 27605,USA Multiple vitellogenins YILMAZ - - a 0373, JAPAN Research building, Lab building, Research * liver liver and ovary , FRANCISCO PRAT , . Tel./Fax; 919

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ACCEPTED MANUSCRIPTgs, 1

in order to to order in body weight once every 7 days. Fish were anesthetized in a bath of clove oil (15 cloveoil of bath a anesthetizedin were Fish days. every7 once weight body Comment citer cedocument: - /or

ACCEPTED MANUSCRIPT Aldrich Corp., St. Louis, MO) dissolved in propylene glycol propylene in dissolved MO) Louis, St. Corp., Aldrich ) assess 4) other farmed fishes (Bromage et al. 1995; Ca 1995; al. et (Bromage fishes farmed other and rm 2 from tained rmty n re t verify to order in trometry

relative abundance of each form each of abundance relative

synthesiswasinduced classi moronids maturationa

received prior study with regard to broodstock management and management broodstock to regard with study prior received - AE odce t ioae rti bns pttv Vg) for Vtgs) (putative bands protein isolate to conducted PAGE later , fy and characterize and fy

Navas et al. Carnevali1998; et al. 2001 - which was finely minced and stored at stored and minced finely was which ya od adult old, year 3 ®

with disparate reproductive life Lf Technologies (Life poelss f ah ye f Lv of type each of proteolysis l )

and in the maletheinfish

not heretoforenot

nuto o vtloeei (see vitellogenesis of induction and to to identify types of Vtg produced Vtg of types identify

in detail the detail in fed ae e bs held bass sea male of Vtg or Vtg of

twice daily twice op, albd C) olwn the following CA) Carlsbad, Corp., e ia E final he bythreeintraperitoneal injections of been been described.

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Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. E (Rocha et 2009).al. t, (Student's E plasma Additionally, shown). not and inclusions ooplasmic such of devoid oocytes transluscent uniformly large with filled was ovary OV OM; to growth oocyte from transition the with associated breakdown or migration vesicle germinal or coalescence droplet lipid did that and ooplasm their in droplets lipid and granules yolk of inclusions obvious with diameter) μm (>750 oocytes large with filled was ovary PV Briefly, 1990). (1988, al. et histologicfor1932) Wolfe, Cleveland using stained and μm, 6 at sectioned paraffin, in embedded 1976), Trump and (McDowell gluteraldehyde formaldehyde:1% 4% in fixed was ovary each of portion at stored and (N=4) females PV the from taken were samples liver and ovary plasma, Additional blotting. Western for used or (2009) al. et Rocha by described as levels plaseparated x 1,500 at centrifuged were samples Blood 7.4). pH TIU/ml, Na mM, was blood proteas of µl the 100 with treated and tubes into collected EDTA, sodium 0.5M with coated syringes ml 1 using vein caudal the from bled post development. at tissue ovary and plasma blood ( seawater template for RT at stored and instructions manufacturer’s 2

(1.5 mg kg mg (1.5 Four ad Four F u year our 2 DA 1 M NaN mM, 11 EDTA ~37.8‰ ult male sea bass from the the from bass sea male ult P sma were stored at smaat were stored < -

- l f old The fish were anesthetized in a bath of 2 of bath a in anesthetized were fish The 1 0.001) as compared to those in OV females (0.84 ng/ml), as ng/ml), (0.84 females OV in those to compared as 0.001) -

PCR generate conducted to body weight) diluted in olive oil, with the injections administered every other day. Four day. other every administered injections the with oil, olive in diluted weight) body )

emale sea bass bass sea emale at the at

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT

Comment citer cedocument:

without a germinal vesicle (nucleus) or overlaying layers of follicle cells (data cells follicle of layers overlaying or (nucleus) vesicle germinal a without ACCEPTED MANUSCRIPT Institute of Aquaculture of Aquaculture of Institute al analysis to identify the stage of ovarian development as described bydevelopmentMayerdescribedovarian as stageof identify tothe analysis al 3

- . m, etmcn . m/l bnaiie 75 M Aprotinin mM, 17.5 benzamidine mg/ml, 2.2 gentamicin mM, 8.5 70 o C until being subjected to enzyme immunoassay (EIA) to measure E being enzymemeasureto subjected until immunoassay (EIA)C to rm sok b stock a from 2

same ees n V eae (.6 gm) ee infcnl elevated significantly were ng/ml) (2.46 females PV in levels - 20 - ielgnc P) n ouae (V sae o stages (OV) ovulated and (PV) vitellogenic vtg Institute of Aquaculture of Institute o C until being used for total RNA extraction to produce a produce to extraction RNA total for used being until C

cDNA direct for sequencing. e inhibitor/bacteriocide/antioxidant mixture (NaCl 150 (NaCl mixtureinhibitor/bacteriocide/antioxidant e e i cpiiy o svrl eeain i flowing in generations several for captivity in red Torre la Sal (Castellón, Spain) were sampled for sampled were Spain) (Castellón, Sal la Torre - phenoxyethanol (0.5% v/v, Sigma v/v, (0.5% phenoxyethanol g

for 30 min at 4 at min 30 for stock were given three injections of injections three given were stock - Wolfe’s trichrome (Cleveland and (Cleveland trichrome Wolfe’s - 70 o C for mass spectrometry. A A spectrometry. mass for C

not exhibit any evidence of evidence any exhibit not is characteristic of sea bass sea of characteristic is o ad lqos f the of aliquots and C - Aldrich) and Aldrich) ovarian f ®

11 6 2

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(VtgC) 1996 followed by

or encoding three forms European of sea bass Vtg (VtgAa, VtgAb and VtgC) were vga )

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Comment citer cedocument: in teleosts vtg ACCEPTED MANUSCRIPT n of and - type" Vtgs, type" (Vtg1 lo lacking also

sequencing.

[ I] or VtgA), and VtgA), or I] h 1996 the . 2 2

were preserved in RNA in preserved were induced males was used as as used wasmales induced

this classification, indicated as gene (protein), is (protein),gene as classification,indicatedthis th

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μl μl o f olive oil only oil olive f has been widelyhas and been adopted vtgAb ; Finn et al. 2009) al. et Finn ;

and RACE (VtgAb) for (VtgAb) ast E ast - - PCR andsequenced directly, or later C cnutd o produce to conducted PCR 2

injection, the

to serve as controls. Fish were not fed not were Fish controls. as serve to as described above and stored at stored and above described as

positive controlpositive primer sets targeted Vtg type vtg2[II] .  With regard to the present the to regard With they were bledtheywere c n Ct and 'c

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familymembers Vtg polypeptides deducedfrom Vtgpolypeptides cDNA were packages, as follows. as packages, available online at at online available DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT sequences obtained for basssea

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o remove any unreliable reads. bymanual alignment using compared to artial cDNA sequences

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Amino acid compos acid Amino - Comment citer cedocument: bin/protparam/protparam ACCEPTED MANUSCRIPT

(ovary samples only), and centrifuged for 10 min at ~13,000 X g and 4°C to 4°C and g X ~13,000 at min 10 for centrifuged and only), samples (ovary

those

http://www.phylogeny.fr/ in other vertebrate species and to confirm their identities as Vtg as identities their confirm to and species vertebrate other in - Results for this phylogenic analysis phylogenic this for Results e otl Atm e a. 2012; al. et (Artimo Portal ce 15% a 15% Vacic et al. was based on based was

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, 2006) with2006) Castresana xpectation (available Hercules, Dereeper

Aa,

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. the using densitometry by proteins marker prestained GeneGenius the to reference with calculated was blots to used were Laboratories)visualization for membranes (Vector Kit Substrate Peroxidase DAB and CA) Burlingame, in incubation sol PBST BSA 3% Anti Goat Purified Affinity Grade (EIA solution PBST in washed were Membranes wer Antisera respectively. dilution, 1:20,000 and the with overnight incubated were Blots anti as to referred henceforth are antisera These 2014b). al. et (Williams et Reading by described as purified in use w antisera The eggs. (OV) ovulated in and females PV of ovary and plasma cephalus,Mugil non reduce to (PBST) 20 Tween % 5 and NaCl) M 0.15 containing PBS M (0.01 saline buffered phosphate min. 30 for 15V Tris (0.025M buffer Semi SD destained in methanol:acetic acid:DW (3:1:6 v/v). Sigma BlueR, (PhastGel Co 0.1% with stained were Gels proteins. separated of mass the estimate to used SDS Prestained 1970). (Laemmli system - Fo specific reactions. specific r Western blotting, proteins in the gels were transferred to PVDF membranes using a Trans a using membranes PVDF to transferred were gels the in proteins blotting, Western r moronids - r Eetohrtc rnfr Cell Transfer Electrophoretic Dry ®

scanner and GeneTools software (Syngene Avidin

Lvs (

Blots were blocked for two h two for blocked were Blots ae uo ter pcfc eonto o te corresponding the of recognition specific their upon based - to. ebae wr wse i PS solution PBST in washed were Membranes ution. Base, 0.195M Base, Amano et 2007abal. -

itn ope (etsan B Kt abt g, etr aoaois Inc., Laboratories Vector IgG, Rabbit Kit ABC (Vectastain Complex Biotin

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Vt - Comment citer cedocument: Aldrich) in methanol:acetic acid:distilled water (DW) solution (4:1:5 v/v) and v/v) (4:1:5 solution (DW) water acid:distilled methanol:acetic in Aldrich) g ACCEPTED MANUSCRIPT - type specific antisera raised against the corresponding purified grey mullet, grey purified corresponding the against raised antisera specific type

as described by the manufacturer. The mass of protein bands on the gels or gels the on bands protein of mass The manufacturer. the by described as

glycine, 20% methanol, pH 8.3) pH methanol, 20% glycine,

al. (2011) and present in present and (2011) al.

- anti - Rabbit IgG (H+L) IgG Rabbit PAGE Standards Broad Range (BioRad) protein markers were markers protein (BioRad) Range Broad Standards PAGE ) were) employed detect to sea bass Vtgs or producttheir YPs in

three times for 30 min and incubated in secondary antibody secondary in incubated and min 30 for times three -

LvAa, anti LvAa, Boa) T (BioRad).

with 3% bovine serum albumin (BSA, Sigma (BSA, albumin serum bovine 3% with

dltd n % S i PS n 02 Ten 20. Tween 0.2% and PBS in BSA 3% in diluted e - LvAb and anti and LvAb USA, Frederick, MD). e e ad ebae ee e wt transfer with wet were membrane and gel he - HRP Conjugate, BioRad) diluted 1:5000 in 1:5000 diluted BioRad) Conjugate, HRP

plasma, and striped bass Vtgs in plasma in Vtgs bass striped and plasma, and - LvC - anti LvAa, the three times for for times three used at 1:40,000, 1:260,0001:40,000, at used previously ere t ransfer was carried out at out carried was ransfer

ht perch white o massie Brilliant Blue Brilliant massie - LvAb and anti and LvAb 5 validated for validated - min Aldrich) in Aldrich) Vtgs dev before - both , - Blot LvC. elop 10 ®

MS and MS/MS data being data MS/MS and MS dditional a dditional - PAGE were prepared for submission to to submission for prepared were PAGE anoLC very rate (FD rate very AFA26670.1 v. 2.3.01 v. liquots - of Vitellogenins and ProteinsYolk by Mass Spectrometry SM dt wr poesd y ACT Mti Sine Ic, otn MA) Boston, Inc., Science, (Matrix MASCOT by processed were data MS/MS - 1D+ system and Nanoflex cHiPLC with an autosampler coupled to a hybrid LTQ hybrid a to coupled autosampler an with cHiPLC Nanoflex and system 1D+ software (NuSep, Bogart, GA) for label free quantitation of the MS data was used was data MS the of quantitation free label for GA) Bogart, (NuSep,software DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT of ) protein database from ENSEMBL (downloaded on 03/17/2011) on (downloaded ENSEMBL from database protein ) R), with a with R), and ) Comment citer cedocument:

ACCEPTED MANUSCRIPT the the pcrl ons rm ACT o ec rpiae ad omlz cut for counts normalize and replicate, each for MASCOT from counts spectral

- l iver and ovary tissue superna tissue ovary and iver SC) for each of the three technical replicates per replicates technical three the of each for SC) tC (GenBank VtgC

ta audne atr, NSAF) factors, abundance ctral interrogated. Because the the Because interrogated. n FDR of <1% of FDR n . - SM dt ws efre uig h trepnd stickleback threespined the using performed was data MS/MS

t wee hy ee nlzd by analyzed were they where ity G. aculeatusG. Fisher

AFA26671.1 being the Mass Spectrometry Facility in the the in Facility Spectrometry Mass

Scientific used to determine to used

D. labrax D. database and thedatabase and tants and tants )

n sqecs for sequences and

- MS/MS) performed using an Eksigent an using performed MS/MS) Raig t al. et (Reading Zbio e a. 06 20) The 2007). 2006, al. et (Zybailov

plasma samplesplasma SM replicates MS/MS genome had genome al cit a ue t cet a create to used was script earl -

MS/MS data to account for the for account to data MS/MS 0.95 being reported. reliable protein identifications.protein reliable

target and reverse sequencesreverse and target biological sample (liver, sample biological

eesd phase reversed not been sequenced to sequenced been not 2013) AFA26669.1

human that were that

. ( .. fs X 3 X fish 4 i.e. combined with combined ah ise or tissue Each eatet of Department keratin

subjected ), VtgAb), HPLC s

with and 11 -

In type each For N transformed average The C Vitellogenin Polypeptide vtg

- (Reading et al. 2009), striped bass striped 2009), al. et (Reading t the analysis of analysis the Rs encoded ORFs

domains, is as typical of

NA,detailed information on - way ANOVA using a mixedmodelStatisticsusingSPSSArmonk,ANOVAa version (IBM way 19; that,NY)

of sea bass sea of - y values were returned, was followed by one by followed was returned, were values

+ 1], where clustered with its orthologues in other in orthologues its with clustered their their

l fv YP five all DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Sequences Comment citer cedocument: ACCEPTED MANUSCRIPT phylogeneticrelationships YP s vtg

. T .

domains VtgC - y , a short stretch of 5' UTR sequence (16 sequence UTR 5' of stretch short a , he S

= N

vle fr ah ilgcl elct (= pr ape ye were type) sample per (N=4 replicate biological each for values C being usually 7b), sea bass sea oan ( domains

sequences. -

SC) to accountSC) to for zero spectral count values in the data set. , and the and , nucleotide positions and length of the the nucleotideof positionsand length

vtgAa

vtgC A e.g. -

type and

3’UTR

ORF was much smaller and did not encode Pv, encode not did and smaller much was ORF NH vtgC Vtgs to other to located 2 - inl peptide Signal is provided in in provided is

(Williams et al. 2014a), and A and 2014a), al. et (Williams ee s follows: as were higher (acanthomorph) teleosts ( teleosts (acanthomorph) higher vertebrate 10 - way ANOVA and Tukey’s HSD post HSD Tukey’s and ANOVA way - 50 bp upstream of the poly the of upstream bp 50 with - Table 1 Table LvH in in Vtg polypeptide sequences, eachpolypeptidesequences,Vtg - 18 bp) was obtained along with along obtained was bp) 18 and between sample types. - Pv e bass sea 5’UTR . -

LvL The - , the ORFincludingthe , β'c sea

tA ws 94% was VtgAa - vtg

- C bass type - , w , ) rsn in present t) Fig. - tail. A vtgAa hite perch hite vtgs

1 ) or β'c or

. from -

The h and F

12 oc or

and - e P oan peet n ah om f Vtg of form each in present domains YP he terminal s terminal D (ReadingDet were identified by aligning by identified were Vtg

length (

eune fo wie ec ( perch white from sequences review: - VtgC derived YPs derived equence Fig s .

(residues)

Carnevali et al. 2006).al. Carnevaliet 1 al. 2009)al. are (Reading e (Reading . in 20a rprs cnevd E cevg st bten h βc n C and β’c the between site cleavage QEY conserved a reports (2007a) Finn ).

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT

of the mature polypeptidemature the of striped bass striped Comment citer cedocument: as follows: cleavage of LvH from Pv from LvH of cleavage follows: as ACCEPTED MANUSCRIPT

at residues 1135/1171, and 1135/1171, residues at from the the from s ed below ed striped bass striped

and , the enzyme , the lar to red seabream,red to lar t

mass al. 2009; Williams et al. 2014 al. et Williams 2009; al. other upeetl al 2 Table Supplemental

VtgC and VtgC the red seabream red , are shown in alignments of the sea bass Vtg sequences with the with sequences Vtg bass sea the of alignments in shown are ,

sea bass sea species of species VtgAb, 93% identical and 96% similar to to similar 96% and identical 93% VtgAb, provided in responsible for primary proteolysis of Vtg into YPsprimaryfollowingforinto responsibleVtg proteolysis of The locations of theselocations of The upeetl i. 1 Fig. Supplemental

iia t ta peeetal cevd y bovine by cleaved preferentially that to similar signal peptidesignal sequence and white perch white

, are highly conserved with respect to the same type of of sametype the to respect with conserved highly are , Vtg

V Pagrus Moronidae t Table 1 g

cleavage of LvL from LvL of cleavage , sequenc

Ab s el s te bohmcl etrs f the of features biochemical other as well as . T .

). and identical 84% and VtgC, major A he

is basedis on h i The

es with es given in in given

that were v were that sea bass sea at residue position 1062/1067 (numbers 1062/1067 position residue at a , conserved CAT D cleavageD conservedCAT ) . xet s tews ntd these noted, otherwise as Except ). V . frain on nformation Particular t gA

N Tab

a VtgC -

. The The . terminal sequences terminal these its erified by Edman degradationEdman by erified le 1 le

predicted cleavage site, andpredictedcleavage site, β’

sequence T attention conserved bass sea -

c at residues 1374/1409 residues at c were predicted based onpredictedbased were he length (residues) and(residues) length he aue e bs Vtg bass sea mature white perch white

VtgAb i was paid was s 95% identical95% s 91% similar to similar 91% cleavage sites

of purified of sites sites

cathepsin was

VtgAb, to to in the in

93% YP 13 - . t

n Vtgr e bass sea

Characteristics of All three All A G Among three the

T A No putative CAT D recognition site has been identified in identified been has site recognition D CAT putative No 1 moronid ) 217 lignment analyses lignment lycosylation ) - ad LLT and a , 1 idn motif binding

1053 and ) are) present in

is that deduced for striped bass by Reading et al. (2009) from consideration of consideration from (2009) al. et Reading by bass striped for deduced that is Vtgs LvH

it appears - vA ad vA a rsde 178 residues at LvHAb and LvHAa Discussion

1056 residues sea bass Q Ab

Vtgs, and the additional ( Supplemental Fig. 1 VVDV lacks the , the latter the , K of LvHAaof at KL o LvHAb for TKDL HLT , h ms o itc LvH intact of mass the Moronidae

LvHs Yolk Yolk Protein D to to be DOI :10.1016/j.cbpb.2015.11.010 ACCEPTEDboth the of other two MANUSCRIPT

LvHC, andneither for further details onthis cleavage site). revealed Comment citer cedocument: ACCEPTED MANUSCRIPT ) andpredicted) mass K

unrelated theto of which is absent from VtgC in sea bass and other teleosts ( teleosts other and bass sea in VtgC from absent is which of have TKDL, TKDL,

residues T

essential species, K conserved cysteine residues that the that 181 ) omains . ) eotd o tilapia for reported glycosylation of LvHAb None of the aforementioned residues are pre

. essential for receptor binding receptor for essential T

the LvH domains each of type of Vtg are nearly identical in lysine residue e corresponding he 794 sequence of the highly conserved, 8 conserved, highly the of sequence

of the glyco A

, Moronidae

C T - - type Vtgs, these include bonds include these Vtgs, type type LvH 819

(114.9 vdne i SDS in evidenced

and N LvH - 8 wt te mn ai sqec LLT sequence acid amino the with 185 >

90%) of sequence identity (Yilmaz 2015). al. et -

sylation sites 115.1 kDa)115.1

1055 and has sequences species. , rohoi aureus Oreochromis

is site

that are involved forming in bonds disulfide predicted for at residueN at glutamine

in in Moronidae

- Supplemental Fig. Supplemental ( for this motif AE and/or PAGE e bass sea Table 1

predicted for bass sea (shown in boldface type) boldface in (shown at 450 the the aligned residues in ; Reading; al. et 2009;

or other or this position

LvHC

at is , -

. predicted for 162 amino acid amino

VtgAb

dicted beto etr bos f yolk of blots Western C - ih h aio acid amino the with C teleost

188 1

L e a. 2003), al. et (Li and indicated in indicated and the the

and (

Supplemental Supplemental LvHC (

Vtg receptor Vtg VtgCs. VtgCs. K

homologous all Ab 204 , RL for TRDL is located is C - all A N C - 90 207 type

he - in

positions 1090 and 1092 The PvsThe of the other two 1081

A being -

type Vtgs type (N 55 ( - 1086 ; ; the 01, 1104 - - the canonical site for cleavage Pv of from Fig. 96% 10.7 kDa)10.7 ( 89 residues; PvAb range 94 moronid re serine,re with

in sea sea in bass VtgAb), which was revealed by alignment analyses bepresentto in all

VtgAb has ) 1 . Moronidae

) of of these , and The basssea Pvs begin with tetrapeptide sequence KKIL DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT - 07 Comment citer cedocument:

Vtg. ACCEPTED MANUSCRIPT

Table 1 and 1109 t

he < 0.02). ir extensive polyserine tracts plus two threonine residues

Pv domains washighly correlated with the predicted mass LvH of domains

- a longer Pv domain of 104 amino acid r sea bass KKI 30 ( -

. A higher variability length in andmass 93, and species, the Pv domains of each type of VtgA are similar in length Moronidae ; Reading; et 2009;al. Williams al. 2014a) et and have 79% PvAa 3 had L sequence in sea bass VtgAa predicted a is N . -

Sea bass

86.2%; PvAb86.2%; range 77.4 However, these

) PvAb had seven predicted another of of these plus onethreonine residue predicted beto -

104 residues) and predicted mass (PvAa range 7.4 species also consist of >50% serine residues (range 49 at s

phorylated (Reading et VtgAa has 447 additional predicted C - C Pv 453 additional

and

LvH

predicted to bephosphorylated N a Pv domain of 73 amino aci (Reading et al. 2009; Finn 2007b). predicted - linked glycosylation site 989 by CAT D (Finn 2007b). Located six - 81.7%), albeit81.7%), not high as as for the other C predictions are uncertain because all -

C prece O 1000 O - linked glycosylation sites at

- al. 2009; Williams et al. 2014a). esidues, 57 (55%) of which are linked glycosylation sites at

in sea bass LvHAbbass sea in ded by KL; this KL is is typical Pvs of - linked glycosylation

at postion d residues,

, primarily due ( a high degree Supplemental

are present in present are - KKIL

O T - 8.8 kDa; he -

linked 1115 - 38 60%), 60%),

, ( motif (  but

that includes that (Finn et al. 2007b) al. et (Finn ’cs 150 ’cs

1272

species, but not other in teleosts (data not shown). species - it is linked glycosylation sites ( ( > C CGLC

- 90%) of sequence identity sequence of 90%) not not Moronidae C -

; these are present in all in present are these ; 151 residues; Cts 123 residues 123 Cts residues; 151 1

species 1293 194 Fig.

or other teleost well , ) ) that is highly conserved amongcomplete forms of

in in and this glycosylation site is conserved in all 3 all in conserved is siteglycosylation this and Yla e a. 2015). al. et (Yilmaz

β′ 1

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT c and C and c sea bass . conserved among other

Comment citer cedocument: ( Also present in the in present Also ACCEPTED MANUSCRIPT Table 1 Table

species, t species, that and - LvLAb t form a disulfide bond in bond disulfide a form predicted s in complete forms of teleost Vtg teleost of forms complete in ; Reading et al. 2009; Williams et al. 2014a) al. et Williams 2009; al. et Reading ;

(data not shown) Williams et al. 2014a) al. et Williams he ah ye of type each .

Supplemental Fig. 1 SupplementalFig. A . 

moronid ll ’c and C and ’c

(Yilmaz et al. 2015). al. et (Yilmaz mass moronid - The  1561 in bass sea VtgAb andthe aligned residues seain bass ’c )

teleost

(LvAs 26.5 (LvAs and The C The domains of the of domains

alak etr o te C the of feature hallmark  - VtgAs and their locations their and VtgAs t domains t ’c C / t ae ery dnia in identical nearly are Vtg . LvL - predicted

vs oti a contain LvLs 1105 of PvAb1105 of are conserved among three all taxa -

t domains of domains t -

t ,

lamprey Lv ( Lv lamprey

and (data not shown) ), but but ), these arewell not conserved among -

26.6 of each type of Vtg of type each of mass they The

sea bass sea kDa; LvCs~22.kDa;

exhibit a exhibit ( species s cluster a is



sea bass sea ar of pair moronid ’c teleost Anderson et al. 1998 al. et Anderson s 16.9 s

and other and

. of moronids of - in in

emnl WD homology vWFD terminal Sea bass very

ihy conserved highly

- Vtg ( LvL 17.1 kDa; Cts ~13.6 Cts kDa; 17.1 VtgAs have one or more or one have VtgAs noted are VtgAs bass sea ,

egh (LvLA length of of and 2 are high degree high - Aa 22.3 Finn 2007b; 14 highly conserved highly 14 moronid they

nearly identical nearly LvL is predicted to be to predicted is

kDa) ( Supplemental Ab also exhibit also

) VtgAs is a is VtgAs among the 238 s and LvLC (94 these and Reading cysteine - 95%) - - 13.9 239

the 16

in a

n oa, 3 nqe rpi ppie drvd from derived peptides tryptic unique 33 total, In

Vtg polypeptides Vtg Mass Spectrometry - derived peptide was detected in detected waspeptide derived

oth sea bass sea oth VtgAa is significantly enriched in in enriched significantly is VtgAa rom PvAb, 9 from LvLAb, 8 from 8 LvLAb, from 9 PvAb, rom

l Y dom YP all omposition

methionine and phenylalanine and methionine plasma and highest in the ovary ( ovary the in highest and plasma A , with alanine with ,

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT ) -

type Vtgs, type . For . of Comment citer cedocument: ACCEPTED MANUSCRIPT ,

is rsn nec tp o sa bass sea of type each in present ains Vitellogen and

corresponded LvHC to and LvLC. e bass sea

for Supplemental Table 3 Table Supplemental sea bass sea

sea bass sea Vtgs, the total normalized spectral counts normalizedspectral total the Vtgs, being most abundant in all three sea bass Vtgs bass sea three all in abundant most being ins ins and ProteinsYolk in Liver,

plasma. For VtgAb, 31 ovarian tryptic peptides were derived fromderived were peptides tryptic ovarian 31 VtgAb,For plasma. tA, 5 nqe rpi ppie acutn fr4.% f the of 43.6% for accounting peptides tryptic unique 55 VtgAb,

( Supplemental Fig. 2 SupplementalFig. Moronidae VtgC, 21 peptides accounting for 22.1% of the polypeptid the of 22.1% for accounting peptides 21 VtgC, VtgC has a lower a has VtgC lnn, leucine, alanine,  'cAb, and 1 from C from 1 and 'cAb,

( l dance values for proteins in the SwissProt database,SwissProt the in proteins for values dance ycine and aspartic acid (data not shown).asparticacid ycine (datanot and Supplemental Fig. 2 Fig. Supplemental

species species i Fig. ). For VtgAa, 22 ovarian tryptic peptides were peptides tryptic ovarian 22 VtgAa, For ). e bass sea are

). ) the most abund most the . n amino acid composition.



proportion of alanine and leucine and alanine of proportion Unique t Unique In same the comparison, sea bass VtgAb

isoleucine 'cAa, and none from C from none and 'cAa, t, xet o PA, ih lot all almost with PvAa, for except Vtg,

tA, conig o 3% f the of 30% for accounting VtgAa, Plasma and Ovary

- tAb. For VtgC, 18 and 3 ovarian 3 and 18 VtgC, For tAb. ( upeetl i. 1 Fig. Supplemental ryptic peptide ryptic ).

n serine and For each form of Vtg, there Vtg, of form each For ant constituents of the the of constituents ant (N - SC)

( Supplemental Fig. Supplemental were lowes were

s sequences were sequences s an - tAa, although 1 although tAa,

dfcet in deficient d Relativeto se also see ; t in the in t and a and sea 17 e

etr blots Western o blots Western In total the of analysis Comparative . Immunologica

VtgAa with VtgAa 3 3 B A e bass sea - PAGE of plasma samples from PV and OV females and also in plasma from males injected with injected males from plasma in also and females OV and PV from samples plasma of PAGE ). ), although it it although ), ximate Vtg type Vtg ximate

LvHAa. ma These values are close to the predicted mass of the the of mass predicted the to close are values These - derived peptides being ~5X more abundant than VtgAa than abundant more ~5X being peptides derived ss of any distinct band distinct any of ss vA plppie ( polypeptide LvHAa the 112 the l Detectionl Vitellogenins of in f lsa rm V n O fmls efre usi performed females OV and PV from plasma of

- fall derived

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT pam fo P ad V eae promd sn anti using performed females OV and PV from plasma f - Comment citer cedocument: - s peptide specific ACCEPTED MANUSCRIPT 113 kDa band representing a major a representing band kDa 113

within the mass range estimated for the bold, diffuse band evident for plas for evident band diffuse bold, the for estimated range mass the within - 175 kDa 175 range ~146 range

. - peptides, and with VtgC with and peptides,

- Fig. 192 kDa), but not control kDa),but male192 PAGE of the same the of PAGE 2 , resulting in a large immunoreactive band on the blot whosemasscouldblotlarge immunoreactivethe , resultinga band on in

3 evident for plas for evident

A - and N ~115 122 kDa, and ~109 and kDa, 122

), Therefore, the 174 Therefore, the), - SC - 186 abundance ratio in liver of 22 of liver in ratio abundance ~112 am, h corresponding the lasma,

VtgAa for each form of sea bass sea of form each for D) respective kDa),

kDa). - 113 kDa 113

Plasma -

samples The ~174 The ma from PV or OV females on the the on females OV or PV from ma n Vt and - derived peptide abundance being abundance peptide derived

that were absent for plasma from control males control from plasma for absent were that

kDa ( kDa - gC and also of also and 175 kDa band is ta is kDaband 175 y ( ly - s.

sea bass sea 175 kDa band kDa 175 - - proteolytic fragment of VtgAa consisting VtgAa of fragment proteolytic eie ppie big lgty higher. slightly being peptides derived eie ppie big ny 2 more ~2X only being peptides derived A bold112Aatband al 1 Table Fig. -

ai ( ratio derived peptides and VtgC and peptides derived 3 Vtg by tissue type ( type tissue by Vtg C VtgAa monomer ( monomer VtgAa

: plasma from males injected with injected males from plasma 113 ng ). ). ) that were that ) ielgnss a massively was Vitellogenesis 7.6:22.6:1.0 anti :1.0 for VtgAa:VtgAb:VtgC, for :1.0 does not exactly match the match exactly not does ken to represent intact searepresent ken intact to - LvAab - - LvAa, 113 kDa was evidentkDawas 113 similar in estimated in similar ws somewhat was ) SDS revealed ~180 Fig.

uh greater, much major - PAGE gel PAGE

kDa) and kDa) ) yielded ) - derived protein protein 18 ma s

gAb monomer gAb (~188 refore, the band the refore, Six The Fig.

protein . anti ( - ing Fig. 189

, which is which , vA, and LvHAb,

(~115 kDa,(~115 - mainly 3 additional degradation additional LvC failed to detect any protein bands in in bands protein any detect to failed LvC

A kDa, ~138 kDa, ~123 kDa, and ~112 and kDa, ~123 kDa, ~138 kDa, ) of the same samples same the of ) 3 bands dete bands . h bn a ~8 ka s ey ls t te rdce ms o the of mass predicted the to close very is kDa ~184 at band The ). D ation, and the remaining faint bands bands faint remaining the and ation,

~143 detect did it but ), with with Proteolysis of Lipovitellins correspond closely or exactly in mass to bands detected in SDS in detected bands to mass in exactly or closely correspond LvHC at ~140 kDa ~140 at interpreted as interpreted

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT~118 at band the D) and kDa) Table 1 Table

Comment citer cedocument: h bo the ACCEPTED MANUSCRIPT . cted in extracts of PV ovary or ovary PV of extracts in cted

ected in the same samples by SDS by samples same the in ected h bn at band the ) is somewhat highersomewhat is ) d ad vdn a ~0 ka ln with along kDa ~109 at evident band ld Table 1 Table is taken to represent to taken is the a products

( major breakdown product of LvHAa. of product breakdown major ad a ~4 ka n ~2 kDa ~123 and kDa ~140 at bands Fig.

e bass sea - ~118 122 ). Therefore, the ~184 kDa band is ta is band kDa ~184 the Therefore, ).

3 A .

The band evident at ~201 kDa in Western blots of plasma of blots Western in kDa ~201 at evident band The kDa ) - 122 vC polypepti LvHC that are very close to the predicted mass of the the of mass predicted the to close very are that

Fig. being - D i coe o h peitd as f the of mass predicted the to close is kDa 113 kDa, respectively), but not not but respectively), kDa, 113

than that of the major immunoreactivemajorba theof that than at at are are

Western blots of of blots Western the intact VtgC monomer wit monomer VtgC intact the 4 ~136 kDa in samples from PV an PV from samples in kDa ~136

its major proteolytic product, proteolytic major its considered to be to considered OV eggs by Western blotting using anti using blotting Western by eggs OV . The ). - PAGE (at ~90 kDa, ~77 kDa, ~48 kDa, ~48 kDa, ~77 kDa, ~90 (at PAGE e ( de predicted ~119 Fig. plasma samples from PV or OV or PV from samples plasma

n lt o etoeie male estrogenized of blots in D) respectively kDa), B

a various VtgAb various a O ) were similar or identical in identical or similar were ) mass of of mass h fit ad t 8 kDa ~88 at band faint the nly this ~93 kDa band was band kDa ~93 this nly ken to represent the intact the represent to ken - PAGE ( PAGE plasma from control from plasma h the band at ~123 at band the h the likely consisting likely e bass sea e bass sea d OV females OV d at at

degradation ( nd presentnd ~13 al 1 Table e bass sea se VtgAb

- a bass a kDa 8 LvAa LvHa 19 ) .

, In Western blots of ovary extracts ovary of blots Western In

I Table 1 Table

al 1 Table 97 kDa 97 4 4 n Western b Western n its its A A ) was detected) in these blots ) and possibly representspossibly and ) ). The ~114 kDa band is close to the predicted mass of the of mass predicted the to close is band kDa ~114 The ). ) degradation product. degradation . Based on its similarity in ma in similarity its on Based .

( Fig. , ), the ~113 kDa band is is band kDa ~113 the ),

) and is taken to represent to taken is and ) in ovary in exceeding that predicted for predicted that exceeding

4C lots performed using anti using performed lots SDS ). ). N -

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT PAGE of the same samples at samples same the of PAGE extracts from PV females and these and females PV from extracts o band corresponding i corresponding band o Comment citer cedocument: ACCEPTED MANUSCRIPT

possibly The major pair of bands detected in egg extracts from OV females stained females OV from extracts egg in detected bands of pair major The

and - vA adis eaie nest fimnsann,te 2 D ba kDa ~27 the immunostaining, of intensity relative its and LvLAa 6ka n ~4 D ( kDa ~24 and kDa 76 LvLC. In Western blots of eggof blots Western InLvLC.

the kDa ~24 band persisted unchanged. . may

- being its proteolytic product proteolytic its being interpreted as representing as interpreted PAGE in the same samples (~106 kDa and ~90 kDa, respectively) kDa, ~90 and kDa (~106 samples same the in PAGE

from PV females PV from ss to that predicted for the sea bass sea the for predicted that to ss simply LvHC, with the ~74 the with LvHC, - LvAb the could but Fig. n mass to that predicted for sea bass sea for predicted that to mass n repre sea bass sea several additional bands of lower mass (~77 kDa, ~48 kDa, (~77 mass lower of bands additional several

( 4 Fig. represent intact represent B sent add ). ). ~106 kDa, ~106

Based upon its estimated mass being identi being mass estimated its upon Based 4 Fig. C

LvLAa performed using anti using performed ), ),

corresponded approximately corresponded only two bands were detected were bands two only 4 itional proteolytic products of - D 76 kDa band being its major degradation major its being band kDa 76 extracts from OV females, the ~114 females, the fromOV extracts ) LvHAb with the band the with LvHAb ~77 kDa, and ~22 and kDa, ~77

. However, . that sea bass sea polypeptide LvLAa persisting through OM, through persisting LvLAa sea bass sea orsodfil eli as to mass in well fairly correspond

LvLC polypeptide ( polypeptide LvLC

these bands were bands these LvHAb polypeptide (~116 polypeptide LvHAb

(~27 kDa, (~27 LvHC - LvC - 23 kDa, respectively kDa, 23

LvLAb (~27 kDa, (~27 LvLAb polypeptide polypeptide three major bands major three below at ~97 kDa ~97 at below Da and ~93 kDa,~93 and Da

Table 1 Table in mass to the to mass in

, LvHAa.

at ~113 kDa ~113 at ~ absent in absent 2 2 ) were ) (

cal to cal

~119

kDa with kDa

20 nd ,

(Finn and Kristoffersen 2007)Kristoffersenand (Finn

ah ye f e bass sea of type Each

confirmed the identities of sea bass VtgAa and VtgAb as paralogous ―complete‖ forms of A of forms ―complete‖ paralogous as VtgAb and VtgAa bass sea of identities the confirmed ;

- terminus,

vtgC bass and white perch white and bass

sebe fo oelpig partial overlapping from assembled moronid very , JQ341410.1

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT high, with each form of sea bass Vtg being Vtg bass sea of form each with high, ) and) lacking ologies of the deduced sea bass Vtg polypeptides with those of those with polypeptides Vtgbass sea deduced the of ologies Comment citer cedocument: omplete cDNA sequences encoding three forms of sea bass Vtg (VtgAa,ompletebasssea Vtg cDNAforms VtgAbsequences encodingthreeof ACCEPTED MANUSCRIPT were commonly found in in found commonly Vtg signal peptides (15 residues) are identical among Vtgs of of Vtgs among identical are residues) (15 peptides signal Vtg

all all the identity of each nucleotide being indepe beingnucleotide each of identity the

β'c and Ct domains, Ct and β'c are eoie i Gnak t h NB ( NCBI the at GenBank in deposited ). 2007a; Reading et al. 2009). al. et Reading 2007a; in adiversein array of

Vtg h Vtg The cleaved from mature the polypeptide atsame the position

( Fig.

, and of sea bass VtgC as a phosvit a as bassVtgC sea of and , itnty lsee wt is rhlge i ohr acanthomorph other in orthologues its with clustered distinctly sea bass sea

1 Moronidae ). Homology among the amongHomology ). the polypeptides "complete" corresponding forms of Vtg in the other two other the in Vtg of formscorresponding VtgAa and VtgAb and VtgAa 2007) as is characteristic of of characteristic is as

species other acanthomorphsother Comparison t vtg . T . A

eue fo tee eune exhibit sequences these from deduced he - type teleost teleost type from amplified cDNAs . The simi The sea bass VtgC lacks a Pv domain Pv a lacks VtgCbass sea The

> possessing all five YP domains (e.g. domains YP five all possessing moronid ass fte aue e bs Vtg bass sea mature the of masses o Vtgs in Other Moronid 93% identical and identical 93% larities inless, ―incomplete‖ form of C ―incomplete‖ formof inless, Vtgs ndently verified 2 ndentlyverified VtgC in other teleosts other in VtgC vtgAa by N

Vtg

(reviews: Matsubara et al. et Matsubara(reviews: extend to numerous othernumerous to extend , -

terminal sequencingterminal of polypeptide sequences polypeptide JQ283441.1 N etat of extracts RNA a diverse array of arraydiverse a > 95% similar to similar 95% the same type type same the - 6 times. The times. 6 as Moronidae s

;

has beenhas (reviws: and has and vtgAb YP 21 - - ,

h Y dmis present domains YP The

( completely (100%) con (100%) completely . T . ~180 kDa, ~180 kDa and ~148 kDa, respectively; kDa, ~148 and kDa ~180 kDa, ~180 - 2 residues derived YPs fro YPs derived

hus, the various the hus, (Finn 2007a) (Finn denatured and reduced and denatured Moronidae peptides present in oocyte and egg extracts that were N were that eggandoocyte extracts present peptides in among the (Folmar - ye ts (~181 Vtgs type predicted apparent DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT , the , m the other two other the m Comment citer cedocument: ACCEPTED MANUSCRIPT Lv and Pv and Lv

species et al.et 1995; Heppel Moronidae

YP cleavage sites sites cleavage YP the the

served among the three the among served ase of VtgAb, disagree with with disagree VtgAb, of ase n h sa as tA polypeptides VtgA bass sea the in cleavage of of cleavage Vtgs perch white purified the of masses striped ba striped

(Reading et al. 2009) al. et (Reading xii a eune atr smlr o ht rfrnily lae by cleaved preferentially that to similar pattern sequence a exhibit - LvLAb cleavage site in striped bass (data not shown; see Reading et Reading see shown; not (data bass striped in site cleavage LvLAb - e f e bs Vga n Vgb ( VtgAb and VtgAa bass sea of re 8 ka ae lo iia t te siae ms o a isolated an of mass estimated the to similar also are kDa) 186 products of each form of VtgA of form each of products sea bass Vtg ( Vtg bass sea

species Moronidae ss VtgAa and VtgAbVtgAa ssand  'c from C from 'c l , as , as has verifiedbeen for

in the A the in et al. 1995). Table 1 Table

as locations for primary YP cleavage (Williams et al. et (Williamscleavage YP primary for locations as

~ species (Reading et al. et (Readingspecies 180 kDa) that, due to the chromatography methods chromatography the to due that, kDa) 180 - Moronidae terminal sequencesterminal of - -

t at a highly conserved QEY motif present in A in present motif QEY conserved highly a at t type Vtgs of sea of Vtgs type and striped bass striped and ) are ) Hiramatsu et al. 2002 al. et Hiramatsu

results of N of results - emns eoe adm as spectrometry mass tandem before terminus

very similar or identical to those predictedthose to identical or similar very signal peptidessignal

species, with the single exception of a of exception single the with species,

( upeetl Fig. Supplemental - are terminallylabeled Morone - terminal sequencing terminal

bass ( bass in their denatured and reduced and denatured their in (Williams aaó e a. 1994a al. et Mañanós expected to be to expected 2009 corresponding Vtgs). Results of Results Vtgs).

species in comparisons of Supplemental Figure 1 Figure Supplemental have been re have been c ; Williams et al. 2014 al. et Williams ; ). The predicted masses predicted The ).

et al. 2014a), al. et ach YP cleavage YP ach

1 prior to tandem priorto very similar in similar very wr mainly were ) purified Vtgs portedbased by the omitting ).

recent which Some 22 a ) ) - .

lapping peptides mapping within the C the within mapping peptides lapping support but no other C other no but Morone chrysops Morone -

derived YP bands commonly detected by SDS by detected commonly bands YP derived those of Kristoffersen et al. (2009) who (2009) al. et Kristoffersen of those s remains be to confirmed experimentally. ge at this site in Western blots of of blots Western in site this at ge ition by CAT D, indicating possible involvement of other proteases in YP production. YP in proteases other of involvement possible indicating D, CAT by ition . However multiple alternative cleavage sites were also discovered also were sites cleavage alternative multiple However .

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT - Morone t - Comment citer cedocument: Verasper moseri Verasper derived peptides were detected in extracts of oocytes or eggs. Likewise, in the in Likewise, eggs. or oocytes of extracts in detected were peptides derived ACCEPTED MANUSCRIPT

, white perch and striped bass (Hiramatsu et al. et (Hiramatsu bass striped and perch white , - Supplemental Figure 1 Figure Supplemental tAb. However, no C no However, tAb.

s did not exhibit a pattern of amino acids with chemical characteristics chemical with acids amino of pattern a exhibit not did s ts ae eetd ad a o na te oiin rdce fr C a for predicted position the near or at bands detected have Vtgs , -

t domain of stri of domain t thus thus . Such alternative cleavage sites are also likely to exist in the sea the in exist to likely also are sites cleavage alternative Such . - t YP was detected during purification of the other YPs in white in YPs other the of purification during detected was YP t , oocyte extracts performed using an antiserum raised against raised antiserum an using performed extracts oocyte , - demonstrating conclusively that the C the that conclusively demonstrating t domain of Atlantic herring, Atlantic of domain t - terminal peptides lab peptides terminal  Morone - t YP was detected in studies of Vtg of studies in detected was YP t 'c and C and 'c employed q employed ped bass VtgAb was found in found was VtgAb bass ped eported detection of a C a of detection eported ; se ; e also

PAGE and/or Western blotting Western and/or PAGE oocyte or egg extracts (Reading et al. 2009; al. et (Reading extracts egg or oocyte - tAb domain was detected in oocyte extractsoocyte in detected was domain tAb - t located at QEY in A in QEY at located t Supplemental Table 3 SupplementalTable - eled at or near QEY were found in the in found were QEY near or at eled TOF mass spectrometry to detect to spectrometry mass TOF se

laining some of the diversity in diversity the of some laining findings in striped bass and sea and bass striped in findings Clupea harengus Clupea 2002 - t protein band in Western in band protein t - d t domain gives rise to rise gives domain t ; Reading et al. 2009; al. et Reading ; - striped bass oocyte bass striped type Vtgs, various Vtgs, type ), but), noother C

- for striped bass striped for derived YPs in YPs derived , Ch , , as also seen also as , vtgAc1 in vtgAc1

two 23 - - t

after endocytosis has been obtained for white perch and striped bass (Reading et al. 2009; Williams2009; al. (Reading basset striped and perchwhite obtainedfor been has endocytosis after tandem mass spectrometry of the protein bands, bands, protein the of spectrometry mass tandem ) vage site has been identified in identified been has site vage

indicate but

the that was N was that ,

Hippoglossus based on apparent mass in SDS in mass apparent on based mass of LvHC evident in SDS in evident LvHC of mass Supplemental Table 3 Table Supplemental

that in .

also (Sawaguchial.,et 2005ab)seabream red and 2006 (Sawaguchi al. et The amino terminus of a VtgC a of terminus amino The DOI :10.1016/j.cbpb.2015.11.010 ACCEPTEDFig. Supplemental MANUSCRIPT Comment citer cedocument: - ACCEPTED MANUSCRIPT terminally labeled before detection by tandem mass spectrometry (Williams et (Williams spectrometry mass tandem by detection before labeled terminally ee eetd n the in detected were Moronidae

hippoglossus es of es - LvC ( LvC ng to LvLC were detected in detected wereLvLC to ng

gener , this position in all three moronid three all inposition this

and perha Fig. 4 Fig.

). It is tentatively concluded that LvHC is cleaved from LvLC fromcleaved LvHCis that concludedtentatively is It ). Moronid 1) ly cu a o na ti site this near or at occur ally

(Finn 2007a), grey mullet (Amano et al. 2007b), mosquito 2007b), al. et (Amano mullet grey 2007a), (Finn was deduced for striped bass from the VtgC polypeptide VtgC the from bass striped for deduced was )

; a ; ovary - same - PAGE ps many teleosts, C other the PAGE and Western blotting of ovary extracts ovary of blotting Western and PAGE

pair of ~24 kDa YPs similar in mass to that predictedthat to mass in similarYPs kDa ~24 ofpair ae t al. 2005a, 2006).2005a, al. t -

derived polypeptide derived or other or or egg extracts. In the present study, a ~114 kDa ~114 a study, present the In extracts. egg or blot

and Western blotting of ovary and egg and ovary of blotting Western and , u these but s, the the VtgCs. The approximate site for cleavage for approximatesite The VtgCs. es partial proteolysis or is or proteolysis partial es in ovary or eggs or ovary in P gnrtd are generated YPs PV PV Evidence for partialEvidence for sea bass bass sea

could VtgC sequences, suggesting thatsuggestingsequences, VtgC

present in gravid striped bass striped gravid in present in in

- ersn LH products. LvHC represent t Moronidae ovary extracts by tandemby ovary extracts may be of either species either of

all

cleaved from vC r LvHC or LvHC

) Hwvr in However, . proteolytically . Collectively,. proteolysis

, extracts

a and nd

24 

no on of 'c

'c domain 'c several 002) and thought to be nearly universally present in chordate Vtgs (Finn et al. 2007b); one of one 2007b); al. et (Finn Vtgs chordate in present universally nearly be to thought and 002) VtgA sequences are otherwise similar to tilapia VtgAb and include the lysine residue ( residue lysine the include and VtgAb tilapia to similar otherwise are sequences VtgA pairs to to

'  signature motif, as typicalis of s be involved in formation of intrachain disulfide bridges responsible for proper fo proper for responsiblebridges disulfide intrachain of formation in involvedbe c Fn 2007a) (Finn 'c

that species, with the ex thespecies, with that is thought to be involved in forming interchain disulfide bonds in the Vtg dimer Vtg the in bonds disulfide interchain forming in involved be to thought is that n i ti psto, and position, this in ine

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT forms a bond thought to stabilize LvH in the vicinity of its Vtgr its of vicinity the in LvH stabilize to thought bond a forms Comment citer cedocument:

ACCEPTED MANUSCRIPT the highly conserved QEY motif at the N the at motif QEY conservedhighly the moronid ' Moronidae

- o

conservative substitution of leucine for histidine in histidine for leucine of substitution conservative s

e ther quences including quences - binding motif reported for tilapia VtgAb (Li et al. 2003) is identical in identical is 2003) al. et (Li VtgAb tilapia for reported motif binding similarities

VtgAs all contain all VtgAs ( ), the ), upeetl i. 1 Fig. Supplemental

, but th but , ception of a conservativea ceptionof teleost typify moronid e sequence CGLCsequence at Supple Vtg

they ewe te three the between LvLC may LvLC

the conserved KL conserved the that type of of type that C s pert b ohrie neae t te A the to unrelated otherwise be to appear

mental Fig. 1 Fig. mental ( tC lc ti rsde wt a non a with residue, this lack VtgCs Reading a series of 14 highly conserved cystein conserved highly 14 of series

for possibly possibly ). ).

diinly fr ah ye f t, the Vtg, of type each for Additionally, t i ohr eess Tee nld the include These teleosts. other in Vtg et et al. the highly conserved CGxC motif presentmotifconserved CGxChighly the

substitution of leucine for met leucine for of substitution ). ). - - KKIL terminus of their Ct dom Ct their of terminus 2009) undergo further degradation shortly degradation further undergo As is typical of A Moronidae

All of All . canonical site for site canonical

the the first residue position, residue first the

pce i structural in species moronid - type teleost Vtgs, - binding domain binding Supplemental - conservative A ain where itwhere ain cleavage of of cleavage - e hionineat type Vtgs type

- residues typeVtg K lding 181 25 )

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. species. related closely in proteins paralogous examining of artifact an is scaling asso is and LvHAa of that than larger always is (Eldridge al. 1983; et Devauchelle and Coves et1997 Bell al. 1988; which Moronidae Vtg Vtg for need the reflect may LvH and correl strong the that and species these in load lipidneutral higher a have may species that have large oil globuleswith ahigh content neutral of lipids their in is eggs, thought it that Vtg ran broad a across Vtg of types complete mass the with correlated shor res serine phophorylated of tracts long of consisting structures amphipathic highly three species similar a and polypeptides (O'Conner and Imperiali 1996; Sola and Griebenow 2009). sorti folding,correct the ensuressite this glycosylationat Presumably, KL the of downsream site unknown. is position this at LvHAs of glycosylation VtgAs bass in given is information ( site glycosylation pred The sea bass Pv polypeptide sequences are sequences polypeptide Pv bass sea The th of All tat o hdpoi residues hydrphobic of tracts t icted yial fud n ees A teleost in found typically solubility in the plasma (Finn 2007b). (Finn plasma the in solubility all

N produce large oil globules containing primarily containing globules oil large produce

- species, is is linked glycosylation site glycosylation linked ,

percentage of serine residues serine percentageof moronid e most highly conserved among other fish taxa fish other among conserved highly

of of these serines upeetl Fig. Supplemental in which up to 80% of VtgA lipids are phosphatidylcholine (Lund et al. 2000) and 2000) al. et (Lund phosphatidylcholine are lipids VtgA of 80% to up which in

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Fig. 1 Fig. - KL sequence KKIL Comment citer cedocument: vA sae h sm two same the share LvHAs es ACCEPTED MANUSCRIPT

f v dmis n h sm Vtg same the in domains LvH of

revealed that revealed - ye Vtg type are are Interestingly, . n all and , s

predicted be to pho

in such species to have larger amp larger have to species such in

ge of teleost taxa teleost of ge 1

(in 07) s rsn i al moronid all in present is 2007a) (Finn s other the in counterparts their to . H ). hr P i cevd rm LvH from cleaved is Pv where only the predicted the only T his does not seem to app to seem not does his

moronid

ciated with a larger Pv domain, but domain, Pv larger a with ciated owever similar in length with length in similar

h mass the Another

(data not shown) not (data predicted vAs hr a additional an share LvHAbs lgmn o te tA eune fr which for sequences VtgA the of alignment sphorylated ( . Because large Pv domains tend domains Pv large Because . of of es wax esters as the neutral lipid component lipid neutral the as esters wax glycosylation site at residue T residue at site glycosylation

highly of of , as as ,

O ). the -

linked In was

ng, stability and cleavage of the Pv the of cleavage and stabilityng, a high degree of degree high a ly to size scaling of Pv of scaling size to ly

conserved Supplemental Fig. 1 Supplemental Fig.

these species, LvHAbthese the domain moronid

eotd y in 20b for (2007b) Finn by reported The functional significance of significance functional The hipathic structures to maintain to structures hipathic Moronidae glycosylation sites sites glycosylation ation betwen the size of Pv of size the betwen ation

( upeetl i. 1 Fig. Supplemental v domains Pv predicted glycosylation predicted

it is possible that this that possible is it de itrutd by interrupted idues

VtgAs predicted

species sequence ie te huge the Given )

794 . to belong to belong to i residues six

All All

are

to LvH in LvH to and in the sea the in N n one and identity

Pvs are - , highly linked

in in 26 all ).

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. Vtg of types different their and 2014a) al. et Williams 2009; al. et (Reading Moronidae Fin by (reviewed development embryonic early for proteolysis limited ( OM during similar, a undergo to appear Lv bass sea of forms three all as Thus, acids. amino only serine and alanine in enriched is VtgAb the whereas the deliver to tailored more average the to relative acids amino noted the with enrichment their to respect with VtgAb and VtgAa bass sea between differences some were There gluconeogenesis. embryonic for substrate major a as role its reflects which polypeptide, entire the of constituting alanine, was Vtgs bass sea three all in acid amino abundant most single the species, 20 Fyhn and Finn (review: generation energy aerobic for substrate between development embryonic early during teleosts several of eggs in sharplydecrease FAA these of Levels OM. duringproteolysis undergoing YPs Vtg from liberated be may they that suggests which 1998), al. et. of(Ronnestad content acid amino 60% for accounting eggs, bass sea spawned newly of pool FAA the dominate quantitatively bass sea the of constituents Alanine, 2010). al. et Lubzens 2007; al. et Cerdà 2003; al. et Matsubara (reviews: influx water driving gradient osmotic the steepens Vtg s a by assisted being maturation during oocytes the of hydration with buoyancy, thisof phenomenon patterns disparate a amon variability dditional - h hg wtr otn (p o 83 to (up content water high The derived YPs, giving rise to an increased content of free amino acids (FAA) in the ooplasm that that ooplasm the in (FAA) acids amino free of content increased an to rise giving YPs, derived

information a te tie bs ad ht prh ae smlr atr o mtrtoa proteolysis maturational of pattern similar a have perch white and bass striped the as , Fig. 4 Fig. g and types and species ), the VtgAa may make a more significant contribution by weight of energy substrates energy of weight by contributionsignificant more a make may VtgAa the ), in teleost Vtgs. onoccurence theof

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Comment citer cedocument:

ACCEPTED MANUSCRIPT in of the the full full t plppie ( polypeptides Vtg yolk lipid lipid yolk

lipid content lipid ecn, eie ioecn, aie n lsn ae h ms abund most the are lysine and valine isoleucine, serine, leucine, pcrm f mn ais oiaig h FA ol f vltd eggs ovulated of pool FAA the dominating acids amino of spectrum

- 2) f h ouae e ovulated the of 92%) deposition is needed is deposition Pv - protein in the Swiss Prot database; the VtgAa is seemingly is VtgAa the database; Prot Swiss the in protein and profiles and LvH sizewithinscalingLvHamongand families fish etlzto ad acig we te sre s major a as serve they when hatching, and fertilization upeetl i. 2 Fig. Supplemental n and Fyhn and n of fish eggs (Mommsen eggs fish of . The sea bass VtgC is not enriched in th in enriched not is VtgC bass sea The . to g o mrn pelagophil marine of ggs 20 shed light on the on light shed 10 10 ). This may be generally the case in case the generally be may This ). ). As typically found in other fish other in found typically As ). ad hs sm aio acids amino same these and ) do

not differ in amino acid amino in differ not econdary proteolysis of proteolysis econdary

and Korsgaard 20 Korsgaard and funtional icess egg increases s significance exhibiting - ~9 derived - 12% 12% total 08 ese 27 ant ) , ,

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. in corresponding bands and VtgAb and VtgAa bass sea intact to mass in corresponding bands protein estroge and females OV and PV from vali rigorously were that 2008a) al. et (Amano Lvs mullet grey corresponding the against raised antisera using blotting orthog Western an by verified was eggs ovulated and ovary PV in and males estrogenized noted, C was ovary in detected not YP only peptides. returned of number and size YP between relationship positive 1 Table s the to due was probably PvAa to mapping peptide tryptic any of absence the trypsin, by recognized not tracts polyserine in detected being all peptides almost with PvAa, for except Vtg, bass sea of type each in present domains YP all from obtained the in YPs as stored and accumulated ovary, for highest 3 ( polypeptide Vtg mature each of portions large for accountingpeptides tryptic detected the with spectrometry, mass tandem by bass sea female PV of ovary 4.2. Detection and Rel Finn2003; al.et 2002b; Finn 2007ab; Kolarevic et al. 2008). nearly the from arises eggs prote maturational ovulated complete in pool FAA the to VtgAa of contribution disproportionate a seawater, in eggsspawningteleostpelagic other manyIn bass.sea orthologues in their composition from . o ec tp o Vg te en N mean the Vtg, of type each For ). h peec o ec om f e bs t o p or Vtg bass sea of form each of presence The three all of presence The

C - with the number of peptides mapping to e mappingtopeptides numberof thewith t is is t likely to be degraded shortly being after cleaved from th s ol b epce i te ts r scee rpdy fe snhss n are and synthesis after rapidly secreted are Vtgs the if expected be would as mall size of this of size mall ative Abundance of Sea Bass Vitellogenins

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT ae a big pcfc o ec mrndVgtre.Wsen lt of blots Western target. Vtg moronid each for specific being as dated Comment citer cedocument: ACCEPTED MANUSCRIPT the the olysis of LvHAa but not of the other forms of Lv of forms other the of not but LvHAa of olysis

types of Vtg, or of their product YPs, was confirmed for liver, plasma and plasma liver, for confirmed was YPs, product their of or Vtg, of types ovary ( ovary nized males performed using performed males nized

Supplemental Table 3 Table Supplemental

- growing oocytes growing YP (~7 kDa). Comparison of the size of YP domains shown in shown domains YP of size the of Comparison kDa). (~7 YP A, n ol oe etd mpig o C to mapping peptide one only and tAa, - C aus ee oet f lowest were values SC ach YP shown in in shown YPach Supplemental Fig. 1 Fig. Supplemental roduct Lv in the in Lv roduct

( Fig. 2 Fig.

). )

. In addition to the presence of presence the to addition In in Liver,in

Unique tryptic peptide sequences were sequences peptide tryptic Unique r ie, nemdae for intermediate liver, or the Supplemental Table 3 TableSupplemental e moronid

Lv lsa f rvd eae and/or females gravid of plasma

; see a see ; As noted, aside from PvAa the PvAa from aside noted, As Plasma andOvary

type H VtgAs. - lso specific antisera detectedantisera specific

(Matsubara et al. 1999, al. et (Matsubara - A ws eetd As detected. was tAb Supplemental Table Supplemental nl methodology, onal

illustratesthe

lsa and plasma extensive

plasma 28

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. stores,YP oocyte contributormajorto the bloodstream and/or the in Vtg form of major the also is VtgAb 2006), al. et (Sawaguchi (Fin seabream red and 2008ab), al. halibut et (Amano mullet grey 2008), Atlantic al. et (Sawaguchi examples, as including, date to examined teleosts below).discussed 2014a; al et (Williamsspectrometrymethodsmass similar total of 18.2% Vtg contributor YP to stores present in fully being abundance peptide VtgAb yielded domains each of form of Vtg the with possible exceptions PvAa of CtAa. and t by up taken are Vtg Sbs 4 LvHs intact be to judged eggs ovulated and ovary 3 Table ( liver the in detected being peptides these of one only and plasma in detected being ovary for extremelylow were N where females same the for results spectrometry mass the by supported strongly is conclusion This males. in estrogen by induced be can but bass sea female males,antiserumusing control this ( not but estrogenized, from samples plasma for detected were proteins, smallerrepresenting bands several females OV or PV anti using fromperformed samples plasma of blots in detected were bands protein No males. control anti using performed th in proteins smaller several representing bands with along LvHs, respective their to mass ; discussed below). discussed ; - h cmaaie nlss fte oa N total the of analysis comparative The derived YP, but that VtgAa and VtgC also make significant contributions, accounting for 27.3% and 27.3% for accountingcontributions, significant make also VtgC and VtgAa that but YP, derived -

derived peptides being ~2X more abundant than VtgAa than abundant more ~2X being peptides derived ). Additionally,unique these). antiseraspectrum specifically a of detectedeach n prxmt Vg type Vtg approximate an Vtg - eie Y, respectively. YP, derived - From these collective results it can be definitively concluded that all three forms of forms three all that concluded definitively be can it results collective these From - VtgC, although bands cor bands although VtgC, VtgAb ( VtgAb

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT plasma and liver liver plasmaand Comment citer cedocument:

ACCEPTED MANUSCRIPT he growing oocytes to form YPs, which include products all of the major YP major the of all products include which YPs, form to oocytes growing he tid f ht o VtgAb. for that of third a i. 3 Fig. - pcfc etd audne ai i P oay f .:.:., with 1.5:3.0:1.0, of ovary PV in ratio abundance peptide specific Fig. 3D Fig. ). All of these bands were absent in blots of plasma samples from samples plasma of blots in absent were bands these of All ).

- grown oocytes, accounting bass sea much as for of 54.5% as total as compared to ovary, with only 3 of the 21 peptides detected indetected peptides 21 the of 3 only with ovary, to compared as ). It is concluded It in thatveryis VtgC levelsarelow ). - C o ah om f e bs t b ise ye ( type tissue by Vtg bass sea of form each for SC responding in mass to intact VtgC and LvHC, along with along LvHC, and VtgC intact to mass in responding oprbe idns ee ae o srpd as using bass striped for made were findings Comparable ( or or possibly hs fnig i findings These - SC attributable to VtgC to attributable SC - LvLs derived peptides, and with VtgC with and peptides, derived )

or their degradation products ( products degradation their or dct ta Vgb s h major the is VtgAb that ndicate In most other acanthomorph other most In 20a, afn flounder barfin 2007a), n

- derived tryptic derived of major YPs in PV in YPsmajor of Supplemental e case of blots of case e PV and OVandPV peptides - derived i. 2 Fig. Fig. 29 )

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. FAA preferentialy utilized by early embryos. aci amino the noted, as because, embr early and/orhydrationoocyte significantly to contribute could VtgCwhereby process a OM, duringproteolysis Moronidae in YP to VtgC of contribution higher comparatively the However, 2011). Sullivan and Reading 2009; FA of pool the to little contribute i der are oocytes vitellogenic in However, they are unusual afor species, such as sea bass, that spawns pelagic eggs is seawater. present YPs the of ~25% which in species, water fresh a 2005ab), al. et (Sawaguchi mosquitofish ovoviviparous the for reported been also having unique, (Williams 26% over of stores YPtotal to VtgC contributionestimatedof an 1.4:1.4:1.0, with be to ovary PV in ratioabundance peptide type Vtg corresponding the revealed bass striped of study spectrometry mass recent typical be may pattern This (~18%). higher much was ovary PV in stores YP the study, present the in However, 2006). al. 9:15: barfin was ratio in this flounder 2008a); al. et (Amano oocytes the by accumulated Lv total of 5.5% only representing LvC with 4:13:1, was ovary vitellogenic in ratio LvAa:LvAb:LvC the mullet, grey In available. were Lv of immunoass which for respectively, waters, marine or brackish in eggs pelagic spawningspecies two for confirmedwas this and 2006) al. et Hiramatsu 2003; al. et Matsubara (reviews: and VtgC being present in only traceamounts. VtgAb with 2008), al. et (Kolaravic YPs of source major the far by is VtgAa which in exception notable mak also VtgAa although ms sc seis Vg i togt o neg ltl dgaain uig M n, hrfr, to therefore, and, OM during degradation little undergo to thought is VtgC species, such most n Inmost teleosts examine oi nutrition. yonic may relate to the fact that, in these species, all three forms of L of forms three all species, these in that, fact the to relate may

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Comment citer cedocument: es a significant contribution. The goldsinny wrasse, an extreme pelagophil, is a is pelagophil, extreme an wrasse, goldsinny The contribution. significant a es ACCEPTED MANUSCRIPT h cnrbto t oct hdain a b mr pyilgcly relevant physiologically more be may hydration oocyte to contribution The d to date, VtgC been has considered to comparatively bea minor form Vtg of 1, with LvC representing only 4.0% of total accumulated Lv (Sawaguchi et (Sawaguchi Lv accumulated total of 4.0% only representing LvC with 1,

et al. 2014a). Such high proportions of YPs emanating from VtgC are not are VtgC from emanating YPs of proportions high Such 2014a). al. et d composition of VtgC is not especially tailored to deliver the types of types the deliver to tailored especially not is VtgC of composition d A present in ovulated eggs or to egg buoyancy (reviews: Reading et al. et Reading (reviews: buoyancy egg to or eggs ovulated in present A

the apparent contribution of sea bass sea of contributionapparent the

vH appear vH

ays for the different forms different the for ays VtgC (primarily LvHC) to to LvHC) (primarily VtgC of to undergo limited undergo to Moronidae vd rm VtgC. from ived - pcfc tryptic specific, As noted, , as the as , 30

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. arising from non corresponding to mass predicted detec was kDa) (~115 LvHAa bass sea intact to mass in corresponding mullet Lvs; t type Vtgperformedtheusing extractsegg and ovaryof blots 4.3. Maturational remain be to discovered. in detected salmonids in present are receptors VtgAa receptor for mechanisms by secondarily of rates by primarily regulated is Vtgs multiple fromderived YPs of accumulationcontrolled the that concluded authors The 2005b). al. et Sawaguchi also see 2008; al. et (Sawaguchi time this during ratios VtgAa:VtgAb:VtgCplasma and liver diffe and in, fluctuation substantial of spite in vitellogenesis throughout (9:15:1) value constant relatively a at maintained is ovary in ratio LvAa:LvAb:LvC the which in flounder, barfin for ( oocytes by Vtg of forms different the of uptake of rates disparate including mechanisms by regulated precisely is Vtgs multiple from derived post through mid maintainedfrom are disparities thesewhich in bass, striped the for reported been have VtgC of abundance relative higher YPs of abundance proportional estimated In blots Western of extracts ovary PV of OV or eggs performed usinganti the Western comparing by bass sea in investigated was proteolysis maturational YP of phenomenon The the in ovary versus plasma and liver bass sea between disparities the underlying mechanisms The -

n VtgAb and Moronidae hus, only proteolysis of the major YPs (LvHs LvLs) and could potentially beevaluated. - vitellogenesis. - specific degradationspecific during storage frommaturational or yolk proteolysis having already

Proteolysis of Sea Bass Lipovitellins - pcfc eetr hv be dsrbd o wie ec ad tie bass striped and perch white for described been have receptors specific

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTEDoocytes in accumulation VtgC substantial the for mechanismsspecial and species MANUSCRIPT

Comment citer cedocument: ACCEPTED MANUSCRIPT

h atos ocue ta pootoa acmlto o yl proteins yolk of accumulation proportional that concluded authors The ~ 93 kDa was taken to be the major LvHAa breakdown product, perhaps product, breakdownLvHAa major the be to taken was kDa 93

- (review: Hiramatsu et al. 2015), no specific VtgC receptor has been has receptor VtgC specific no 2015), al. et Hiramatsu (review: derived tryptic peptides in ovary, are unknown. Similar observations Similar unknown. are ovary, in peptides tryptic derived eai snhss n scein f h dfeet ye o Vg and Vtg of types different the of secretion and synthesis hepatic

Williams et al. 2014b). This phenomenon was originally reported originally was phenomenon This 2014b). al. et Williams - eitd pae f h Vg it octs Hwvr although However, oocytes. into Vtgs the of uptake mediated derived from the different forms of Vtg, especially the much the especially Vtg, of forms different the from derived

- specific antisera raised against purifiedgreyagainstraisedantisera specific e ad h bn o highest of band the and ted - VtgAa, noprotein band ecs between, rences - vitellogenes

an VtgC d

31 is

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. its be to assumed band kDa ~97 a with along detected was kDa) (~116 LvHAb bass sea to mass in closely corresponding Reith et al. 2001; Finn et al. 2002ab; Sawaguchi et al. Finn2006; 2007ab; Kolarevic al. 200 et 2003; 1999, al. et (Matsubara seawater in eggs pelagic spawning teleosts acanthomorph other some in bass 2002d). al. et (Hiramatsu proteolysis limited undergo chrysops SDS various from Evidence time. this during fragments smaller into processed are but OM during FAA into degradation complete survive sma slightly several and LvHAa intact that revealed experiments These 2014a). al. et (Williams spectrometry mass tandem by analysis SDS before tags isobaric products. extracts from female PV (Willia Western experiment similar blotting a of results on based bass striped for postulated been has as OM, through persisting therefore, could, bands two These 2014a). al. et (Williams bass striped and 2006) al. et r in occur to reported been have as VtgAa, of product cleavage LvLa alternative corresponds limited that LvHAa indicating of degradation eggs, OV of extracts for evident were mass lower the only occurreportedbeen hasto recognition site,as D CAT canonical the fromdownstream location alternate an at VtgAa bass sea fromdomain LvHAa the of fe PV in initiated been In Western blots of extracts from PV PV from extracts of blots Western In and other moronids other and ,

~ The YPs present in striped bass oocyte and egg extracts were labeled were extracts egg and oocyte bass striped in present YPs The oye udron OM undergoing oocytes 93 kDA band was apparent in blots of PV ovary extracts, ovary PV of blots in apparent was band kDA 93

in mass to mass in degradation product degradation males by the time of sampling. It is also possible that this band representscleavageband this that possiblealso is It sampling.of time the bymales

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT intact sea bass LvLAa (~27 kDa) (~27 LvLAa bass sea intact occurs during OM leading up to ovulation. to up leading OM during occurs s. - Comment citer cedocument: differs from differs

PAGE and excision of putative LvHA protein bands for trypsin digestion and digestion trypsin for bands protein LvHA putative of excision and PAGE ACCEPTED MANUSCRIPT Therefore, we ms et al 2014a), but it is unclear why unclear is it but 2014a), al et ms ller LvHAa products are present in striped bass oocytes, and that they that and oocytes, bass striped in present are products LvHAa ller - n vivo in AE n Wsen ltig xeiet on experiments blotting Western and PAGE the the

or a result of alt of result a or tentatively nearly com nearly sea bass bass sea

and for LvHAafor n vitro in ovary performed using anti using performed ovary judge these bands be to plete degradation of this YP this of degradation plete The app The ernative cleavage ernative

ugss ht vA (ye o seiid also specified) not (type LvHAs that suggests and in striped striped in a smaller ~23 kDa band could represent an represent could band kDa ~23 smaller a arent limited arent they mn hs adtoa ad, one bands, additional these Among bass (Williams et al. 2014a).(WilliamsWhile bassal. et it

would then be absent for ovary for absent be then would and several additional bands of bands additional several and of LvHAb from VtgAb, from LvHAb of additional degradation of of degradation - VtgAb, a ~113 kDa band kDa ~113 a VtgAb, ed seabream (Sawaguchiseabream ed (incomplete)

N their on that occurs during OM during occurs that ht bass, white LvHAa breakdown represent LvLAa represent LvHAa in in LvHAa - termini with termini 8) proteolytic .

Morone as has sea 32

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. LvLCmaturation,oocytewhereas during proteolysis limited undergoes LvHC bass sea that indicating as interpreted are results These unchanged. bands protein smaller of several appearance the with band kDa ~114 the of dissapearance the by evidenced was LvHC bass sea of proteolysisMaturational LvLC. bass sea of mass predicted the near band kDa ~24 a with along product, ~74 a basskDA)LvHCand sea massof (~119 the to close white bass. Wil 2009; al. et (Reading bass Among1999). al. flounderbarfinthis proteolysis LvHAb of involves aboutthird oneofintact the polypeptide (Matsubara et 2008a al. et Amano 2006; al. et Sawaguchi 2002ab; al. et Finn 2001; maturational. et oocyte (Reith during proteolysis limited only undergoes LvHAb the which in seawater, in eggs consisten generally are findings collective These 2014a). al. et (Williams OM during degraded further are that YPs domain VtgAb of N of spectrometry mass from Evidence bass. sea in size this near YP intact an as stored be not may LvLAb that possible ( experiment, produced also are fragments LvHAb smaller several that i nearly OM survives LvHAb some that revealed spectrometry fract before extracts egg and oocyte bass striped in YPs of (range~68 bandsprotein smaller several as well extracts blot in bands these of dissappearance the by evidenced was OM during LvHAb bass been etr bos f V vr etat promd sn anti using performed extracts ovary PV of blots Western reported for LvHAb for reported

and

no band corre their replacement by bands corresponding to slightly lower masses (~111 and ~93 kDa) as kDa) ~93 and (~111 masses lower slightly to corresponding bands by replacement their

at positionsat alternative the to canonical CAT D recognition site mayyield much smaller Moronidae - emnly aee Ys n tie bs octs suggests oocytes bass striped in YPs labeled terminally DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED pelagic spawning teleosts acanthomorph other several from observations with t MANUSCRIPT Comment citer cedocument:

sponding mass in sea to ACCEPTED MANUSCRIPT in striped bass (Williams et al. 2014a). Limited maturational proteolysis of sea of proteolysis maturational Limited 2014a). al. et (Williams bass striped in

species, a similar phenomenon has been described for LvHAb from striped fromLvHAb for described been has phenomenonsimilar a species,

(range ~26 (range liams et al. 2014a) and 2014a) al. et liams

- possibly 111 kDa) while the ~74 the while kDa) 111 - 82 kDa). 82 bass LvLAb the wasin detected Western may not. The apparent proteolysisapparent The may not. , as noted, as , The experimentsinvolvingTheN - 76 kDa band taken t taken band kDa 76 oain y SDS by ionation - tC eetd ~1 ka rti band protein kDa ~114 a detected VtgC ilas t l 2014a) al. et Williams mass molecular high as ntact

for LvH for - 76 kDa and ~24 kDa bands persisted bands kDa ~24 and kDa 76 A

(type not specified) from the the from specified) not (type - ht cle that o be its major degradationmajor its o be AE n tne mass tandem and PAGE

o ) Fr xml, in example, For b). f sea bass LvHCbass sea f vg o te LvL the of avage s of ovulated egg ovulated of s - I te present the In . terminal labeling terminal

blots,

for sea bass sea for protein but protein and it it is 33

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. 199 al. et Bell 1988; Coves esters wax amounts mechanism. this via buoyancy egg adjust 2002b flounderbarfinMatsubara (~18%;Fyhn goldsinney2010), andFinn (~19%; Koyawrasse 1997) eg pelagic spawning fish other a contain to oocytehydration,2007).eggs degree bass lesser keepingof seaIn the Babin with al. et 2007; 2 Krisofferson, and Finn 2003; al. et Matsubara (reviews: LvHAa mainly YPs, derived FAAgeneratedbymaturational of drivenbya largepool proteolysisaccumulationofbuoyancy ofis Vtg 2015 al. et Yilmaz by (reviewed seawater in eggs pelagic spawning fish in seen typically hydration oocyte of degree the undergo not do ( water fresh in eggsdemersal or bass) (striped water Moronidae partial der LvHs the that suggest study this in antisera specific maturationlargely oocyte survive to appears it but 2014a), al. et Williams 2009; al. et (Reading bass striped other Among study. evident extent apparent the to not although maturation, oocyte during degraded partly or nicked proteolytically is VtgC 2002a), al. et (Finn halibut Atlantic and 2006) al. et (Sawaguchi 200 al. et Sawaguchi 2007b; al. et (Amano intact largely proteolysis yolkmaturational survives LvHC) (including VtgC mosquitofish, and mullet grey In above. cited studies the inacanthomorphs other some for reported situation the unlike The The limited extentof maturational yolk sea proteolysis in bass SDS of results collective The ). ).

proteolys intact thein white

species that spawn pelagic eggs in seawater (sea bass), (sea seawater in eggs pelagic spawn that species lower content of FAA on a dry weight basis (~7.5%; Naz 2008 Naz (~7.5%; basis weight dry a on FAA of content lower is during OM during is , which may comprise 40 comprise may which , moronids

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Comment citer cedocument: perch (Reading andSullivan 2011). ACCEPTED MANUSCRIPT 7)

b. oee, n afn ludr Mtuaa t l 20) rd seabream red 2003), al. et (Matsubara flounder barfin in However, 5b).

and Vg (r vC i prily erdd uig oye auain n the in maturation oocyte during degraded partially is LvHC) (or VtgC , gs in seawater including, as examples, Atlantic halibut (~18%; Finn and Finn (~18%; halibut Atlantic examples, as including, seawater in gs t and , n hc hdain f oye laig o custo o poe egg proper of acquisition to leading oocytes of hydration which in ), -

PAGE and Western blotting of ovary and egg extracts using Lv type Lv using extracts egg and ovary of blotting Western and PAGE which his limited his

have low specific gravity compared to other types of egg lipids egg of types other to compared gravity specific low have In

moronids - 90% of egg lipids lipids egg of 90% ,

non - white bass, w bass,white specific , egg buoyancy is enhanced by storage of copious of storage by enhanced is buoyancy egg , ived from all three forms of seabass of forms three all from ived LvH proteolysis appears to be common to to common be to appears proteolysis LvH

( may hite perch). hite Eldridge et al. 1983; 1983; al. et Eldridge nearly neutrally buoyantneutrallynearly be ass ) than seen seen than ) ociated a with Additionally,t

for sea bass VtgC in this in VtgC bass sea for 007; Cerdà et al. et Cerdà 007; Devauchelle and Devauchelle in in

lesser need to eggs of some of eggs

eggs in fresh in eggs Vtg undergo Vtg he are reportedare moronids et al.et 34 - -

Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. DOI : 10.1016/j.cbpb.2015.11.010 VersionVersion postprintpreprint Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 194-195, 71-86. vitellogenins andproduct yolkproteins inEuropean seabass(Dicentrarchus labrax): Molecular Yilmaz, O.,Prat,F.,Ibáñez, A.J.,Köksoy, S.,Amano, H.,Sullivan, C.V.(2016). Multiple characterization, quantification in plasma, liverand ovary, andmaturational proteolysis. ty these for known feature structural major every to respect other two of those to compared and cDNA from 4.4. Summary and Conclusions hydration to adjust egg buoyancy. condition degenerate a represents OM in LvHAa of proteolysis preferential for mechanisms specialized of lack the that 2008). al. et (Amano intact largely remain LvHC and LvHAb the while FAA to degraded similar 1986) al. et a undergo oocytes the 1967), Schlenk eg whose mullet, grey the In th em that to 2003). tend al. et (Bergey which globules oil smaller waters, having column, water turbulentthe in eggs more maintain in spawned eggs with spawn, they which in watershed fresh globules oil egg their of size by habitats American North spawning freshwater of invasion the to prior ancestor common a from control buoyancy extant that likely therefore is It 1994). bass, yellow bass; (white spawning freshwater seabass, spotted bass; (sea mouths river with associatedspawning coastal fromranging salinity, low and habitat spawning All challenge. greater a is control buoyancy encoun commonly that eggs buoyant produce ova their into esters wax of ( e.g. a t exhibit a lesser degree of oocyte hydration does not preclude disproportionate proteolysis of LvHAa. of disproportionateproteolysis preclude not does hydration oocyte of degree lesser a exhibit t In the presentIn study, polypeptidethe sequencesencoding basssea VtgAa,anddeduced VtgAbwere VtgC phospholipids, triglyceride phospholipids, p loyment of the dual system for regulating egg buoyancy (i.e. hydration, wax esters) in species in esters) wax hydration, (i.e. buoyancy egg regulating for system dual the of loyment Morone Dicentrar to to that DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT

Comment citer cedocument: (Secor 2002). (Secor ACCEPTED MANUSCRIPT

seen in sea bass ( bass sea in seen hs punctatus chus

and, therefore, wax ester content, depending on the physical energy of the of energy physical the on depending content, ester wax therefore, and, g lipids lipids g

). ).

As noted by Vetter et al. (1983), species incorporating large quantities large incorporating species (1983), al. et Vetter by noted As

modest also

Interestingly, striped bass of different stocks appear to adjust the adjust to appear stocks different of bass striped Interestingly, ht may that

moronid , o auttv aarm (tie bs) ad obligatory and bass), (striped anadromy facultative to ), oti a ih ecnae f a etr (0; yna and Iyengar (70%; esters wax of percentage high a contain 1 percentage increase in volume during OM during volume in increase percentage 96 Morone Moronidae

%; see Yilmaz et al. 2015 al. et Yilmaz see %; Moronidae aris ihrtd hs unique this inherited s e

mississipiensis n pce wt a oe iie ne fr oocyte for need limited more a with species in

pce so a ls ascain between association close a show species

species, striped bass and white perch, with perch, white and bass striped species, e o Vg. ah om f e bs Vtg bass sea of form Each Vtgs. of pes ; white perch)(Pickett and Pawson and perch)(Pickett white ; and ), and the and ), e rdcd aiiis where salinities reduced ter lxbe method flexible Moronidae

It should be noted be should It LvHAa is mostly is LvHAa (171%; Greel (171%;

It is possible is It

undergoing

f egg of 35 e y

ales and/or estrogenized males was confirmed by Western blotting using Lv type Lvusingblotting Western confirmedby was estrogenizedmalesand/or ales We acknowledgeWe the staff of the e hydration in in hydration e orth Carolina -

as as a preliminary in report proceedingsthe for that SC values, VtgAb makes the dominant contribution to sea bass egg YPs (~54.5%), about (~54.5%), YPs egg bass sea to contribution dominant the makes VtgAb values, SC Symposium Reproductive the on Physiology Fish, of 25 s ing the Lv type Lv the ing Some of results this study werer ly en n te teleosts. other in seen ally

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT

Agricultural Foundation, Inc.

moronids Comment citer cedocument: ( ACCEPTED MANUSCRIPT Yilmaz 2013) al. et Program for Moronidae -

5 September, 2013,Ghent, Belgium, andare included in the book of - may be linked to their additional use of high levels of wax esters to esters wax of levels high of use additional their to linked be may specific antisera, appears to involve limited proteolyticdegradationoflimitedinvolve to appearsantisera, specific

species seem not to vary in spite of their different egg types and types egg different their of spite in vary to not seem species

Dr. C.V. doctoral research . ance with deployment various of

Selected results also were presented at the 10th e bs m bass Sea Sullivan eported at the 6th Fish and Shellfish Larviculture Ozlem

Structural modeling of Vtgs wassupported by ’s in in Unitedthe States,

laboratory in Biologythe of Department trtoa yl poelss a rvae by revealed as proteolysis, yolk aturational Vtg, with the exceptions of PvAa and CtAa.and PvAa of exceptions the with Vtg, Yilmaz from the

meeting (Yilmaz 2015). al. et ach form of sea bass Vtg in the in Vtg bass sea of form ach adm as pcrmty Tu, the Thus, spectrometry. mass tandem - 30 May, 2014,Olhão, Portugal, and International Fulbright

and laboratory protocols and to Craigto V. Sullivan - specific antisera specific This This

plasma of plasma 36

DOI :10.1016/j.cbpb.2015.11.010 ACCEPTED MANUSCRIPT Comment citer cedocument: ACCEPTED MANUSCRIPT

versity, Antalya, Turkey (Yilmaz 2013 Faculty of Fisheries,Faculty of Postgraduate ) .

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