Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization

Submi ed 7 June 2017 Accep ed 10 Oc obe 2017 Published 3 No embe 2017 Co esponding au ho s Leand o Rod íguez-Vie a, [email p o ec ed], [email p o ec ed] E ick Pe e a, [email p o ec ed], [email p o ec ed] Academic edi o K is in Ham e Addi ional In o ma ion and Decla a ions can be ound on page 16 DOI 10.7717/pee j.3975 Copy igh 2017 Rod íguez-Vie a e al. Dis ibu ed unde C ea i e Commons CC-BY 4.0 OPEN ACCESS Ca bohyd a es diges ion and me abolism in he spiny lobs e (Panuli us a gus): biochemical indica ion o limi ed ca bohyd a e u iliza ion Leand o Rod íguez-Vie a1,2,*, E ick Pe e a3,*, Vi ian Mon e o-Alejo4, Rolando Pe domo-Mo ales4, Tsai Ga cía-Galano1, Gonzalo Ma ínez-Rod íguez5 and Juan M. Mance a2 1Cen e o Ma ine Resea ch, Uni e si y o Ha ana, Ha ana, Ha ana, Cuba 2Facul y o Ma ine and En i onmen al Sciences, Campus de Excelencia In e nacional del Ma (CEIMAR), Uni e si y o Cadiz, Pue o Real, Cadiz, Spain 3Nu igenomics and Fish G ow h Endoc inology, Ins i u e o Aquacul u e To e de la Sal, IATS-CSIC, Cas ellón, Valencia, Spain 4Depa men o Biochemis y, Cen e o Pha maceu icals Resea ch and De elopmen , Ha ana, Cuba 5ICMAN-CSIC, Ins i u o de Ciencias Ma inas de Andalucía, Pue o Real, Cadiz, Spian *These au ho s con ibu ed equally o his wo k. ABSTRACT As o he spiny lobs e s, Panuli us a gus is supposed o use p e e en ially p o eins and lipids in ene gy me abolism, while ca bohyd a es a e well diges ed bu poo ly u ilized. The aim o his s udy was o e alua e he e ec o die a y ca bohyd a e le el on diges ion and me abolism in he spiny lobs e P. a gus. We used complemen a y me hodologies such as pos - eeding lux o nu ien s and me aboli es, as well as measu emen s o α-amylase exp ession and ac i i y in he diges i e ac . Lobs e s eadily diges ed and abso bed ca bohyd a es wi h a ime-cou se ha is dependen on hei con en in die . Lobs e showed highe le els o ee glucose and s o ed glycogen in di e en issues as he inclusion o whea lou inc eased. Modi ica ions in in e media y me abolism e ealed a dec ease in amino acids ca abolism coupled wi h a highe use o ee glucose as ca bohyd a es ise up o 20%. Howe e , his e ec seems o be limi ed by he me abolic capaci y o lobs e s o use mo e han 20% o ca bohyd a es in die s. Lobs e s we e no able o igh ly egula e α-amylase exp ession acco ding o die a y ca bohyd a e le el bu exhibi ed a ma ked di e ence in sec e ion o his enzyme in o he gu . Resul s a e discussed o highligh he limi a ions o inc easing ca bohyd a e u iliza ion by lobs e s. Fu he g owou ials a e needed o link he p esen ed me abolic p o iles wi h pheno ypic ou comes. Subjec s Aquacul u e, Fishe ies and Fish Science, Ma ine Biology Keywo ds α-amylase, Ca bohyd a e, Ene gy me abolism, C us aceans, Glycemia, Gene exp es- sion, Panuli us a gus, Lobs e , Ca bohyd a e diges ion INTRODUCTION Despi e signi ican achie emen s made on he la al p opaga ion o spiny lobs e s (Ba na d, Johns on & Phillips, 2011;Pe e a & Simon, 2014), a majo in e es emains on How o ci e his a icle Rod íguez-Vie a e al. (2017), Ca bohyd a es diges ion and me abolism in he spiny lobs e (Panuli us a gus): biochemical indica ion o limi ed ca bohyd a e u iliza ion. Pee J 5:e3975; DOI 10.7717/pee j.3975 he g owou o lobs e s based on he cap u e o wild seed (Williams, 2007;Pe e a & Simon, 2014;Radhak ishnan, 2015), especially o as -g owing opical species (e.g., Panuli us a gus,Panuli us o na us) (Je s & Da id, 2003;Williams, 2007;Nguyen, Long & Hoc, 2009). Howe e , he absence o app op ia e die s is so a he main impedimen o he sus ainable expansion o his ac i i y (Williams, 2007); du ing g owou , spiny lobs e s a e cu en ly ed wi h ash ish (Pe e a & Simon, 2014;Radhak ishnan, 2015), wi h downs eam nega i e e ec s such as en i onmen al pollu ion, poo eed con e sion, appea ance o eme ging diseases, and o e p essu e on wild ish s ocks (Pe e a & Simon, 2014;Radhak ishnan, 2015). Al hough he nu i ional equi emen s o some spiny lobs e s ha e been e alua ed, g ow h a es wi h o mula ed die s a e s ill low o mos species (C ea e al., 2000;Glenc oss e al., 2001;Smi h e al., 2003;Wa d e al., 2003;Johns on e al., 2003;Smi h, Williams & I in, 2005;Simon & Je s, 2008). I is ecognized ha p oblems o eeding spiny lobs e s wi h o mula ed die s a e pa ially due o gaps in ou knowledge on hei diges i e physiology and me abolism (Pe e a & Simon, 2014). Di e en s udies in he spiny lobs e P. a gus diges i e physiology ha e been ocused on p o ein diges ion (Pe e a e al., 2008a;Pe e a e al., 2008b;Pe e a e al., 2010a;Pe e a e al., 2010b;Pe e a e al., 2012a;Pe e a e al., 2012b), while diges ion o o he nu ien s has ecei ed less a en ion. F om a die de elopmen pe spec i e, ca bohyd a es (CHs) would p o ide a cheap sou ce o ene gy, which is assumed o be ad an ageous in e m o g ow h and p o i abili y. Howe e , he e is no e idence o a signi ican use o CHs o ene gy in spiny lobs e s, hough some ene gy appea s o be de i ed om glycogen o he diges i e gland (DG) du ing sho e m as ing (Simon & Je s, 2013). P. a gus is cu en ly supposed o use p e e en ially p o eins and lipids in ene gy me abolism (Pe e a e al., 2005), and s udies in his and o he spiny lobs e species ha e p o ided indica ion ha se e al CHs a e well diges ed bu poo ly u ilized (Simon, 2009a;Simon, 2009b;Simon, 2009c;Simon & Je s, 2013;Rod íguez-Vie a e al., 2014). The me abolism o o he c us aceans such as penaeid sh imps is also di ec ed o he use o p o eins and lipids o ene gy, bu CHs can spa e die a y p o eins o a ce ain ex en (Cuzon e al., 2000). P e ious s udies indica ed ha na i e whea lou (∼70% s a ch) esul s in a g adual diges ion and libe a ion o glucose o he hemolymph and may ha e he majo po en ial o op imizing ene gy me abolism o lobs e s (Simon, 2009a;Simon, 2009b;Simon, 2009c; Simon & Je s, 2011;Simon & Je s, 2013;Rod íguez-Vie a e al., 2014). Al hough di e en ac o s a ec ing s a ch hyd olysis come om he CH sou ce i sel (e.g., g anule size and shape, amylose con en ), he ue diges ion a e a ises om he in e ac ion be ween CHs and diges i e ca bohyd ases. As in o he c us aceans (Pa aso ic e al., 2004), adap a ion o α-amylase o die a y CHs has been demons a ed in he spiny lobs e J. edwa dsii. The α-amylase ac i i y in his species signi ican ly dec eased wi h inc easing inclusion o CHs in die (Simon & Je s, 2011;Simon & Je s, 2013). P elimina y gene exp ession analysis in P. a gus sugges ed ha die a y egula ion o α-amylase ac i i y in spiny lobs e s may be exe ed a he ansc ip ional le el (Rod íguez-Vie a e al., 2016). Howe e , his lexibili y in gene exp ession o P. a gus α-amylase seems no su icien o con ol CHs diges ion when Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 2/21 lobs e s a e ed on o mula ed die s, p obably due o low Kmo he enzyme (Rod íguez-Vie a e al., 2016) and i s high ac i i y in he condi ions o he gas ic juice (Pe e a e al., 2008a; Pe e a e al., 2008b). The aim o his s udy was o e alua e he e ec o CH le el on diges i e α-amylase ansc ip ional egula ion, CH diges ion and me abolism in he spiny lobs e P. a gus. Complemen a y me hodologies such as pos - eeding luxes o nu ien s and me aboli es, as well as assessmen s o ac i i y and gene exp ession o α-amylase in he diges i e ac we e used. Resul s sugges ha egula o y mechanisms o diges i e α-amylases in he lobs e a e no well de eloped a he ansc ip ional le el, wi h mo e complexi y added a he le el o sec e ion o he enzymes. Fu he mo e, his is p obably he i s s udy p o iding biochemical e idence o he p o ein spa ing e ec o die a y CHs in spiny lobs e s. Howe e , his e ec seems o be limi ed by he me abolic capaci y o lobs e o use die -de i ed glucose, wi h no imp o emen wi h inc emen s in die a y CHs beyond 20%. MATERIALS & METHODS Expe imen al die s and eeding ial Th ee expe imen al die s we e o mula ed o con ain di e en inclusion le els o CHs (6%, 20%, and 35%) (Table 1). All eeds u s we e ob ained om comme cial supplie s (Table 1). Pelle s we e made as desc ibed in a p e ious wo k (Pe e a e al., 2012a;Pe e a e al., 2012b). Spiny lobs e s we e collec ed in he Gul o Ba abanó, Cuba, unde pe mission o he Fishe ies Regula o Depa men om he Minis y o he Fishing Indus y o Cuba. The eeding ial was conduc ed a he Cen e o Ma ine Resea ch o he Uni e si y o Ha ana, Cuba, in a acili y equipped eci cula ed sea wa e , cons an ae a ion, and pho ope iod o 12 h ligh : 12 h da kness. Wa e quali y was moni o ed wice a week: ∼26 ◦C, pH∼8.0, salini y 36 ups, oxygen ∼6.0 mg/L, and ammonia-N∼0.07 mg/L. Each expe imen al die was so ed a andom o six lobs e s (∼250 g), housed indi idually in 60 L anks. Only in e mol indi iduals (D ach & Tchemigo z e , 1967;Lyle & MacDonald, 1983) we e used. Lobs e s we e acclima ized o one week o expe imen al die s by g adually educing ish lesh as ood un il hey consumed only he pelle s. The a ion was p og essi ely adjus ed o 2% o body weigh pe day (BW day−1). This a ion is su icien o lobs e s o eed close o sa ia ion (Simon, 2009a;Simon, 2009b;Simon, 2009c). A e his pe iod, lobs e s we e as ed o wo days and hen hey we e p o ided wi h a 2% BW a ion o he expe imen al die s o se ial collec ion o gas ic juice and hemolymph. Se ial collec ion o gas ic juice and hemolymph Samples o gas ic luid we e ob ained h ough he o al ca i y using insulin sy inges wi h a plas ic cannula o e he needle as desc ibed be o e (Pe e a e al., 2012b). Gas ic juice was no sampled be o e eeding as his a ec s eed in ake. Se ial samples (∼100 µL) o gas ic juice we e aken a 2, 6, 12, 24 and 30 h a e inges ion, cen i uged a 10,000×g o 10 min, ozen in liquid ni ogen and s o ed a −80 ◦C. Samples we e apidly aken (less han 1 min) o a oid excessi e s ess. Hemolymph was no sampled p io o eeding, as his is known o a ec eed in ake in o he spiny lobs e species (Simon, 2009a) and by p e ious obse a ion in ou labo a o y (Rod íguez-Vie a e al., 2014). Hemolymph sampling began 2 h a e Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 3/21 Table 1 Fo mula ion (%) and p oxima e composi ion o he expe imen al die s. Ing edien s 6% 20% 35% Fish meala35 32 28.7 Squid mealb15 15 15 Gela inc5 5 5 Whea lou d3 22.2 42.4 Fish oile1.9 1.9 1.9 Leci hin 2 2 2 Choles e olg1 1 1 Vi & Min P emixh1 1 1 Phospha e/ca bona ei2 2 2 A ac an sj1 1 1 Talcd33.1 16.9 – To al 100 100 100 P oxima e composi ionk C ude p o ein 41.70 45.13 43.47 C ude lipid 9 9 10 Ca bohyd a e 5.75 19.82 36.45 Ash 43.65 27.05 9.08 No es. Pelle s con ained 10–12% o wa e . aP o azul 65: 65% p o eins, 12% lipids, 5% mois u e. bImpe ial Bai s Ca p ack p oduc s: 70% p o eins, 15% lipids, 6% mois u e. cG2500; Sigma-Ald ich, S . Louis, MO, USA. dComme cially a ailable egula eeds u . eFishe ies Resea ch Cen e Labo a o y, Ha ana, Cuba. Calbiochem (429415); Me ck Chemicals L d., Bille ica, MA, USA. gSigma-Ald ich (C8667). hVi amins and Mine als P emix om DIBAQ-Aquacul u e, Sego ia, Spain, con aining (pe kg o eed): i amin A 15,000 IU, i amin D3 3000 IU, i amin E 180 mg, i amin K 15 mg, i amin B1 37.5 mg, i amin B2 37.5 mg, i amin B6 24.75 mg, i a- min B12 0.045 mg, i amin H 1.14 mg, D-pan o henic acid 120 mg, nico inic acid 225 mg, i amin C 300 mg, olic acid 11.24 mg, Inosi ol 112.5 mg, zinc 75 mg, selenium 0.3 mg, magnesium 86.25 mg, coppe 2.25 mg, manganese 22.5 mg, iodine 7.5 mg, i on 3 mg, cobal 0.3 mg. iDicalcium phospha e/Calcium ca bona e (1:2); San a C uz Fish Feed Fac o y, Camagüey, Cuba. jTau ine (T0625; Sigma-Ald ich, S . Louis, MO, USA) 500 mg/kg die , Glycine (G8898; Sigma-Ald ich, S . Louis, MO, USA) 500 mg/kg die . kMeasu ed as desc ibed be o e in Rod íguez-Vie a e al. (2014). eeding, wi h addi ional samples a 6, 12, 24, and 30 h. Se ial sampling o hemolymph in lobs e s has li le e ec on hemolymph glucose concen a ion (Rad o d e al., 2005; Rod íguez-Vie a e al., 2014). Hemolymph samples (500 µL) we e aken om he sinus o he 4 h walking legs (Pe domo-Mo ales e al., 2007) in 1 mL py ogen ee sy inges con aining 500 µL o p ecooled an icoagulan solu ion (400 mM NaCl, 10 mM KCl, 10 mM HEPES, 20 mM EDTA, pH 7.3) (He nandez-Lopez e al., 2003). An addi ional g oup o six lobs e s we e ed wi h esh ish as con ol and sampled as abo e. Time-cou se o p o eins and glucose in gas ic juice and hemolymph Soluble p o ein in gas ic juice was measu ed as a sign o solubiliza ion o die a y p o ein and enzyme sec e ion in o he o egu . Gas ic juice glucose was measu ed as indica o o he a e o CHs hyd olysis in he o egu . The glycemic p andial esponse was analyzed Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 4/21 as indica o o diges ibili y and abso p ion o die a y CHs in lobs e s (Rod íguez-Vie a e al., 2014;Simon, 2009a;Rad o d e al., 2005) and p o ein in he hemolymph as a sign o hei diges ion and abso p ion. Soluble p o ein concen a ions we e quan i ied by he Low y me hod using bo ine se um albumin as s anda d (Low y e al., 1951). Glucose le el was de e mined using a HELFA R RapiGluco-Tes glucose oxidase ki (Quime a Biological P oduc s Inc., Ha ana, Cuba). Amylase ac i i y Amylase ac i i y was measu ed as desc ibed be o e (Rod íguez-Vie a e al., 2016) in a mix u e composed o 5 µL o DG ex ac o gas ic juice and 200 µL o assay bu e (50 mM MES (2-(N-mo pholino) e hanesul onic acid), pH 5.5), wi h 0.5 mM 2-Chlo o-4- ni ophenyl-a-D-mal o ioside (CNP-G3) as he subs a e. CNP eleased was measu ed (a 405 nm and 37 ◦C) kine ically o 10 min in an ELx808IU mic opla e eade . Ini ial eloci ies we e ob ained using he so wa e KC4 e sion 3.4 (BioTek Ins umen s, Winooski, VT, USA). The ex inc ion coe icien o p-ni ophenol a 405 nm ( eac ion olume o 205 µL) was 9.774 mM−1cm−1. A uni o amylase ac i i y was de ined as he amoun o enzyme ha p oduces 1 µmol p-ni ophenol/minu e. Amylase ac i i y was exp essed pe olume o gas ic juice (µL) o DG weigh (mg). Me aboli es in diges i e gland and muscle A e he 30 h ime-cou se sampling o gas ic juice and hemolymph, lobs e s we e ed o one mon h wi h he co esponding die s, le un ed o 48 h, and hen ed again wi h he espec i e die s (Rod íguez-Vie a e al., 2014). They we e killed 24 h la e in ice-cold wa e o emo e DG, muscle, and hemolymph samples, which we e immedia ely ozen in liquid ni ogen and eeze-d ied o me aboli e and me abolic enzyme measu emen s. Samples o ∼20 mg we e homogenized in 1 mL wa e , cen i uged (30 min, 10,000×g, 4 ◦C), and he supe na an aken o assess issue me aboli es. Be o e cen i uga ion, an aliquo was aken o iglyce ide (TG) de e mina ion. Soluble p o ein and glucose concen a ions we e measu ed as de ailed abo e. F ee amino acid concen a ion was assessed colo ime ically by he nynhid in me hod (Yemm, Cocking & Ricke s, 1955;Rosen, 1957) wi h L-alanine as he s anda d. TG and lac a e concen a ions we e measu ed using he comme cial ki s TAG (Spin eac , Gi ona, Spain) and Lac a e (Spin eac , Gi ona, Spain), espec i ely. Glycogen concen a ion was assessed by he b eakdown o glycogen by amyloglucosidase (Kepple & Decke , 1974) and he de e mina ion o esul an glucose by a comme cial ki (Spin eac , Gi ona, Spain) as in ou p e ious wo k (Rod íguez-Vie a e al., 2014). Me abolic enzymes in HP and muscle The ac i i ies o enzymes om di e en me abolic pa hways we e quan i ied in wo key issues o lobs e me abolism, DG and muscle. Lyophilized samples o DG and muscle we e homogenized in 10 olumes o ice-cold bu e (50 mM imidazole hyd ochlo ide, pH 7.5, 1 mM 2-me cap oe hanol, 50 mM sodium luo ide, 4 mM EDTA, 250 mM suc ose, and 0.5 mM PMSF). Homogena es we e cen i uged o 30 min a 10,000×g and supe na an s used o assays. Enzymes ac i i ies measu ed we e: hexokinase (HK, EC 2.7.1.11), glyce ol- 3-phospha e dehyd ogenase (G3PDH, EC 1.1.1.8), py u a e kinase (PK, EC 2.7.1.40), Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 5/21 L-lac a e dehyd ogenase (LDH, EC 1.1.1.27), uc ose 1,6-biphospha ase (FBPase, EC 3.1.3.11), glycogen phospho ylase (GPase, EC 2.4.1.1), glucose-6-phospha e dehyd ogenase (G6PDH, EC 1.1.1.49), aspa a e ansaminase (AST, EC 2.6.1.1), alanine ansaminase (ALT, EC 2.6.1.2), glu ama e dehyd ogenase (GDH, EC 1.4.1.2), and 3-hyd oxyacyl-CoA dehyd ogenase (HOAD, EC 1.1.1.35). The amoun o sample was se o ensu e ini ial eloci ies. Condi ions o enzyme assays (e.g., bu e composi ion, co ac o s, addi ional enzymes o coupled enzyme assays, and elec on dono (NADH) o accep o s (NADP, NAD)) we e acco ding o Laiz-Ca ión e al. (2003) and Sangiao-Al a ellos e al. (2003). Subs a es we e as in ou p e ious wo k (Rod íguez-Vie a e al., 2014): 5 mM D-glucose o HK and PK, 0.2 mM dihyd oxyace one phospha e o G3PDH, 6.25 mM lac ic acid o LDH, 0.1 mM uc ose-1,6-bisphospha e o FBPase, 5 mg/mL glycogen o GPase, 1 mM glucose-6-phospha e o G6PDH, 10 mM L-aspa a e o AST, 7.5 mM L-alanine o ALT, 1.40 mM α-ke oglu a a e o GDH, and 0.1 mM ace oace yl-CoA o HOAD. Reac ions wi hou subs a es we e pe o med as nega i e con ols. Reac ion a es o enzymes we e de e mined in duplica e by he inc ease o dec ease in abso bance a 340 nm and 37 ◦C, as a esul o NADPH p oduc ion o HK, LDH, FBPase, GPase, and G6PDH ac i i ies, o NADH consump ion o G3PDH, PK, AST, ALT, GDH, HOAD ac i i ies, espec i ely. All assays we e pe o med using a Bio-Tek Powe Wa e 340 Mic opla e spec opho ome e using KCjunio Da a Analysis So wa e (Bio-Tek Ins umen s, Winooski, VT, USA). One uni o enzyme ac i i y (U) was de ined as he amoun o enzyme needed o ans o m 1µmoL o subs a e o p oduce 1 µmoL o p oduc pe min. Enzyme ac i i y was exp essed as U/mg o soluble p o ein. E ec s o ca bohyd a e le el on lobs e α-amylase gene exp ession and ac i i y Amylase gene exp ession and ac i i y we e assessed in lobs e s (n=6 pe die ) acclima ed o he h ee expe imen al die s and esh ish o one mon h, le un ed o 48 h, hen ed again wi h he espec i e die s, and hen killed 24 h a e las inges ion. Fo amylase ac i i y de e mina ion, DGs we e homogenized wi h chilled Milli-Q R wa e (90 mg/500 µL) using a glass pis on homogenize and he homogena es we e cen i uged a 10,000×g, 30 min a 4◦C. The esul an uppe lipid laye s we e disca ded and he emaining supe na an s s o ed a −80 ◦C. Samples o gene exp ession analyses we e immedia ely placed in RNAla e a 4◦C o 24 h and hen s o ed a −20 ◦C un il o al RNA ex ac ion. To al RNA was isola ed om indi idual DGs using an Ul a-Tu ax R T25 (IKA R - We ke) and he illus aTM RNAspin Mini Ki (GE Heal hca e, Do ns ad , Ge many). Concen a ion o o al RNA was measu ed a 260 nm wi h he BioPho ome e Plus (Eppendo ), and i s quali y was de e mined in an Agilen 2100 Bioanalyze (Agilen Technologies, San a Cla a, CA, USA) using he Agilen RNA 6000 Nano Ki . Speci ic p ime s (Table 2) we e designed using he so wa e P ime 3 .0.4.0 (h p:// odo.wi.mi .edu/) o assessing he ela i e exp ession o α-amylase and elonga ion ac o 1 alpha (e 1a) as he in e nal e e ence gene (Pe e a e al., 2010a;Pe e a e al., 2010b). e 1a showed low a iabili y (less han 0.20 C ) among expe imen al g oups. P ime s we e syn hesized by IDT (In eg a ed DNA Technologies, Leu en, Belgium). Fi s , 500 ng o o al RNA we e Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 6/21 Table 2 P ime s used in his s udy o quan i y he ela i e exp ession (qPCR) o α-amylase om P. a - gus. qPCR p ime s Nucleo ide sequence Ampli ied size EF1- αFw 50-CCAGTAGACAAACCACTTCG-30532–551 EF1- αR 50-CATACCTGGCTTCAAGATGC-30620–639 Pa-qPCR-AMY-Fw 50-GAGTGACGGAGTTCAAGTACGG-30841–862 Pa-qPCR-AMY-R 50-GTCGTGGTTGTCGATGAAGAC-30980–1,000 e e se- ansc ibed in a 20 µL eac ion using he qSc ip TMcDNA syn hesis ki (Quan a BioSciences) o 5 min a 22 ◦C, 30 min a 42 ◦C, and 5 min a 85 ◦C. qPCR condi ions we e op imized (Rod íguez-Vie a e al., 2016), and di e en amoun s o cDNA we e used in iplica e (6 poin s o se ial 1/5 dilu ions om 10 ng o 3.2 pg pe eac ion) as empla es o check he assay linea i y (R2) and ampli ica ion e iciency (E) (Rod íguez-Vie a e al., 2016). Assay was linea along all six se ial dilu ions (R2=0.999, E=98.6), hus 10 ng o cDNA pe eac ion we e u he used in qPCR eac ions; qPCR was pe o med wi h CFX Connec TM Real-Time Sys em (BIO-RAD, Mad id, Spain). Each 10 µL eac ion mix u e con ained 0.5 µL a 400 nM o each speci ic o wa d and e e se p ime , and 5 µL o Pe eCTa SYBR R G een Fas MixTM (Quan a Biosciences, Gai he sbu g, MD, USA) in Ha d-Shell R PCR Pla es, 96 wells, hin-wall, co e ed wi h Mic oseal R ‘B’ seal ilm (BIO-RAD). Con ol eac ions wi h RNAse- ee wa e (NTC) and RNA ins ead o cDNA (NRT) we e included o ensu e he absence o con amina ion o genomic DNA. qPCR he mal p o ile was: 95 ◦C, 10 min; (95 ◦C, 20 s; 60 ◦C, 35 s) X 40 cycles; mel ing cu e (60 ◦C o 95 ◦C, 0.5 ◦C/5 s)) (Rod íguez-Vie a e al., 2016). Rela i e quan i ica ion was pe o med using he 2−11CT me hod (Li ak & Schmi gen, 2001) co ec ed o e iciency o he s anda d cu e (P a , 2001). S a is ical analyses Only esul s om lobs e s in in e mol s age C we e analyzed as mol s age has been ound o a ec diges i e enzyme ac i i ies in P. a gus (Pe e a e al., 2008b). All da a we e checked o no mali y and homogenei y o a iance using Kolmogo o –Smi no and Le ene’s es s, espec i ely, wi h P≤0.05. Me abolic enzymes and me aboli es in diges i e gland, hemolymph, and muscle 24 h a e inges ion we e analyzed by one-way ANOVA (P≤0.05). Da a om he ime-cou se in gas ic juice and hemolymph a e inges ion we e subjec ed epea ed measu es ANOVA (P≤0.05), wi h sampling ime as he wi hin subjec ( epea ed measu e) ac o , and die as he be ween g oup ac o . Addi ionally, one-way ANOVA analyses (P≤0.05) we e pe o med o each ime o compa e he single main e ec die . The Tukey’s es (P≤0.05) was used o de e mine di e ences among means. The so wa e package S a is ica 7.0 (S a So Inc., Tulsa, OK, USA) was used o all es s and igu es we e p oduced by G aphPad P ism 5.00 (G aphPad So wa e, Inc., San Diego, Cali o nia, US) (Rod íguez-Vie a e al., 2016). Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 7/21 Figu e 1 Soluble p o ein (A) and α-amylase ac i i y (B) in he gas ic juice o Panuli us a gus a e eeding. Die s we e named acco ding o he le el o CH hey con ained (6%, 20%, 35%), and a con ol wi h esh ish muscle. Each alue is he mean ±SEM (N=6 lobs e s pe die ). Di e ences among di- e s h oughou he 30 h s udied a e ma ked by di e en supe sc ip le e s in legend (P≤0.05). Fo each sampling ime, s a is ically di e en (Tukey’s es , P≤0.05) die a y ea men s a e indica ed by as e isks. Full-size DOI: 10.7717/pee j.3975/ ig-1 RESULTS Soluble p o eins and amylase ac i i y in gas ic juice Soluble p o eins in he gas ic juice did no a y among die s (Repea ed measu es ANOVA, F=0.83, P>0.05). Time and ime x die in e ac ion we e no signi ican ac o s (Repea ed measu es ANOVA, F=1.39, P>0.05) ei he . Howe e , wo appa en peaks o soluble p o eins we e ound in he gas ic juice a 6 and 24 h a e inges ion, excep wi h he die con aining 6% CH (Fig. 1A). Di e ences among die s (one-way ANOVA, F=8.52, P≤0.05) in soluble p o ein concen a ion o he gas ic juice 6 h a e inges ion we e obse ed, being signi ican ly highe in he die s wi h 35% CH and wi h esh ish (Tuckey’s es , P≤0.05) (Fig. 1A). Thi y hou s a e inges ion, basal alues o soluble p o ein we e only achie ed by lobs e ed esh ish (one-way ANOVA, F=5.10, P≤0.05) (Fig. 1A). Lobs e s om all ea men s showed simila alues a 12 and 24 h (Fig. 1A). Amylase ac i i y pe olume o gas ic juice signi ican ly a ied among die s (Repea ed measu es ANOVA, F=5.61, P≤0.05), and h ough ime (Repea ed measu es ANOVA, F=3.21, P≤0.05). Lobs e s inges ing low CH die s (i.e., 6% CH die and esh ish) exhibi ed highe amylase ac i i y in he gas ic juice, especially du ing he i s hou s pos -inges ion. A e 30 h, only lobs e ed he esh ish dec eased amylase ac i i y in he gas ic juice (Fig. 1B). Acco dingly, he in e ac ion ime x die esul ed signi ican (Repea ed measu es ANOVA, F=2.32, P≤0.05). Amylase ac i i y was highe o he 6% CH die 2 h a e inges ion (Tuckey’s es , P≤0.05) (Fig. 1B). Time-cou se o glucose in gas ic juice and hemolymph a e eeding F ee glucose concen a ion in gas ic juice was a ec ed by die s (Repea ed measu es ANOVA, F=4.91, P≤0.05) bu no in e ac ion ime x die was ound (Repea ed measu es ANOVA, F=1.96, P>0.05). The main e ec ime had he majo impac on he libe a ion o glucose in o he gas ic juice (Repea ed measu es ANOVA, F=6.77, P≤0.001), wi h a peak 2 h a e inges ion (Fig. 2A). As expec ed, die s wi h 20 and 30% CH p oduced Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 8/21 Figu e 2 Glucose in gas ic juice (A) and hemolymph (B), o Panuli us a gus a e eeding. Die s we e named acco ding o he le el o CH hey con ained (6%, 20%, 35%), and a con ol wi h esh ish mus- cle. Each alue is he mean ±SEM (N=6 lobs e s pe die ). Di e ences among die s h oughou he 30 h s udied a e ma ked by di e en supe sc ip le e s in legend (P≤0.05). Fo each sampling ime, s a is i- cally di e en (Tukey’s es , P≤0.05) die a y ea men s a e indica ed by as e isks. Full-size DOI: 10.7717/pee j.3975/ ig-2 highe glucose le els han he 6% CH die and esh ish (Tuckey’s es , P≤0.05) (Fig. 2A). Howe e , 6 h a e inges ion, ee glucose le els in gas ic juice did no di e among ea men s. On he o he hand, he e we e no di e ences in hemolymph ee glucose le els due o he single main e ec die (Repea ed measu es ANOVA, F=2.91, P>0.05) (Fig. 2B). Howe e , signi ican a ia ion we e ound h ough ime (Repea ed measu es ANOVA, F=15.08, P≤0.001) and a signi ican in e ac ion ime x die was ound (Repea ed measu es ANOVA, F=2.03, P≤0.001). Concen a ion o glucose in hemolymph inc ease wi h maximal alues a 6 h ( o 6% and 20% CH die s and esh ish) o 12 h ( o 35% CH die ) a e inges ion (Fig. 2B). The concen a ion o glucose in he hemolymph 12 h a e inges ion was signi ican ly highe (one-way ANOVA, F=3.41, P≤0.05; Tuckey’s es , P≤0.05) in 35% CH ed lobs e s han in lobs e s ha inges ed he o he die s o esh ish. Me aboli es and me abolic enzymes in lobs e issues Twen y- ou hou s a e he inges ion o expe imen al die s and esh ish he concen a ion o glucose (one way ANOVAs, F=0.69, P>0.05), lac a e (F=2.51, P>0.05), and amino acids (F=0.42, P>0.05) in muscle did no a y among die a y ea men s, and di e ences we e ound in TG (F=16.54, P≤0.05) and glycogen con en s (F=3.76, P≤0.05). Highe glycogen con en in muscle was ound o he whea die a 35% (Table 3). In DG, di e ences we e ound among die a y ea men s in con en o glucose (F=8.88, P≤0.05), glycogen (F=3.90, P≤0.05), lac a e (F=3.51, P≤0.05), amino acid (F=3.22, P≤0.05), bu no in TG con en (F=0.28, P>0.05) (Table 3). The highes di e ence was obse ed be ween 6% and 35% CH die s, while he 20% CH die showed in e media e alues (Table 3). A his sampling ime, 24 h a e inges ion, no signi ican di e ences we e ound in he concen a ion o glucose (F=0.73, P>0.05), lac a e (F=0.41, P>0.05), and TG (F=0.29, P>0.05) in he hemolymph (Table 3). Di e ences we e ound in amino acid con en in he hemolymph (F=3.97, P≤0.05), wi h majo di e ences obse ed be ween lobs e s inges ing esh ish and he o mula ed die s (Tuckey’s es , P≤0.05) (Table 3). Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 9/21 b oaden his issue, as i may lie behind he inabili y o lobs e o con ol he in ensi y and ime-cou se o CHs diges ion when ed o mula ed die s. Gi en ha lobs e s a e no able o igh ly egula e α-amylase exp ession in esponse o a wide ange o die a y CH inclusion, high enzyme ac i i y in gas ic juice may ha e a isen as an adap a ion o die s wi h ew o mode a e CH con en . Howe e , e en when some an icipa o y esponse (acco ding o he usual CH con en in die ) in gas ic juice α-amylase ac i i y was obse ed in his s udy, his may be no sui ed o con ol diges ion o highly diges ible CHs o high CH o mula ed die s, likely because o uncon olled sec e ion o α-amylase in o he o egu du ing as ing and i s high ac i i y in he condi ions o he gas ic juice (Pe e a e al., 2008a;Pe e a e al., 2008b;Rod íguez-Vie a e al., 2016). ACKNOWLEDGEMENTS Special hanks o Láza o Macias o echnical suppo du ing expe imen s. ADDITIONAL INFORMATION AND DECLARATIONS Funding This wo k was pa ially suppo ed by Minis y o Economy and Compe i i eness h ough P ojec AGL2013-48835-C2-R and AGL2016-76069-C2-1-R g an ed o Juan M. Mance a, UCA-In e na ional ellowship g an ed o Leand o Rod iguez-Vie a, and AUIP g an in he amewo k o he Academic Mobili y P og am be ween Andalusian and Ibe o-Ame ican Uni e si ies associa ed o he AUIP g an ed o Leand o Rod íguez-Vie a. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . G an Disclosu es The ollowing g an in o ma ion was disclosed by he au ho s: Minis y o Economy and Compe i i eness: AGL2013-48835-C2-R, AGL2016-76069-C2- 1-R. AUIP g an . Compe ing In e es s The au ho s decla e he e a e no compe ing in e es s. Au ho Con ibu ions •Leand o Rod iguez-Vie a and E ick Pe e a concei ed and designed he expe imen s, pe o med he expe imen s, analyzed he da a, w o e he pape , p epa ed igu es and/o ables, e iewed d a s o he pape . •Vi ian Mon e o-Alejo and Rolando Pe domo-Mo ales pe o med he expe imen s, con ibu ed eagen s/ma e ials/analysis ools, e iewed d a s o he pape . •Tsai Ga cía-Galano pe o med he expe imen s, e iewed d a s o he pape . •Gonzalo Ma ínez-Rod íguez pe o med he expe imen s, analyzed he da a, con ibu ed eagen s/ma e ials/analysis ools, e iewed d a s o he pape . Rod íguez-Vie a e al. (2017), Pee J, DOI 10.7717/pee j.3975 16/21 •Juan M. Mance a concei ed and designed he expe imen s, analyzed he da a, con ibu ed eagen s/ma e ials/analysis ools, w o e he pape , e iewed d a s o he pape . Da a A ailabili y The ollowing in o ma ion was supplied ega ding da a a ailabili y: The aw da a has been uploaded as Supplemen a y Files. 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