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Changes in metabolic substrates during early development in anchoveta Engraulis ringens (Jenyns 1842) in the Humboldt Current

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We assessed the ontogenetic changes in protein content and free amino acids (FAA) in eggs and early larvae of Engraulis ringens (anchoveta) off central Chile on different dates during the spawning season. On all sampling dates, a reduction in
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  Mar Biol (2010) 157:1137–1149DOI 10.1007/s00227-010-1395-7  1 3 ORIGINAL PAPER Changes in metabolic substrates during early development in anchoveta  Engraulis ringens  (Jenyns 1842) in the Humboldt Current M. C. Krautz · S. Vásquez · L. R. Castro · M. González · A. Llanos-Rivera · S. Pantoja Received: 13 March 2009 / Accepted: 18 January 2010 / Published online: 11 February 2010 ©  Springer-Verlag 2010 Abstract We assessed the ontogenetic changes in proteincontent and free amino acids (FAA) in eggs and early lar-vae of  Engraulis ringens  (anchoveta) o V   central Chile ondi V  erent dates during the spawning season. On all samplingdates, a reduction in embryonic yolk-sac volume, proteinsand FAA concentrations occurred during development.Protein electrophoresis (SDS–PAGE) of eggs and larvaeshowed at least 22 protein bands: 11 were consumed earlyand not detected after hatching. The proportion of essentialFAA (EFAA) was higher than the proportion of non-essen-tial FAA (NEFAA) in early eggs and in 7day-old larvae(82.5-73% EFAA respectively). During egg development,the FAA pool was dominated by leucine, alanine andlysine, three amino acids contributing 35–44% of the totalFAA in eggs. During larval development, histidine was themost abundant FAA. In July, total FAA constituted 13–18% of the egg dry weight. A similar proportion (45–51%)occurred in July between protein plus FAA and total lipids.The di V  erences in egg size during the spawning seasonalong with variability in batch composition suggests thatthe female spawning condition is a major factor determin-ing egg quality and early o V  spring success. Introduction The anchoveta (  Engraulis ringens  Jenyns 1842) is a smallpelagic W sh endemic to the Humboldt Current that supportsone of the world’s most important W sheries and plays a keytrophodynamic role by transferring energy from primaryproduction up the food web. Anchoveta is distributed incoastal upwelling areas over a broad range in latitudes fromnorthern Peru (4°S) to southern Chile (42°S, Serra etal.1979). O V   central Chile, this iteroparous species distributesits eggs mainly between 0 and 50m depth within the 10nautical miles of the coast, exhibiting a main spawningpeak at the end of austral winter (July–August) and a sec-ond, more variable and less conspicuous one, at the end of summer (February–March) (Cubillos and Arancibia 1993). Recent studies on anchoveta early life stages have demon-strated that egg volume increases with latitude anddecreases during the spawning season (Castro etal. 2002; Llanos-Rivera and Castro 2004). The impact of di V  erencesin egg volume on larval size, yolk volume, and larval Communicated by M. A. Peck.M. C. Krautz ( & ) · S. Vásquez · L. R. Castro · A. Llanos-RiveraLaboratorio de Oceanografía Pesquera y Ecología Larval (LOPEL), Departamento de Oceanografía, Universidad de Concepción, Casilla 160-C, Concepción, Chilee-mail: ckrautz@udec.clM. C. KrautzPrograma de Doctorado en Oceanografía, Departamento de Oceanografía, Universidad de Concepción, Casilla 160-C, Concepción, Chile Present Address: S. VásquezInstituto de Investigación Pesquera (INPESCA), Casilla 350, Talcahuano, ChileL. R. Castro · S. PantojaDepartamento de Oceanografía y Centro de Investigación Oceanográ W ca en el Pací  W co Sur Oriental (COPAS), Universidad de Concepción, Concepción, ChileM. GonzálezDepartamento de Bioquímica Clínica e Inmunología, Facultad de Farmacia, Universidad de Concepción, Concepción, ChileA. Llanos-RiveraUnidad de Biotecnología Marina, Facultad de Ciencias Naturales y Oceanográ W cas, Universidad de Concepción, Concepción, Chile  1138Mar Biol (2010) 157:1137–1149  1 3 growth rates have also been documented (Llanos-Riveraand Castro 2004, 2006). Moreover, Castro etal. (2009) reported that egg lipid contents were correlated withchanges in hatching success during the spawning season.These combined observations suggest that energetic adjust-ments made by spawning females in response to environ-mental variability in key factors (e.g. prey availability/ quality, turbulence, and/or temperature) might result inchanges in the biochemical composition of the maternalreserves provided to the progeny.The anchoveta spawns transparent, ovoid, pelagic eggswithout any visible oil globules in the yolk. In this type of egg, nitrogenous metabolites (proteins and free aminoacids, FAA) have been considered quantitatively importantas metabolic fuels, mostly in the egg and during yolk saclarval development (Rønnestad etal. 1998). Several studies have also suggested that FAA may play an importance roleas osmolytes (Thorsen etal. 1996; Finn etal. 2002). Rønnestad etal. (1999) reported FAA concentrations in eggs ranging from 150 to 200mM, representing 50% of theyolk osmolality and 40–50% of the total amino acid pool.Other molecules such as proteins and carbohydrates havedensities of about 1.3g/cm 3  and act to decrease egg buoy-ancy in seawater (Craik and Harvey 1987). Dietary protein requirements decrease with age and size in teleosts, beinghighest during early larval development. Egg FAA aresrcinated from the partial hydrolysis of a fraction of vitel-logenin (lipovitellin, MW » 100kD), a glycophospholipo-protein produced by hormonal stimulation and incorporatedin oocytes by pinocytosis before oocyte hydration. Vitello-genin is a very high-density lipoprotein (VHDL) consti-tuted by 80% lipids and 20% proteins. Two-thirds of thelipids are phospholipids, and one-third corresponds to tria-cylglycerols (Carrillo etal. 2000).Lipids have been considered to be the main energeticsubstrate for biosynthesis and reproduction in marine W sh(Greene and Selivonchick 1987; Sargent etal. 1999). Lip- ids and water are the most signi W cant chemical componentsof W sh eggs, which have a density lower than sea water(Craik and Harvey 1987). Pelagic eggs are characterized by high water content ( » 90–92%) and moderate lipid ( » 15–30%) concentrations. According to Rønnestad etal. (1999), eggs without oil droplets, such as anchoveta eggs, meet30% of their energy requirements through the consumptionof triacylglycerols and phospholipids and the remaining70% from amino acid catabolism.In this study, we determined the biochemical composi-tion of early stages (eggs and larvae) of  E. ringens  collectedat two sites and at two times during the reproductive season(2004–2005) in a coastal area of the upwelling system inthe Humboldt Current o V   central Chile. Additionally, wereported results of an electrophoretic pro W le of eggs andlarvae obtained in experiments performed in 2003. Weassessed the changes in proteins and FAA occurring duringegg and early larval development, considering complemen-tary data on lipid and TAG concentrations in anchovetaeggs. Finally, we discuss the implications of these W ndingsfor anchoveta reproductive ecology. Methods Egg collections and experimental designExperiments were performed during the main spawningseasons from 2003 to 2005. Samples obtained in 2003(eggs and larvae) were used for electrophoresis proteinanalyses and those from 2004 and 2005 for total and yolk volumes of eggs and biochemical analyses (egg and larvaldry weight, water content, FAA, total proteins and lipids).All ichthyoplankton samples were collected in ColiumoBay (36.5°S, 72.9°W, Fig.1).In August 2003, samples were collected from the W eldby gentle oblique tows with a bongo net (300   m mesh,60cm diameter). Samples were rapidly transported (<2h)to the Dichato Marine Biology Station (University of Con-cepción), where the anchoveta eggs were separated andincubated in W ltered sea water at 12°C. During these exper-iments, samples eggs ( n =50) and young larvae (15–20)samples were taken from the incubators at di V  erent times.To simplify comparisons between experiments, eggs were Fig.1 Sampling area.  Black circle  shows  Engraulis ringens  eggs col-lection area located in Coliumo Bay (South-Central Chilean coast) 73.2 73.1 73 72.936.836.636.4Coliumo BayConcepcion BayTALCAHUANO PACIFICOCEAN Itata River ºS ºW Dichato  Mar Biol (2010) 157:1137–11491139  1 3 classi W ed into three groups according to their developmentstage: stage I eggs (no embryo), stage II (early embryo),and stage III (late embryo) corresponding to phases 1–3,4–7 ( » 52h), and 8–12 ( » 88h), respectively, of Moser andAlhstrom (1985), (see Fig.2). Newly hatched larvae were unfed and examined at six di V  erent ages (1, 3, 5, 7, 9, and11days post hatch). Samples were stored in vials at ¡ 20°Cuntil protein analysis by electrophoresis SDS–PAGE.Anchoveta eggs were also collected from plankton sam-ples obtained on 13 (batch I), 20 October 2004 (batches IIand III) and 27 July 2005 (batch IV). Stage I eggs wereeither immediately frozen at ¡ 20°C for biochemical analy-ses (50–100 eggs) or incubated at 12°C (sea surfacetemperature) in 5-L glass containers (100eggsL ¡ 1 ) in atemperature controlled room using a 12:12 light regime.Di V  erent numbers of replicates were incubated on eachdate, depending on eggs availability in plankton samples.Egg stages I, II, and III were sampled from batches I, II,and IV, while egg stage I and larval stages (early yolk saclarvae and larvae without yolk) were collected from batchIII.From batches I, II, and IV, 950 stage I eggs wereremoved for biochemical analyses. Two groups of 100 eggswere used for protein analysis, three groups of 50 eggs forfree amino acid analysis, two groups of 50 eggs for dryweight determination, and 100 eggs for volume estima-tions. Other two groups of 200 eggs were used for lipidanalysis. Because the low number of eggs, batch III (Octo-ber 2004) considered only FAA analysis. Additionally, dur-ing October 2004 (batches I and II), we obtained eggs inthree stages of egg development from the plankton sample.They were called “ W eld collected eggs natural conditions”(e.g. non-incubated conditions) and were only utilized fortotal protein determination. All eggs stored (incubated andnon-incubated conditions) for biochemical analyses wereplaced in vials and preserved at ¡ 20°C. Eggs selected tomake total and yolk volume determinations were preservedin 5% formaldehyde.In order to allow comparisons between eggs and larvaeand because we were not able to obtain dry weights foranchoveta larvae, the results from all analyses areexpressed (standardized) as  gg ¡ 1  wet tissue. The dryweight of eggs is mentioned in the “Results” and “Discus- sion” sections and in Table2. FAA results were also reported in nmolindividual ¡ 1  (eggs or larvae).  Egg volume Egg volume (mm 3 ) was estimated for preserved eggs,assuming a half-ellipsoid shape according to the formula:volume=4   abc/6 where a , b , and c  correspond to ellipseradii (Llanos-Rivera and Castro 2004). We also estimatedyolk volume (mm 3 ) in the three developmental stagesof eggs in batches I, II, and IV using the same function.Egg and yolk volume were measured using the softwareOPTIMAS 6.0. Water content  To determine wet weight, two groups of 50 anchoveta eggswere sorted, gently dried on absorbent paper, and weighedon an analytical balance ( § 0.0001g). The samples werethen dried at 60°C for 24h, and the dry weights were regis-tered. Proteins Anchoveta egg and larva samples were macerated in icecold PBS (Phosphate-Saline Bu V  er 10mM, pH 7.4) using aglass tissue homogenizer. Each tube was centrifuged at Fig.2 Stages of development of  Engraulis ringens  eggs considered inthis study. a  Stage I: eggs without embryo, less than 12h from spawn-ing. b  Stage II: eggs with early embryo, 12h to 2.2days from spawn-ing. c  Stage III: eggs with late embryo, from 2.2 to 3.7days fromspawning.  Bar   represents 0.1mm  1140Mar Biol (2010) 157:1137–1149  1 3 8,000rpm for 5min (4°C), and then the supernatant wasremoved and frozen at ¡ 20°C. Total W sh egg proteins werequanti W ed after Lowry etal. (1951). The absorbance of  each sample was measured in a spectrophotometer at490nm. Proteins were separated by SDS–PAGE electro-phoresis following Laemmli (1970). The same amount of  total protein (10   g of egg or larvae protein) was loadedwithin each lane of the gel, and protein bands were visual-ized with silver stain. Free amino acids Free amino acids (FAA) were determined in pooled sam-ples of 50 eggs or 20 larvae. FAA were identi W ed and quan-ti W ed using RP-HPLC after precolumn derivatization witho-phthaldialdehyde (OPA) and 2-mercaptoethanol (Lind-roth and Mopper (1979), as described by Pantoja and Lee (1999)). Fifteen amino acids were quanti W ed, 10 classi W edas essential FAA (EFAA) to W sh: leucine, valine, isoleu-cine, lysine, threonine, phenylalanine, arginine, histidine,methionine, and tyrosine; and W ve were classi W ed as non-essential FAA (NEFAA): aspartic acid, glutamic acid,serine, glycine, and alanine (Wilson 1985).  Lipids Egg samples were macerated in PBS 10mM (pH 7.4) usinga glass tissue homogenizer. Macerated eggs were trans-ferred to 10-mL glass tubes for analyses. Lipid extractionwas performed after Bligh and Dyer (1959). After addingthe solvents, tubes were centrifuged at 3,000rpm for 5minto separate phases. The chloroform phase was transferred topreviously weighted glass vials. Samples were W ltratedthrough glass wool to remove any impurities. Glass vialswere placed over a thermo block at 36°C to avoid the accu-mulation of humidity. Chloroform was completely evapo-rated using N 2  (g). The vials were cooled on silica gel toavoid humidity and weighed on an analytic balance( § 0.0001g). The triacylglycerols (TAGs) were determinedusing an enzymatic kit TG PAP 150 (bioMérieux), and ana-lyzing aliquots of 10–20   l of dry lipids extracts reconsti-tuted with 300   L isopropanol.All statistical analyses were performed using the freestatistical software PAST (Hammer etal. 2001). Results Egg volume and yolk consumptionThe egg yolk volume was estimated in batches I and II(October 2004) and batch IV (July 2005, Fig.3). Weobserved signi W cant di V  erences in mean egg volume (one-way ANOVA P <0.05) between batches I, II (October2004), and IV (July 2005, Table1). Batch IV showed highermean volume (0.369mm 3 , SD 0.046) than October batches Iand II. In egg stage I, mean yolk volume was signi W cantlydi V  erent between batches I and II and also between batches Iand IV ( P <0.05, one-way ANOVA). No signi W cant di V  er-ences ( P >0.05) were observed between batches II (October2004) and IV (July 2005). Within egg batches I and II, yolk volume between egg stages I and II signi W cant decreased (by39 and 45%, respectively) between egg stages I and II( P <0.001, one-way ANOVA), and the yolk volume reduc-tion in stage III eggs was 65 and 69% (batches I and II,respectively). In batch IV, it was possible to obtain eggs onlyat stages I and II, in which we observed the same decreasingtrend between stages (56%).Water contentWater content in the eggs was similar among batches. Inbatch I (October 2004), water content ranged from 90.4 Fig.3 Changes in  Engraulis ringens  egg yolk volume (mm 3 ) duringembryonary development. Gray bar   batch I (126 determinations, Octo-ber 2004), white bar   batch II (74 determinations, October 2004) and white dashed bar   batch IV (407 determinations, July 2005) 00.050.10.150.20.25Sta g e ISta g e IISta g e III E gg  sta g e    E      g     g    y  o   l   k  v  o   l      u   m  e   (  m  m     3    ) Table1 Variation of egg volume (mm 3 ) during development of anchoveta  Engraulis ringens Number between parentheses corresponds to number of determina-tionsEgg stageEgg volume (mm 3 )October 2004July 2005Batch IBatch IIBatch IVAverageSDAverageSDAverageSDStage I0.355 (100)0.0290.326 (52)0.0360.370 (204)0.046Stage II0.344 (100)0.0320.331 (50)0.0350.348 (214)0.050Stage III0.330 (81)0.0440.320 (81)0.042––  Mar Biol (2010) 157:1137–11491141  1 3 (stage II) to 89.7% (stage III), whereas in batch II X uctuatedbetween 92.1% (egg stage I) and 88.4% (egg stage III).During July 2005 (batch IV), water content ranged from89.9 (stage I) to 90.4% (stage III) (Table2).ProteinsFrom egg stages I–II, mean ( § SD) protein content inbatches I, II, and IV decreased from 29.4 ( § 3.2) to 18.9( § 4.4), 35.6( § 3.7) to 23.4( § 0.1), egg stage III), and 31.9( § 5.5) to 30.2 ( § 2.4)  gmg wet tissue ¡ 1 , respectively(Table2). Incubated (laboratory) and W eld collected eggs(not incubated) exhibited very similar protein contents(Table2). Mean protein concentrations in batches I and IIwere between 30.9 ( § 3.4) and 37.3 ( § 3.9)   gmg ¡ 1  in eggstage I and 19.9 ( § 4.9) and 24.6 ( § 0.1)   gmg ¡ 1  in eggstage III, respectively.Electrophoresis SDS–PAGEAt least 22 silver-stained bands were detected after proteinelectrophoresis with some bands rapidly disappearing dur-ing anchoveta egg and larval development (Fig.4). The W rstgroup includes several bands that are consumed during thedevelopment of the egg stage. During egg stage I, the bandscorresponded to: 56.8, 35, and 12.4kD; during egg stages Iand II: » 175 and 30.7kD; and during egg stages I–III:58.7, 51.6, 44.6, 42.5, 38.5, and 19.5kD. A second groupshowed decreased concentrations from stage I eggs to3-day-old larvae (21.5kD and a band<6.5kD). A thirdgroup of bands (66.8, 60.6, 25.3, 23.3, 17.2kD) decreaseduntil 5-day-old larvae, after this point (complete yolk absorption), they were not visible. A high molecular weightband (>175kD) occurred only in 3- and 5-day-old larvae.The bands in a fourth group (76, 69, 49.9kD) decreasedin concentrations from stage I eggs to 11-day-old unfedlarvae.FAAIn batches I, II, and III (October 2004), mean ( § SD) totalFAA contents in stage I eggs were 13.0 ( § 4.84), 18.1( § 1.95), and 11.5 ( § 1.8)   gg ¡ 1  wet tissue and 13.1 Table2 Changes in water content, total proteins, total lipids and triacylglycerides during development of anchoveta eggs in batches I, II (October2004) and IV (July 2005) ( W eld and incubations data)Numbers between parentheses correspond to number of samples. All data from stages I (initial conditions for all experiments) was obtained from W eld samples. All other data was obtained from rearing experiments (unless indicated; i.e. total proteins)Month/batchEgg stage% Water (mean)SDDry weight (  g)SDTotal protein  gmg ¡ 1  wet tissue(experimental conditions)SDTotal protein  gmg ¡ 1  wet tissue ( W eld collected eggs natural conditions)SDTotal lipids  gmg ¡ 1  wet tissueSDTAGs  gmg ¡ 1  wet tissueSDOctober 2004Batch IEgg I29.4 (2)3.230.9 (2)3.4––Egg II90.4 (2)0.331 (2)1.426.0 (2)3.127.3 (2)3.2––Egg III89.7 (2)0.930 (2)2.818.9 (2)4.419.9 (2)4.7––Batch IIEgg I92.1 (2)5.9434 (3)8.635.6 (2)3.737.3 (2)3.9––Egg II90.3 (1)32 (2)4.730.2 (4)5.033.6 (2)8.0––Egg III88.4 (1)22 (1)23.4 (2)0.124.6 (2)0.1––July 2005Batch IVEgg I89.9 (3)0.635 (3)1.231.9 (2)5.544.7 (2)14.34.6 (2)0.2Egg II90.2 (2)0.133 (2)1.418.0 (2)5.942.4 (2)15.43.7 (2)0.7Egg III90.4 (3)0.533 (3)2.330.2 (2)2.422.0 (2)10.73.5 (2)1.1 Fig.4 Changes in anchoveta eggs proteins during egg and larvaedevelopment detected by SDS PAGE. Experiment was carried out inAugust 2003. Protein bands were visualized with silver stain.  MWM  molecular weight marker,  E I  –  III   eggs on stage I, I and III, 1 - 5 dL  yolk sac larvae (1, 3 and 5days age), 7  - 11 dL  larvae in starvation (7, 9 and11days age) 175 (kD) 83 62 47.5 32.5 25 16.5 6.5 MWME-IE-IIE-III1dL3dL5dL7dL9dL11dL

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