Mar Biol (2010) 157:1137–1149

DOI 10.1007/s00227-010-1395-7

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) oV central Chile ondiVerent 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 essential

FAA (EFAA) was higher than the proportion of non-essen-tial FAA (NEFAA) in early eggs and in 7 day-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 diVerences 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 oVspring success.

Introduction

The anchoveta (Engraulis ringens Jenyns 1842) is a smallpelagic Wsh endemic to the Humboldt Current that supportsone of the world’s most important Wsheries 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 et al.1979). OV central Chile, this iteroparous species distributesits eggs mainly between 0 and 50 m 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 ofsummer (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 et al. 2002;Llanos-Rivera and Castro 2004). The impact of diVerencesin 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.cl

M. 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, Chile

L. R. Castro · S. PantojaDepartamento de Oceanografía y Centro de Investigación OceanográWca en el PacíWco Sur Oriental (COPAS), Universidad de Concepción, Concepción, Chile

M. GonzálezDepartamento de Bioquímica Clínica e Inmunología, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile

A. Llanos-RiveraUnidad de Biotecnología Marina, Facultad de Ciencias Naturales y OceanográWcas, Universidad de Concepción, Concepción, Chile

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1138 Mar Biol (2010) 157:1137–1149

growth rates have also been documented (Llanos-Riveraand Castro 2004, 2006). Moreover, Castro et al. (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 ofegg, nitrogenous metabolites (proteins and free aminoacids, FAA) have been considered quantitatively importantas metabolic fuels, mostly in the egg and during yolk saclarval development (Rønnestad et al. 1998). Several studieshave also suggested that FAA may play an importance roleas osmolytes (Thorsen et al. 1996; Finn et al. 2002).Rønnestad et al. (1999) reported FAA concentrations ineggs ranging from 150 to 200 mM, representing 50% of theyolk osmolality and 40–50% of the total amino acid pool.Other molecules such as proteins and carbohydrates havedensities of about 1.3 g/cm3 and act to decrease egg buoy-ancy in seawater (Craik and Harvey 1987). Dietary proteinrequirements decrease with age and size in teleosts, beinghighest during early larval development. Egg FAA areoriginated from the partial hydrolysis of a fraction of vitel-logenin (lipovitellin, MW » 100 kD), 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 et al. 2000).

Lipids have been considered to be the main energeticsubstrate for biosynthesis and reproduction in marine Wsh(Greene and Selivonchick 1987; Sargent et al. 1999). Lip-ids and water are the most signiWcant chemical componentsof Wsh eggs, which have a density lower than sea water(Craik and Harvey 1987). Pelagic eggs are characterized byhigh water content (»90–92%) and moderate lipid (»15–30%) concentrations. According to Rønnestad et al. (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 oV central Chile. Additionally, wereported results of an electrophoretic proWle of eggs andlarvae obtained in experiments performed in 2003. We

assessed 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 Wndingsfor anchoveta reproductive ecology.

Methods

Egg collections and experimental design

Experiments 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 yolkvolumes 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 Weldby gentle oblique tows with a bongo net (300 �m mesh,60 cm diameter). Samples were rapidly transported (<2 h)to the Dichato Marine Biology Station (University of Con-cepción), where the anchoveta eggs were separated andincubated in Wltered 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 diVerent 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 72.936.8

36.6

36.4

Coliumo Bay

Concepcion Bay

TALCAHUANO

PACIFIC OCEAN

Itata River

ºS

ºW

Dichato

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Mar Biol (2010) 157:1137–1149 1139

classiWed 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 (»52 h), and 8–12 (»88 h), respectively, of Moser andAlhstrom (1985), (see Fig. 2). Newly hatched larvae wereunfed and examined at six diVerent ages (1, 3, 5, 7, 9, and11 days 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 (100 eggs L¡1) in atemperature controlled room using a 12:12 light regime.DiVerent 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 “Weld 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 were

placed 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 �g g¡1 wet tissue. The dryweight of eggs is mentioned in the “Results” and “Discus-sion” sections and in Table 2. FAA results were alsoreported in nmol individual¡1 (eggs or larvae).

Egg volume

Egg volume (mm3) 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 (mm3) 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 (§0.0001 g). The samples werethen dried at 60°C for 24 h, and the dry weights were regis-tered.

Proteins

Anchoveta egg and larva samples were macerated in icecold PBS (Phosphate-Saline BuVer 10 mM, 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 12 h from spawn-ing. b Stage II: eggs with early embryo, 12 h to 2.2 days from spawn-

ing. c Stage III: eggs with late embryo, from 2.2 to 3.7 days fromspawning. Bar represents 0.1 mm

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1140 Mar Biol (2010) 157:1137–1149

8,000 rpm for 5 min (4°C), and then the supernatant wasremoved and frozen at ¡20°C. Total Wsh egg proteins werequantiWed after Lowry et al. (1951). The absorbance ofeach sample was measured in a spectrophotometer at490 nm. Proteins were separated by SDS–PAGE electro-phoresis following Laemmli (1970). The same amount oftotal 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 identiWed and quan-tiWed 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 quantiWed, 10 classiWedas essential FAA (EFAA) to Wsh: leucine, valine, isoleu-cine, lysine, threonine, phenylalanine, arginine, histidine,methionine, and tyrosine; and Wve were classiWed as non-essential FAA (NEFAA): aspartic acid, glutamic acid,serine, glycine, and alanine (Wilson 1985).

Lipids

Egg samples were macerated in PBS 10 mM (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 3,000 rpm for 5 minto separate phases. The chloroform phase was transferred topreviously weighted glass vials. Samples were Wltratedthrough 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 N2 (g). The vials were cooled on silica gel toavoid humidity and weighed on an analytic balance(§0.0001 g). 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 et al. 2001).

Results

Egg volume and yolk consumption

The egg yolk volume was estimated in batches I and II(October 2004) and batch IV (July 2005, Fig. 3). Weobserved signiWcant diVerences in mean egg volume (one-

way ANOVA P < 0.05) between batches I, II (October2004), and IV (July 2005, Table 1). Batch IV showed highermean volume (0.369 mm3, SD 0.046) than October batches Iand II. In egg stage I, mean yolk volume was signiWcantlydiVerent between batches I and II and also between batches Iand IV (P < 0.05, one-way ANOVA). No signiWcant diVer-ences (P > 0.05) were observed between batches II (October2004) and IV (July 2005). Within egg batches I and II, yolkvolume between egg stages I and II signiWcant 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 content

Water 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 (mm3) duringembryonary development. Gray bar batch I (126 determinations, Octo-ber 2004), white bar batch II (74 determinations, October 2004) andwhite dashed bar batch IV (407 determinations, July 2005)

0

0.05

0.1

0.15

0.2

0.25

Stage I Stage II Stage III

Egg stage

Egg

yol

k vo

lum

e (m

m 3 )

Table 1 Variation of egg volume (mm3) during development ofanchoveta Engraulis ringens

Number between parentheses corresponds to number of determina-tions

Egg stage

Egg volume (mm3)

October 2004 July 2005

Batch I Batch II Batch IV

Average SD Average SD Average SD

Stage I 0.355 (100) 0.029 0.326 (52) 0.036 0.370 (204) 0.046

Stage II 0.344 (100) 0.032 0.331 (50) 0.035 0.348 (214) 0.050

Stage III 0.330 (81) 0.044 0.320 (81) 0.042 – –

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Mar Biol (2010) 157:1137–1149 1141

(stage II) to 89.7% (stage III), whereas in batch II Xuctuatedbetween 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) (Table 2).

Proteins

From 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) �g mg wet tissue¡1, respectively(Table 2). Incubated (laboratory) and Weld collected eggs(not incubated) exhibited very similar protein contents(Table 2). Mean protein concentrations in batches I and IIwere between 30.9 (§3.4) and 37.3 (§3.9) �g mg¡1 in eggstage I and 19.9 (§4.9) and 24.6 (§0.1) �g mg¡1 in eggstage III, respectively.

Electrophoresis SDS–PAGE

At least 22 silver-stained bands were detected after proteinelectrophoresis with some bands rapidly disappearing dur-ing anchoveta egg and larval development (Fig. 4). The Wrstgroup includes several bands that are consumed during thedevelopment of the egg stage. During egg stage I, the bandscorresponded to: 56.8, 35, and 12.4 kD; during egg stages Iand II: »175 and 30.7 kD; and during egg stages I–III:58.7, 51.6, 44.6, 42.5, 38.5, and 19.5 kD. A second groupshowed decreased concentrations from stage I eggs to3-day-old larvae (21.5 kD and a band < 6.5 kD). A thirdgroup of bands (66.8, 60.6, 25.3, 23.3, 17.2 kD) decreased

until 5-day-old larvae, after this point (complete yolkabsorption), they were not visible. A high molecular weightband (>175 kD) occurred only in 3- and 5-day-old larvae.The bands in a fourth group (76, 69, 49.9 kD) decreasedin concentrations from stage I eggs to 11-day-old unfedlarvae.

FAA

In 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) �g g¡1 wet tissue and 13.1

Table 2 Changes in water content, total proteins, total lipids and triacylglycerides during development of anchoveta eggs in batches I, II (October2004) and IV (July 2005) (Weld and incubations data)

Numbers between parentheses correspond to number of samples. All data from stages I (initial conditions for all experiments) was obtained fromWeld samples. All other data was obtained from rearing experiments (unless indicated; i.e. total proteins)

Month/batch Egg stage

% Water (mean)

SD Dry weight (�g)

SD Total protein �g mg¡1 wet tissue(experimental conditions)

SD Total protein �g mg¡1 wet tissue (Weld collected eggs natural conditions)

SD Total lipids �g mg¡1 wet tissue

SD TAGs �g mg¡1 wet tissue

SD

October 2004

Batch I Egg I 29.4 (2) 3.2 30.9 (2) 3.4 – –

Egg II 90.4 (2) 0.3 31 (2) 1.4 26.0 (2) 3.1 27.3 (2) 3.2 – –

Egg III 89.7 (2) 0.9 30 (2) 2.8 18.9 (2) 4.4 19.9 (2) 4.7 – –

Batch II Egg I 92.1 (2) 5.94 34 (3) 8.6 35.6 (2) 3.7 37.3 (2) 3.9 – –

Egg II 90.3 (1) 32 (2) 4.7 30.2 (4) 5.0 33.6 (2) 8.0 – –

Egg III 88.4 (1) 22 (1) 23.4 (2) 0.1 24.6 (2) 0.1 – –

July 2005

Batch IV Egg I 89.9 (3) 0.6 35 (3) 1.2 31.9 (2) 5.5 44.7 (2) 14.3 4.6 (2) 0.2

Egg II 90.2 (2) 0.1 33 (2) 1.4 18.0 (2) 5.9 42.4 (2) 15.4 3.7 (2) 0.7

Egg III 90.4 (3) 0.5 33 (3) 2.3 30.2 (2) 2.4 22.0 (2) 10.7 3.5 (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. MWMmolecular weight marker, E I–III eggs on stage I, I and III, 1-5 dL yolksac larvae (1, 3 and 5 days age), 7-11 dL larvae in starvation (7, 9 and11 days age)

175

(kD)

83 62

47.5

32.5

25

16.5

6.5

MWM E-I E-II E-III 1dL 3dL 5dL 7dL 9dL 11dL

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1142 Mar Biol (2010) 157:1137–1149

(§ 3.37) �g g¡1 wet tissue in July 2005 (batch IV). Thetotal FAA constituted from ca. 15.1% (batch I, estimatedusing the dry weight obtained in egg stage II), 18% (batchII), and 13.6% (batch IV) of the dry weight of the ancho-veta eggs. Equivalence in nmol egg¡1 or larvae¡1 is shownin Table 3. SigniWcant diVerences in FAA contents of stageI eggs were recorded (P < 0.05, Kruskal–Wallis test)between batches I and II, and between batches II and IV.No signiWcant diVerences in FAA contents of stage I eggswere detected between batches I and IV. All experimentsshowed decreasing FAA concentrations from egg stagesI–III. In batch I, this trend was maintained until stage III(a reduction of 40–46% of the FAA contained in stage Ieggs). On batch II, the reduction from stage I to II wasaround 25% FAA, but a slight increase occurred from stageII to III. Batch IV showed a decreasing trend similar tobatch I (Table 3).

Details of the changes in FAA concentrations in ancho-veta eggs are shown in Fig. 5. All batches showed reduc-tions in serine, glycine, arginine, alanine, methionine,valine, isoleucine, and leucine with egg development. Therest of the FAA did not show a clear trend with develop-ment.

Of the 15 FAA quantiWed, three amino acids (lysine, leu-cine, alanine) constituted between 35 and 44% of the totalFAA in the three egg development trials. The EFAA lysinehad the highest concentrations in batches I and II. Lysineconcentration in the batches I and II (2004) was 2.68 and2.94 �g g¡1 (6.73 and 8.51 nmol egg¡1) and 1.53 �g g¡1

(3.73 nmol egg¡1) in batch I. In July 2005 (batch IV),another EFAA, histidine, had the highest concentration of2.03 �g g¡1 (4.6 nmol egg¡1).

We observed decreasing concentrations of EFAA as wellas NEFAA with egg development. For eggs in stage I, theEFAA made up 73.8% (batch I), 68% (batch II), and 71%(molar, batch III) of the total FAA. In batches I and II,the EFAA concentration decreased from 27.3 to33.3 nmol egg¡1 (stage I) to 15.2–27 nmol egg¡1 (stage III)respectively. In batch IV, this trend was similar; a decreasewas observed from stage I (25.7 nmol egg¡1) to stage III(15.9 nmol egg¡1). NEFAA showed higher decreases(around 50%) than EFAA in all egg development incuba-tions (Fig. 6).

Changes in the FAA concentration occurred throughoutlarval development (Fig. 7, batch III) with 13 of 15 mea-sured FAA decreasing with larval development. Total con-centration of FAA showed the same decreasing trend(nmol ind¡1, Table 3). Only aspartic and glutamic acid con-centrations increased from egg to larval stages. Threonineshowed a clear decrease from egg to the yolk sac stage andan increase after that. For egg stages and yolk sac larvae,the most important FAA were histidine (11.0 and 9.6 mol%total FAA respectively), alanine (12.7 and 8.0 nmol %), andleucine (11.6 and 14.3 nmol %); whereas aspartic acidshowed the lowest concentration (0.21 mol% in egg phaseand 2.21 mol% in yolk sac stage). In 7-day-old larvae, themost concentrated FAA was threonine (15.1 nmol%),followed by histidine (12.4 nmol%) and glutamic acid(9.4 nmol%). Methionine showed the lowest concentration(0.79 nmol%).

Figure 8 shows the EFAA and NEFAA concentrationsfrom the larval development experiment (batch III,October 2004). EFAA concentrations were higher in allstages (8.77 �g g¡1 equivalents to 82.5% in eggs and3.73 �g g¡1 equivalents to 73% of total FAA in the oldestlarval stage). Unlike EFAA, NEFAA concentrationsincreased slightly from eggs to yolk sac larvae, followedby a noticeable consumption until reaching an older larvalstage.

Lipids

A decreasing trend was observed in lipid concentrationsduring the egg development in batch IV (July 2005). Eggsin this batch had a lipid concentration between 44.7 and22 �g mg wet weight¡1 (Table 2). Triacylglycerols (TAG)exhibited the same decreasing trend as total lipids. In batchIV, the stage I eggs had a mean (§SD) concentration of 4.6(§ 0.3) �g mg¡1 which decreased to 3.4 (§1.1) �g mg¡1 instage III eggs (Table 2). The percentage of total lipids con-stituted by TAG was 10.3, 8.7, and 12.6% in egg stages I,II, and III, respectively.

Table 3 Changes in total free aminoacids in anchoveta eggs andlarvae

Batches I, II and III correspond to October 2004, batch IV correspondsto July of 2005. YSL: yolk sac larvae

Batch/month Stage FAA �g mgwet weight¡1

FAA nmol ind¡1

Mean SD Mean SD

October 2004

Batch I Egg I 13.0 4.8 36.9 10.6

Egg II 8.8 1.0 27.9 3.4

Egg III 7.8 1.1 19.7 3.0

Batch II Egg I 18.1 1.9 49.1 4.5

Egg II 12.6 2.6 31.3 6.6

Egg III 14.7 2.4 37.6 7.1

Batch III Egg 11.5 1.8 31.1 5.5

YSL 1d 12.1 2.9 12.4 3.0

Larvae 7d 4.6 0.5 9.0 1.4

July 2005

Batch IV Egg I 13.1 3.4 36.2 10.1

Egg II 11.7 1.8 32.1 5.1

Egg III 8.0 0.9 21.4 2.6

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Mar Biol (2010) 157:1137–1149 1143

Fig. 5 Changes in FAA concentration (nmol egg¡1) during anchoveta egg development. Black bar batch I (October 2004), gray bar batch II(October 2004), white dashed bar batch IV (July 2005)

EFAA

NEFAA

0

1

2

3

4

5

nmol

FA

A e

gg-1

Arginine

0

1

2

3

4

5

nmol

FA

A e

gg-1

Arginine

0

1

2

3

4

5Tyrosine

0

1

2

3

4

5

nmol

FA

A e

gg-1

Methionine

0

1

2

3

4

5Phenilalanyne

0

1

2

3

4

5

6

7Isoleucine

0

1

2

3

4

5

6

7Leucine

0

2

4

6

8

10

12

Egg I Egg II Egg III

nmol

FA

A e

gg-1

Lysine

0

1

2

3

4

5Valine

0

1

2

3

4

5Histidine

0

0.2

0.4

0.6

0.8Aspartic acid

0

0.5

1

1.5

2

nmol

FA

A e

gg-1 Glutamic acid

0

1

2

3

4Serine

0

1

2

3

4Glycine

0

2

4

6

8

10

nmol

FA

A e

gg-1

Alanine

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

Egg I Egg II Egg III

nmol

FA

A e

gg-1

123

1144 Mar Biol (2010) 157:1137–1149

Substrate comparison

During October 2004, we were not able to obtain enougheggs to determine lipid concentration. In batch IV (July2005), the sum of FAA and proteins was almost equivalentto the total lipid concentration in all egg developmentstages. In egg stages I and II, lipids were the most concen-trated substrate available for egg development (�g mg¡1).In stage III, proteins were the most important. Lipids, pro-teins, and FAA were consumed throughout egg develop-ment, but FAA and lipid consumption were the highest.

Discussion

Anchoveta is a small pelagic Wsh that plays a key role in theHumboldt Current upwelling ecosystem. In central Chile,this species spawns in coastal zones associated withupwelling centers, where eggs and larvae can occur in highabundance. The water temperature during the main spawn-ing and larval growth periods varies between 11 and 12°Cin winter (July–September) and 14°C at the end of spring

(Castro et al. 2002). Adult females and their larvae face avariable, complex, and sometimes (i.e. in mid-winter) harshenvironment. To enhance survival until the time of Wrstfeeding, females should provide eggs and young larvaewith suYcient energy reserves. In our study, we measuredkey physical (e.g., size, yolk volume, % water) and bio-chemical (e.g., proteins, FAA, TAG) attributes of ancho-veta eggs and report on inter-batch variability (within andbetween collection dates) and developmental changes thatoccurred through hatching and prior to Wrst feeding.

Inter-annual and inter-seasonal variability in mean eggsize can result from diVerences in a variety of factorsincluding female size and/or metabolic adjustments chang-ing the relationship between somatic growth and reproduc-tive output (Wootton 1990). OV central Chile, anchovetaegg size has been reported to decrease with increasing timeduring the main spawning period and exhibit inter-annualvariability (Llanos-Rivera and Castro 2004). More recently(2003 and 2004) and during a longer period that includesthe months assessed in our study, mean egg volume alsodecreased during the main spawning season (Castro et al.2009). In the present study, diVerences were observed in

Fig. 6 Changes in total FAA concentration in amino acids during egg development. a, b Correspond to batch I and c, d correspond to batch II from October 2004. e, f Correspond to batch IV from July 2005. Amino acids were grouped in essential (EFAA, gray bars) and non-essential amino acids (NEFAA, white bars). Left plots EFAA and EFAA (nmol egg¡1), right plots percentage of total amino acids which represent EFAA or NEFAA

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Fig. 7 Changes in FAA concentration (nmol individual¡1) in anchoveta larval development (batch III, October 2004)

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egg and yolk volume among batches spawned within thesame month as well as among diVerent months. DiVerenceswere detected in egg volume between batches I and II(October 2004) and batch IV (July 2005). These diVerencesmight have resulted from changes in the size (age) structureof spawning females during the season or changes ingonadal condition related to increasing egg batch numberduring the spawning season. In terms of reductions in yolkoccurring during egg development, changes observed here(56–65% between egg stages I–III at 12°C) are similar tothe value (40%) reported by Finn et al. (1996) turbot(Scophthalmus maximus L.) at 15°C. In that study, totalyolk absorption occurred in larvae at 8 days after hatch.Llanos-Rivera and Castro (2006) reported complete ancho-veta larval yolk depletion (and eye pigmentation) at5.22 days post hatch (12°C).

The pelagic eggs of marine teleosts are characterized byhigh water content, and our results for anchoveta (89.9–92.1%) agree well with results of studies on eggs of otherspecies. For example, mean (§SD) water contents rangingfrom 90.8 (§1.6) to 93.0 (§0.5)% have been reported forspecies such as sole (Solea solea), halibut (Hippoglossushippoglossus), turbot (Scophthalmus maximus L.), haddock(Melanogrammus aegleWnus), and plaice (Pleuronectesplatessa) (Craik and Harvey 1987; Skjærven et al. 2003).Similar to the present study, Finn et al. (1995a) alsoreported changes in the percentage water content of Atlan-tic cod (Gadus morhua) eggs between early fertilization(92.9%) and just prior to hatching (93.1%). Ecologically,these changes in water content could impact buoyancy andthe vertical position of eggs in the water column, poten-tially altering advective transport and retention in nurseryareas or exposure to the oxygen minimum layer, whichis very shallow in the late austral spring in the HumboldtCurrent.

Proteins constituted »30–35% of the dry weight in stageI anchoveta eggs in all sampled dates. A trend of a decreasein protein concentration with egg development (expressedas wet weight) occurred in all batches (less marked in batch

IV, Table 2) and was observed in both incubated samplesand Weld collected batches. The results of the electrophore-sis SDS–PAGE are consistent with this decrease in proteinconcentration, which resulted from protein and peptidehydrolysis. A catabolism based on proteins and FAA ischaracteristic in eggs without oil drops in the egg yolk.Thorsen et al. (1996) reported the presence of at least Wvedense bands (14, 27, 51, 64, 72 kDa) in vitellogenic oocytesof brackish and marine water cod very similar to those thatwe observed in the anchoveta gels. In our gels, most ofthese bands disappeared during egg development (e.g., low-weight bands in stage I eggs, or stages I and II eggs, gradualdecline in the group of bands <58.7 kD prior to hatching).Previous studies of anchoveta eggs also reported ontoge-netic changes (Krautz et al. 2003), but changes were not aspronounced compared to the present study. In the presentstudy, we detected the presence of two high weight bands(»175 and <175 kD) visible in the early egg (stages I andII) and in two larval stages that could be attributed to amonomeric form of vitellogenin (Bailey et al. 2002). Work-ing with sea bream (Pagrus major), Sawaguchi et al. (2006)reported the presence of a 182-kD bands in vitellogenic fol-licle homogenates and presumed that these could be attrib-uted to intact monomeric vitellogenin A or B. Similarbands have been reported in the serum of rainbow trout(Oncorhynchus mykiss) by Babin (1987) and also in theoocyte stage and serum of pollock (Theragra chalco-gramma) by Bailey et al. (2002). Additionally, we detectedtwo bands of 76 and 23.3 kD, very similar to the molecularweight (73 and 23 kD) reported by Ohkubo et al. (2006) forwalleye pollock eggs and yolk sac homogenates. The originof these bands appears related to the cleavage of a mainband (95 kD) that is highly similar to the heavy chain oflipovitellin B found in haddock (Reith et al. 2001). Twolow-weight bands (30.7 kD, detected from egg stage I toegg stage II and 21.5 kD, detected from egg stage I to3 days larvae) were comparable to bands of 30 and 21.5 kDattributed to light chain of mullet (Mugilidae) vitellogeninA and C, whereas a band of 25.3 kD is similar to a band of

Fig. 8 Changes in total FAA concentration from egg stage (I) throughlarval development (batch III, October 2004). Amino acids weregrouped in essential (EFAA, gray bars) and non-essential amino acids

(NEFAA, white bars). Left plot EFAA and EFAA (nmol egg¡1), rightplot percentage of total amino acids which represent EFAA or NEFAA

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25 kD attributed to light-chain lipovitellin B of ovulatedeggs of red seabream (Sawaguchi et al. 2006).

In the present study, FAA constituted 13.6–18% ofanchoveta eggs dry weight, values that are comparable tothe value (12%) reported by Rønnestad et al. (1999) forDicentrarchus labrax eggs and larvae and is close to that(25%) measured shortly after fertilization in Atlantic codeggs (Finn et al. 1995a). In our study, FAA and proteinstogether accounted for 48% of the suitable substrates forembryo development in batches II and IV. According toFinn et al. (1995b), in Wsh eggs without oil drops (such asanchoveta), FAA and proteins meet at least half of theenergy demands in egg development. In addition, FAAcould act as important regulators of pelagic egg buoyancy(Craik and Harvey 1987). We observed high variability inFAA consumption prior to hatching, with »46.7 and 40%in batches I and IV and »22.6% in batch II. During larvaldevelopment (batch III, larva 7 days), there was a consump-tion of 70% of the FAA contained in stage I eggs. During asimilar developmental period (5 days post-fertilization tohatching), Ohkubo et al. (2006) reported 28% consumptionof the initial FAA content in pollock eggs. Rønnestad et al.(1992) reported 65% consumption of the FAA pool beforehatching in turbot eggs and about 60% (53 nmol ind¡1) inEuropean sea bass (Dicentrarchus labrax) eggs (Rønnestadet al. 1998).

As in other pelagic eggs, the most concentrated FAA inanchoveta eggs were mainly neutral or positively chargedamino acids. Most showed rapid consumption during theegg phase. Essential amino acids showed the highest con-centrations (about 70% of total amino acids, expressed innmol egg¡1) but lower consumption than NEFAA. Theseresults were slightly higher than those recorded for Gadusmorhua eggs by Finn et al. (1995a). In Atlantic cod, EFAArepresent 58% of the total FAA and showed little variationbefore hatching. After hatching, EFAA showed rapid con-sumption. In our anchoveta study, EFAA reduction duringegg development before hatching was between 19 and44.4%, and the NEFAA reduction was 33.1 and 52.5%. Incod, NEFAA consumption was about 20% before hatchingand remained almost constant during larval development.

During anchoveta egg development, the most concen-trated EFAA were lysine and leucine, whereas alanine wasthe most concentrated NEFAA. These three amino acidsaccounted for between 35 and 45% of the FAA pool (nmol)and, together with glycine and valine, total more than 51%of the FAA pool. These results are similar with thoseobserved by Matsubara and Koya (1997) in barWn Xounder(Verasper moseri) and Rønnestad et al. (1993) in Atlantichalibut (Hippoglossus hippoglossus), and with amino acidproWle propose by Finn (2007) for which lysine, leucine,and alanine quantitatively dominated the FAA pool. Duringour larval development experiments, we detected signiW-

cant decreases in the EFAA and NEFAA concentrationsfrom the egg stage to the yolk sac larvae (1-day-old, Fig. 6).Our results did not show any selective use during embryo-genesis, contrasting with the preferential consumption ofNEFAA observed in walleye pollock by Ohkubo et al.(2006). In the larval stage from 1- to 7-day-old larvae, ourresults showed a preferential consumption of EFAA overNEFAA.

The consumption of FAA during embryonic develop-ment continued after hatch but at a lower rate. After hatch(yolk sac larvae, 1-day-old) consumption had reached 60%of the FAA contained in egg stage I, whereas consumptionat 7-day-old larva reached 70%. Likewise, Rønnestad et al.(1992) and Finn et al. (1996) in turbot observed rapid FAAdepletion during the Wrst few hours after hatching (ca. 65%of initial content of FAA), followed by increased consump-tion of proteins and lipids. In our experiments, neutral(leucine, threonine) and polar (histidine) amino acids werethe most concentrated, both in eggs and in yolk sac larvae.Phenylalanine and glutamic acid, showed high concentra-tions in yolk sac larvae and 7-day-old larvae, respectively,suggesting an amino acid transformation in the old unfedlarvae. Our results on the most concentrated neutral FAAagree well with those of Finn et al. (1995b) for Atlantichalibut, although higher concentration of histidine wasfound in anchoveta eggs and yolk sac larvae in the presentstudy. In adults, histidine has been recognized to increasethe buVering capacity in plasma and muscle tissue (Szebed-inszky and Gilmour 2002). In our study, an adjustment ofthe amino acid composition of each egg batch to local envi-ronmental conditions may have occurred during the spawn-ing season in response to changes in salinity associatedwith changes in upwelling intensity (increased water salin-ity, batch III October 2004) or the amount of fresh waterdischarge due to rainfall and riverine input (decreasedsalinity, batch IV July 2005).

The importance of lipids to egg development has beenrecognized by several authors (Sargent et al. 1999; Tocher2003; Bell et al. 2003). Tocher (2003) suggested two clas-ses based on lipid composition: low lipid eggs (<5% wetweight of total lipids) dominated by polar lipids (>60%,mainly phospholipids such as phosphatidylcholine), andhigh lipid eggs (>5% wet weight) dominated by neutral lip-ids such as TAGs and containing one or several oil drops.TAG concentrations in our anchoveta eggs (1.6 �g egg¡1 inthe early stage of anchoveta development, batch IV) weresimilar to those reported in pollock (Ohkubo et al. 2006). Thisconcentration is equivalent to 10% of the total anchovetalipids and is in the range of values reported by Falk-Peter-sen et al. (1989) for eggs of Atlantic halibut (Hippoglossushippoglossus), a species that, similar to anchoveta, has eggsthat do not contain oils drops. Because of the TAG concen-tration (equivalent to 4.6% of dry weight), anchoveta eggs

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seem to belong to the Wrst group, but exhibit a variablerange of total lipid concentrations during the spawning sea-son (Castro et al. 2009). Lipid content corresponding tobatch IV was around 45% egg dry weight which is slightlyless than the 65% reported by Harrell and Woods (1995) oneggs of striped bass (Morone saxatilis).

In conclusion, this study quantiWed endogenous ener-getic substrates within anchoveta eggs collected near theaustral latitudinal limit of the distribution of this species inthe Humboldt Current. Our study focused on examiningdevelopmental changes and batch (seasonal) diVerenceswith special emphasis on FAA and proteins. On average,protein and FAA were responsible for 50% of the egg dryweight (July 2005, batch IV), while lipids were »45% ofthe dry weight. During development from egg stage I–III,our composite results included: (1) 65–69% consumptionof yolk (batches I and II) and rapid consumption of FAA,(2) the attenuation of 21 of 22 observed protein bands andthe absence of eleven of those bands after hatching (2003experiment), and (3) losses of 50% of the lipid and 24% ofTAG content. A comparison among spring egg batchesindicated signiWcant diVerences in maternally-derived fea-tures (such as egg size, yolk volume, FAA and protein con-tents). Egg volume was variable among spring batches andwas higher in July (batch IV) compared to October (batchesI and II). Protein-FAA and total lipid contents were foundin similar proportion (dry weight-based) during July. Theseegg batch diVerences illustrate the important role thatmaternal eVects play in determining the level of endoge-nous energy reserves (and potentially diVerences in starva-tion potential and oVspring success) of anchoveta. Furtherstudies are needed to assess.

Acknowledgments The authors want to thank our colleagues atLOPEL for their cooperation in experimental and Weld work, R.R.González for his important support in analytical issues, L. Nuñez forher kind cooperation with free amino acid analyses, and R. Veas for histimely collaboration in statistical analyses. We are also grateful to theR/V Kay Kay crew for support during Weldwork and to J. Marileo andC. Valenzuela for help throughout the experimental stage of this study.Financial support and equipment were obtained from the InternationalFoundation of Science (IFS Grant AA 3643-1, to MCK) and FONDE-CYT Grants 1070502 and 1030819 (LRC and G. Claramunt). MCKwas partially supported by the Graduate School of the Universidad deConcepcion, a CONICYT and a COPAS doctoral fellowships and athesis support grant (AT- 24080047).

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