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Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

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Seasonal variations of biochemical, pigment, fatty acid, and sterol compositions infemale Crassostrea corteziensis oysters in relation to the reproductive cycle

Miguel A. Hurtado a,b,1, Ilie S. Racotta a,c,1, Fabiola Arcos a, Enrique Morales-Bojórquez a, Jeanne Moal c,Philippe Soudant d, Elena Palacios a,d,⁎a Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexicob Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa, Paseo Claussen s/n, CP 82000, Mazatlán, Sinaloa, Mexicoc Ifremer, UMR M100, Laboratoire de Physiologie des Invertébrés, BP 70, Centre de Brest, 29280 Plouzané, Franced UMR/CNRS Université de Bretagne Occidentale; InstitutUniversitaire Européen de la Mer LEMAR—Laboratoire des sciences de l'environnement marin (UMR 6539) Technopole Brest 17 Iroise, Place Nicolas Copernic, 29280 Plouzané, France

⁎ Corresponding author at: Centro de Investigacio(CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa RitTel.: +52 612 123 8508; fax: +52 612 125 3625.

E-mail address: epalacio@cibnor.mx (E. Palacios).1 Current address: Centro de Investigaciones Biológic

Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S

1096-4959/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.cbpb.2012.05.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 June 2011Received in revised form 14 May 2012Accepted 14 May 2012Available online 18 May 2012

Keywords:ARADHAPigmentsFood availabilityVitellin

Wild female Crassostrea corteziensis oyster (n=245) were analyzed over one year to understand the mainecophysiological events associated to gonad development. Different indicators (mainly biochemical) wereanalyzed to infer: i) utilization and accumulation of energy reserves (e.g. neutral lipids, carbohydrates, pro-teins; vitellogenin), ii) membrane components provided by the diet as essential nutrients and indicative ofcell proliferation (e.g. highly unsaturated fatty acids linked to phospholipids, sterols), iii) indicators of foodavailability (chlorophyll a in water, pigments in tissues, specific fatty acids and sterols), iv) gonad develop-ment (e.g. gonad coverage area, vitellin). A PCA analysis was applied to 269 measured variables. The firstPC (PC1) was composed of total carbohydrate and lipid concentration, percentage of esterified sterols, fattyacids specific of diatoms; 16:1n−7/16:0, 20:5n−3 in neutral lipids with positive loadings and non methylene−interrupted fatty acids (NMI) in neutral lipids with negative loadings. The second PC (PC2) was composed of18:4n−3 in lipid reserves and the concentration of zeaxanthin, a pigment typical of cyanobacteria with positiveloadings and the proportion of 20:4n−6 in polar lipids with negative loading. The third PC (PC3) was composedof gonad coverage area (GCA) and the concentration of vitellin. Variation inGCA confirms that gonaddevelopmentbegan in April with an extended period of spawning and rematuration from April to November. The PCA furthershows that a second period of minimal maturation from November to March corresponds to the accumulationof reserves (PC1) together with an initial high availability of food (PC2) at the beginning of this period. Thesetwo periods are in accordance with the classical periods of allocation of energy to reserves followed by gonad de-velopment reported for several mollusks.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

The oyster Crassostrea corteziensis is distributed from the Gulf ofCalifornia to Panama (Keen, 1971). It has a widespread gonad devel-opment and spawning activity and a short period of quiescencethroughout a year (Rodríguez-Jaramillo et al., 2008). In contrast, oys-ters from temperate zones have a short but intensive spawning peri-od and accumulate biochemical reserves seasonally to sustain thegametogenic process (Deslous-Paoli and Héral, 1988; Matus de la

nes Biológicas del Noroestea, La Paz, B.C.S. 23090, Mexico.

as del Noroeste (CIBNOR), Mar. 23090, Mexico.

rights reserved.

Parra et al., 2005) that usually concludes in a single spawning insummer (Heffernan et al., 1989; Lango-Reynoso et al., 2006).

In oysters from temperate zones, the main reserve, glycogen, isstored during the end of autumn and winter and thereafter is usedas a source for lipid synthesis and to sustain energy demand. This isa conservative strategy of the storage cycle and use of energy (Gabbott,1975; Barber andBlake, 1991). In contrast,C. corteziensis,whichmaintainsreproductive activity throughout most of the year, is expected to acquireenergy for reproduction through recently ingested food, particularly car-bohydrates and lipids, as do other bivalves that use an opportunistic strat-egy for reproduction in food-rich sites (Racotta et al., 2008). To infer foodbioavailability, chlorophyll a concentration is generally used as a proxyfor phytoplankton biomass and therefore together with the patterns ofgonad development and accumulation of reserves provides a usefulgeneral picture of the need of mobilization of endogenous reserves inrelation to environmental food availability (Arellano-Martínez et al.,2004). In addition, biochemical biomarkers like pigments themselves

173M.A. Hurtado et al. / Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

or their metabolites and some fatty acids and sterols can also be used toinfer particular abundances or ingestion of particular algae majorgroups, particularly if separated into neutral lipids and phospholipids(Soudant et al., 1996; Desvilette et al., 1997; Soudant et al., 1998).

Bivalve mollusks have a limited ability for the elongation anddesaturation of PUFA (Ackman and Kean-Howie, 1995) and for cho-lesterol synthesis, or its conversion from phytosterols (Teshima andPatterson, 1981; Holden and Patterson, 1991). Therefore, these lipidsare considered essential and have to be acquired by ingesting phyto-plankton (Idler and Wiseman, 1972). In temperate species with con-servative strategies for maturation, such as Crassostrea gigas, theselipids can be stored in tissues when there are phytoplankton blooms.However, in opportunistic species we expect that availability of suchlipids could affect sustained reproductive performance, because witheach spawning PUFAs and sterols are lost with the eggs.

In a previous study using histological and quantitative histochemicalapproaches in a different subset of females of the same location, it wasconcluded that mature C. corteziensis were found most of the year witha short resting period from December to January and two strongspawning periods one in summer and the other in autumn (Rodríguez-Jaramillo et al., 2008). The aim of the present study was to understandthe main ecophysiological events associated to gonad development inwild female C. corteziensis oysters over one year. For this purpose, differ-ent indicators (mainly biochemical) were analyzed to infer: i) utilizationand accumulation of energy reserves (e.g. neutral lipids, carbohydrates,proteins; vitellin), ii) membrane components provided by the diet as es-sential nutrients and indicative of cell proliferation (e.g. highly unsatu-rated fatty acids linked to phospholipids, sterols), iii) indicators of foodavailability (chlorophyll a in water, pigments in tissues, specific fattyacids and sterols), iv) gonad development (e.g. gonad coverage area,vitellin).

2. Material and methods

2.1. Sampling and general analyses

Thirty wild C. corteziensis oysters were collected monthly frommangle roots in an intertidal zone near an oyster farm in Laguna deCeuta, Sinaloa, in northwestern Mexico from April 2005 to April2006 (n=460 oysters), as described in Rodríguez-Jaramillo et al.(2008). The oysters were shipped packed in ice (b24 h elapsed fromcollection to arrival) to the laboratory in La Paz, B.C.S., Mexico. Theywere scrubbed on arrival to remove epibionts and the length, width,total weight (shell on), and wet tissue weight (soft tissue) of eachspecimen were recorded. A transversal portion of the gonad and di-gestive gland was dissected and fixed in Davidson's solution for his-tological analysis (Gonad Coverage Area, GCA), as described byRodríguez-Jaramillo et al. (2008). Only females were selected (n=245female oysters) and analyzed after defining sex based on histologicalanalysis. Another sample of gonad tissue was obtained for vitellin analy-sis (see below). The remainingwholefleshwas frozen at−80 °C and thefrozen tissue of each oyster was choppedwhile frozen in a cooled porce-lain mortar to obtain a grounded tissue for lipid analysis. Part of thisgrounded tissue was weighed (about 100 mg) and lyophilized for 36 h,then weighed again for the estimation of the water content. It wasthen homogenized in 1 mL of cold saline solution (35% NaCl) to obtaina crude extract for total protein (Bradford, 1976) and carbohydrate(Roe, 1955) analyses. The sea surface temperature and chlorophyll awere determined daily using satellite-derived estimates detected fromthe waters outside the lagoon, as described in Rodríguez-Jaramillo etal. (2008).

2.2. Vitellin

The gonad tissue samples, stored at −80 °C, were individually ho-mogenized in equal volumes of extraction buffer [0.05 M Tris, 0.5 M

NaCl, 5 mM EDTA, pH 7, (3:1; v/v buffer:tissue)] and a protease inhib-itor cocktail (0.003% ref. P2714, Sigma-Aldrich, St Louis, MO, USA),using a glass Potter–Elvehjem homogenizer (Sartorius, Gottingen,Germany) on ice. The homogenate was centrifuged at 10 000 g for15 min at 4 °C (Beckman ultracentrifuge, Pasadena, CA, USA), andthe supernatant was stored at −80 °C until quantification of totalprotein and vitellin–vitellogenin (Vn–Vtg). Total proteins (Bradford,1976) were determined in the crude gonad homogenate after diges-tion with 0.1 N NaOH. The Vn–Vtg concentrations were assessed inthe resulting supernatant by quantitative enzyme-linked immunosor-bent assay (ELISA) according to Arcos et al. (2009). Polyclonal anti-bodies against C. corteziensis Vn–Vtg were prepared. Rabbits wereimmunized subcutaneously with 50 mg of pure Vn–Vtg of C. cor-teziensis emulsified with an equal volume of Freund's complete adju-vant (Difco, Lawrence, KS, USA). One and 4 weeks later, animals wereinjected with 120 mg of pure Vn–Vtg emulsified with an equal vol-ume of Freund's incomplete adjuvant (Difco). Blood (70 mL) was col-lected 40 and 48 days later, and the serum obtained was centrifuged(2500 g at 4 °C for 10 min) and stored at −80 °C for purification ofantibodies by HPLC. The specificity of this antibody was tested byWestern blot analysis and an immune diffusion test against the anti-gen (pure Vn–Vtg, hemolymph, and gonad samples of females andmales). The Vn–Vtg concentration was determined on the centrifugedhomogenate of gonads with a quantitative enzyme-linked immuno-sorbent assay (ELISA). A standard curve was made with the purifiedVn–Vtg and a linear regressionwas calculated to assess the relationshipbetween optical density (OD) and the amount of purified Vn–Vtg. Alldeterminations for the calibration curve and tissue samples weremade in triplicate. The assay precision was tested by determination ofthe intra- and interassay coefficients of variation (CVs) by running thestandard curve five times on different days (interassay CV). On eachplate every point of the standard curve was run eight times (intraassayCV). Serial dilutions of vitellogenic female hemolymph, vitellogenic fe-male gonad, spawned eggs, purified Vn–Vtg, and a control sample ofmale hemolymph were assayed by ELISA to assess parallelism with astandard curve.

2.3. Pigments

Pigments in the oyster tissue powder were analyzed according toVidussi et al. (1996). The extraction of the pigmentwasmade in 100% ac-etone HPLC grade at−20 °C for 24 h. Samples were centrifuged (1200 g,5 °C for 15 min) and passed through 0.45-mm filters (GF/F Whatman).Samples were analyzed in a Hewlett‐Packard HPLC 1100, equippedwith Hypersil MOS C8 (10 cm×0.45 cm, 5-μm thick film) silica column(Agilent, USA) and photodiode array detector (190–900 nm). The elutionwas run at a flow rate of 1 mL min−1 using solvent A (MeOH:0.5 N am-monium acetate, 70:30 v/v) and solvent B (MeOH). The elution proce-dure was (min;solv. A, solv. B) (0; 75:25), (1; 50,50), (15; 0, 100),(18.5; 0, 100), and (19, 75, 25). Pigments were identified according tospectral absorption at 350–750 nm and the comparison of retentiontimeswith external standards (DHI Lab. Products, Hoersholm, Denmark).

2.4. Lipids

Lipidswere extracted from the grounded oyster tissue in chloroform:methanol (2:1). Neutral lipids (sterols, acylglycerides, free fatty acids,and esterified sterols) and polar lipids (mainly phospholipids)were sep-arated in a silica gel micro-column (Marty et al., 1992). The fatty acidsfrom each fraction were analyzed by gas chromatography according toPalacios et al. (2005). Free and esterified sterols from the neutral lipidfraction were separated using a Hewlett‐Packard HPLC 1100, equippedwith a Nucleosil C18 120A (25 cm×4.6 cm, 5-μm thick film) column(Phenomenex, USA) and a UV multiple wavelength detector. Elutionwas run at a flow rate of 1 mL min−1 using isopropanol and hexane(3:97 v/v). Each fraction was identified at 280 nm and separated into

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Table 1Eigenvalues, variance, and cumulative variance obtained by principal componentsanalysis. PC means principal component.

Eigenvalue Variance(%)

Cumulativeeigenvalue

Cumulative variance(%)

PC1 4.8 43.6 4.8 43.6PC2 2.1 18.7 6.9 62.3PC3 1.1 9.9 7.9 72.2PC4 0.9 7.9 8.8 80.0PC5 0.6 5.8 9.4 85.8PC6 0.5 4.3 9.9 90.1PC7 0.3 2.8 10.2 93.0PC8 0.3 2.4 10.5 95.4PC9 0.2 2.1 10.7 97.5PC10 0.2 1.5 10.9 99.0PC11 0.1 1.0 11.0 100.0

174 M.A. Hurtado et al. / Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

different vials. Sterols from esterified sterol fraction were released usingsodium methoxide (0.5 M in methanol) at ambient temperature for90 min (Soudant et al., 1998). The sterols were analyzed by gas chroma-tography according to Palacios et al. (2007).

2.5. Gonad coverage area

The area occupied by the gonad (GCA) was determined as describedby Rodríguez-Jaramillo et al. (2008) by using an image system analyzer(Image Pro Plus v. 4.) at 4× (7.9 mm2) from a mean of three differentsections of a slide covering three area of 7.9 mm/area, from each speci-men. The area reported as the gonadal coverage area (GCA) was calcu-lated as GCA=(gonad occupation area/total area)×100.

2.6. Statistical analysis

A one-way ANOVA was used to analyze significant differences be-tween sampling dates (12 levels), followed by a post-hoc Tukey testfor unequal number of data (n) to assess significant differences be-tween means (Pb0.05). We estimated the orthogonal empirical func-tions to analyze variation of different data related to the reproductivecycle in wild female C. corteziensis. The orthogonal empirical func-tions were estimated using the multivariate analysis of the principalcomponents analysis (PCA) (Tabachnick and Fidell, 1989), with thevectors computed from PCA having the characteristic of being orthog-onal and are uncorrelated, avoiding the regression on residuals(Mardia et al., 1989; Milstein, 1993). The PCA permitted a rankingand simplification of the new variables called principal components,determining the total variation of the data Xj, and explaining themwith a few principal components (factor loadings≥0.7). Only eigen-values>1.0 showed the principal component to be statistically signif-icant (Krzanowski, 1993; Manly, 1994). Each principal component(Pci) was a linear combination of the biochemical composition, mor-phometric, and reproductive variables (defined as variables Xj) in fe-males of C. corteziensis, the Pci was expressed as:

Pci ¼ ∑jλij Xj−�Xj

� �s−1j

h i

Where λij is the eigenvector's j-th coordinate of the i-th principalcomponent. It therefore represents the contribution of each variableXj (standardized with mean and variance values ) (Tabachnick andFidell 1989). In order to analyze significant variation of PC throughthe year, we used them as new variables in a one-way ANOVAfollowed by Tukey post-hoc testing, as already done by Delaporte etal. (2005). All statistical analyses were made using StatisticaTM v. 6.0.

3. Results

3.1. Environmental variables

The daily sea surface temperature during the sampling period isreported in Fig. 1A. The temperature varied around 20 °C from December2005 to March 2006 and peaked above 30 °C from August to October2005. The chlorophyll a showed an inverse relationship with tempera-ture, with two initial peaks in April and May 2005, low values betweenJune and October 2005, and several peaks duringwinter and early spring,namely at themiddle of November, oscillatory high values from Februaryto April 2006 with a particular prominent peak in April (Fig. 1B).

3.2. Principal component analysis (PCA)

Data of PCA are presented first in order to describe the majortrends of the variables analyzed in the present work, allowing specialattention to the main temporal patterns of physiological events.

Thereafter, detailed presentation of all data is also shown for a com-plete description of all variables analyzed.

The PCA showed twelve variables that summed the whole varia-tion from the original data set analyzed in each female (n=245total females) and which are presented in Table 1, indicating the rel-ative importance of the principal component; from these, three prin-cipal components were required to describe most (72%) of thevariance contained in the original data set (eigenvalues>1.0). Thefirst principal component explained 44% of the variance. The factorloadings, presented in Table 2, revealed that there were significantcontributions to PC1 (factor loadings≥0.7) of the concentration oftotal carbohydrates (0.88) and total lipids (0.82), the proportion ofesterified sterols (0.88), the proportion of 20:5n−3 (0.75) in neutrallipids and the ratio 16:1n−7/16:0 (0.85) in neutral lipids, all withpositive loadings, and the proportion of total NMI (−0.78) in neutrallipids with a negative loading.

The second principal component explained 19% of the variance. Thefactor loadings indicated that there was a significant (factor load-ings≥0.7) contribution to PC2 of the proportion of 18:4n−3 in neutral

Table 2Factor loadings estimated by principal component analysis. PC means principalcomponent.

Variable PC1 PC2 PC3

Gonad coverage area (% of total tissue area) 0.22 −0.06 0.76Vitellin (mg g−1, dry weight) −0.29 −0.09 0.73Total carbohydrate (mg g−1, dry weight) 0.88 −0.09 −0.08Total lipids (mg g−1, dry weight) 0.82 0.03 0.12Total esterified sterols (% of free+esterified sterols) 0.88 0.02 −0.01Zeaxanthin (ng mg−1, dw) −0.20 0.77 0.1318:4n−3 in neutral lipids (% total fatty acids) 0.11 0.87 −0.2320:5n−3 in neutral lipids (% total fatty acids) 0.75 0.32 −0.15Sum of total NMI in neutral lipids (% total fatty acids) −0.78 −0.15 0.24Sum of 20:4n−6 in polar lipids (% of total fatty acids) −0.51 −0.70 0.27Ratio 16:1n−7/16:0 in neutral lipids 0.85 −0.08 0.12

NMI=non methylen interrupted FA. Factor loadings ≥0.7 were considered significant.

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Fig. 2. Monthly variations of A) PC1, B) Ratio of 16:1n−7/16:0 (right axis, white dia-monds), and proportion of 20:5n−3 (left axis, black circle) and total NMI in neutrallipids (left axis, white circles), C) Total carbohydrates (left axis, black circle), total lipids(right axis, white circle) and proportion of esterified sterols (right axis, black triangle).Values are shown as mean±standard deviation. Different lowercase letters indicatesignificant differences between sampling months.

175M.A. Hurtado et al. / Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

lipids (0.87) and the concentration zeaxanthin (0.77) with a positiveloading and the proportion of 20:4n−6 in phospholipids (−0.70) witha negative loading (Table 2).

The third principal component explained 10% of the variance, witha significant contribution of vitellin (0.73), and of the gonad coveragearea (0.76) (Table 2).

The variable association given by the PCA was essential for a betterunderstanding of the data, especially because of the number of variablesanalyzed for each female. Each principal component was analyzed by aone-way ANOVA, using the sampling dates as the independent variable.The analysis showed that differences in the selected three principalcomponents were affected by sampling month (Pb0.01).

The first principal component (PC1) was high in spring 2005, de-creased in summer 2005, and then reached lowest values in fall. It in-creased again in December to values similar to summer, and thenfurther increased in January 2006 to reach levels thereafter similar tospring 2005 (Fig. 2A). Carbohydrates levels presented a very similartrend to PC1 with a progressive decline from April/May to November2005 followed by an increase till reaching maximum values, similar toMay 2005, in February 2006 and thereafter. Total lipids showed a simi-lar trend to carbohydrates, but the decrease started from September.The proportion of esterified sterols also showed a decrease inNovember2005 and higher levels in spring 2005 and 2006 (Fig. 2C). The propor-tion of 20:5n−3 in neutral lipids followed this pattern, with the highestvalues in April 2005 and February–March 2006 and the lowest ones inSeptember–October 2005 (Fig. 2B). In contrast, the proportions ofNMI in neutral lipids followed an opposite trend with lower values atthe beginning and end of the period analyzed (April–June 2005 and Jan-uary–April 2006) and the highest values in October 2005. The ratio of16:1n−7/16:0 showed higher levels in spring, decreased in summerand showed the lowest values in November (Fig. 2B).

The second principal component (PC2) showed a slow but steadydecrease from spring to summer 2005 till reaching lowest levels in Sep-tember, then peaked in November and decreased slightly to intermedi-ate levels in December, remaining stable thereafter throughout thesampling period (Fig. 3A). The proportion of 18:4n−3 in the neutrallipids presented the lowest values in September–October 2005, peakedin November and then remained at intermediate levels till the end ofthe sampling period (Fig. 3B). The proportion of 20:4n−6 in phospho-lipids followed an opposite trend, since it increased from April 2005 toreach its maximum in September and October, and then decreased toreach the minimal values in January 2006 and thereafter (Fig. 3B). Thelevels of zeaxanthine in tissues was very low, and sometimes belowthe detection levels, before November 2005, when it peaked to its max-imum. Then levels decreased slowly till reaching in January 2006 levelssimilar to summer 2005. It increased again in February 2006 andremained stable till the end of the sampling period (Fig. 3C).

The third principal component (PC3) showed a peak in May 2005,decreased in June and then increased again in July, thereafter slowly

decreasing until reaching its lowest levels from January to March2006, and then increasing to its highest levels in April 2006(Fig. 4A). The gonad coverage area (GCA) followed very closely thepattern of PC3 with the highest values in May 2005 and April 2006,a first steep decrease in June 2005, increased in July and then had aslow decrease till reaching the lowest values in January and February2005 (Fig. 4B). The lowest values of vitellin were observed betweenJanuary–March 2006, and the highest in April 2006 (Fig. 4C).

3.3. Biomass, water content and biochemical composition

Tissue wet mass decreased to minimum values from June toDecember (9.0±0.1 g) and then increased to highest value in April2006 (29.2±1.7 g) (Table 3). The lowest water content in the tissuewas measured in April 2005 (72%) and highest from September toNovember 2005 (83%–85%). Low values of protein content werefound in April–May 2005 (b359 mg g−1dw), increased to the highestlevels in November 2005 (555±22 mg g−1dw), and thereafter gradual-ly decreased again till April 2006. The opposite trend was observed fortotal carbohydrates and lipids than decreased between April–May2005 to November and then increased attaining higher values betweenFebruary and April 2006 (Table 3).

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Fig. 4. Monthly variations on A) PC3, B) gonad coverage area (% of total tissue), and C)vitellin (mg g−1 dw). Values are shown as mean±standard deviation. Different lower-case letters indicate significant differences between sampling months.

176 M.A. Hurtado et al. / Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

3.4. Pigments

The main general pattern of pigments and their metabolites inoyster tissues revealed low values between April and October 2005,followed by in an increase in November with oscillatory high valuestill April 2006. This pattern was observed for total concentration ofpigments and in general for all pigment individually, with a majorparticipation of pheophorbide a as the most abundant metabolitefrom chlorophyll a (Table 3).

3.5. Total free and esterified sterols

Cholesterol (sum of the free and esterified fractions) was the majorsterol in oyster tissues, ranging from 32% to 45%, followed bybrassicasterol (13%–16%) and isofucosterol (9%–13%). Among the sterolsthat accounted for less than 10% of total sterols were campesterol (3%–7%), 24–methylenecholesterol (4%–7%), stigmasterol (4%–6%), ergoster-ol (4%–5%), β–sitosterol+fucosterol (5%–6%), t–dehydrocholesterol(4%–6%), c–dehydrocholesterol (1%–2%), and desmosterol (1%–3%).

Sterols were mostly found in their free form (56% to 93%) throughoutthe sampling period.

The concentration of total free sterols increased more than twofold from April to September 2005 and then it progressively declineduntil reaching its lowest levels in March 2006. In contrast, esterifiedsterols decreased in October and November 2005 by 50% or morecompared to previous months and then increased again reachingtheir highest values in April 2006 (Table 4).

3.6. Fatty acids

The proportion of the 22:6n−3 fatty acid (FA) in neutral lipidswas fairly constant although significantly higher values (>25%)were observed during reproductive quiescence in January 2006,whereas the lowest values were observed at the end of the reproduc-tive period in November 2005. A similar pattern was observed for thisfatty acid content in phospholipids. Whereas the proportion of 20:5n−3 in neutral lipids closely matched PC1 related to the accumulationof reserves, its content in phospholipids were higher in February–March 2006 and decreased in autumn, with intermediate levels inspring of both years. The 20:4n−6 in neutral and polar lipids were

Table3

Morph

ometricpa

rameters,prox

imal

compo

sition

,and

pigm

entco

nten

tsin

femaleCrassostreacorteziensissampled

from

April20

05to

April20

06in

Lagu

nade

Ceuta,

Sina

loa,

Mex

ico.

2005

2006

April

May

June

July

Septem

ber

Octob

erNov

embe

rDecem

ber

Janu

ary

Februa

ryMarch

April

Water

conten

t(%

)71

.8±

0.6b

77.2±

3.4a

b80

.2±

2.1a

b81

.8±

2.0a

b85

.1±

2.9a

83.0±

1.8a

84.8±

1.5a

82.1±

2.6a

b76

.5±

2.1a

b78

.9±

2.6a

b75

.2±

2.8a

b75

.7±

2.4a

b

Wet

biom

ass(g)

16.0±

1.5b

18.3±

0.5b

20.9±

1.8a

b15

.5±

2.1b

12.4±

3.2c

9.3±

1.3c

9.0±

0.7c

9.1±

0.4c

13.8±

0.8b

17.4±

1.8b

16.2±

2.2b

29.2±

1.7a

Bioche

mical

compo

sition

Proteins

(mgg−

1dw

)34

6±

16e

335±

16e

414±

31c

376±

28d

499±

38b

518±

38ab

555±

22a

425±

41bc

388±

31d

376±

31d

401±

16cd

366±

20ed

Carboh

ydrates(m

gg−

1dw

)27

8±

13b

320±

5a24

9±

18b

261±

15b

198±

29c

163±

18cd

137±

7d24

2±

17bc

270±

6b30

4±

19a

295±

13a

318±

14a

Lipids

(mgg−

1dw

)83

±6b

c10

7±

7a76

±9b

c93

±7a

60±

6c57

±6d

59±

3c75

±7b

c88

±4b

c95

±6a

90±

6ab

89±

6b

Pigm

ents

(μgg−

1dw

)Ch

loroph

ylla

0.07

±0.02

b0.16

±0.07

bN.D.

N.D.

0.06

±0.04

b0.02

±0.01

b1.8±

1.0a

0.73

±0.5a

b0.11

±0.10

b0.11

±0.08

b0.08

±0.05

b0.05

±0.03

b

Pheo

phorbide

a2.4±

1.3e

8.4±

3.1d

4.2±

1.3e

5.1±

1.1e

d3.5±

1.0e

12.1±

4cd

30.2±

8bc

43.6±

13ab

31.5±

8b27

.0±

5c43

.7±

14ab

53.5±

16a

Pheo

phythina

0.01

7±

0.01

bN.D.

0.03

±0.02

bN.D.

0.75

±0.70

b0.11

±0.03

b5.4±

1.2a

1.6±

0.7a

b0.28

±0.11

b0.28

±0.07

b0.81

±0.44

b0.38

±0.18

b

Pyroph

eoph

ythina

0.25

±0.20

bN.D.

0.02

±0.01

cN.D.

0.11

±0.01

c0.07

±0.04

c3.9±

0.9a

3.0±

1.8a

b0.17

±0.02

bc

0.14

±0.05

c0.25

±0.10

b0.21

±0.09

b

Fuco

xanthin

0.01

±0.00

5e1.42

±1.30

cd0.49

±0.40

dN.D.

0.03

±0.02

e0.02

±0.01

e5.3±

1.9a

b4.2±

2.2b

c3.15

±1.92

c3.5±

2.1c

4.8±

1.9b

5.6±

1.9a

Zeax

anthin

N.D.

0.41

±0.28

b0.04

±0.02

b0.06

±0.04

b0.01

±0.00

3b0.03

±0.01

b10

.0±

4.2a

4.6±

3.7a

b0.59

±0.34

b3.0±

2.1a

b2.5±

1.3b

0.96

±0.52

b

Lutein

N.D.

0.03

±0.02

b0.01

±0.00

4b0.03

±0.02

b0.01

±0.00

2b0.01

±0.00

9b4.4±

1.9a

2.4±

1.9a

b0.04

±0.03

b0.03

4±

0.03

bN.D.

0.24

±0.22

b

Totalp

igmen

ts2.7±

1.4d

19.9±

11.5

c8.7±

4.6c

d8.8±

3.7c

d4.0±

1.6d

10.4±

3.6c

d55

.2±

16ab

88.4±

33a

49.0±

15b

44.3±

14bc

76.0±

28a

85.1±

29a

Resu

ltsarethemea

n±

S.E.,a

ndwerean

alyz

edwithaon

e-way

ANOVA,followed

byaTu

keypo

st-h

ocan

alysis

toassert

differen

cesbe

twee

nmea

ns;mea

nsno

tsh

aringthesamesu

perscriptin

arow

aresign

ificantly

differen

t(P

b0.05

).

177M.A. Hurtado et al. / Comparative Biochemistry and Physiology, Part B 163 (2012) 172–183

highest in September–October, and lowest from January 2006 till theend of the sampling period (Tables 5 and 6).

4. Discussion

As previously analyzed by classical histology and by histochemis-try (Rodríguez-Jaramillo et al., 2008), we observed that gonad devel-opment began in April with an extended period of spawning andrematuration from April to November and a short resting periodfrom December to March. The principal component analysis (PCA)further shows that this period of minimal maturation (PC3) corre-sponds to the accumulation of reserves (PC1) together with an initialhigh availability of food at the beginning of this period (PC2). The twoperiods are in accordance with the classical periods of allocation ofenergy to reserves followed by gonad development reported for sev-eral mollusks as a conservative strategy (Barber and Blake, 1991;Strohmeier et al., 2000).

A principal component (PC2) was composed of the 18:4n−3 per-centage in neutral lipids and the zeaxanthin content in tissues. Severaldirect (chlorophyll a levels inferred from satellite) and indirect indica-tors (biomarkers in oyster tissues such as specific pigments, sterols,and fatty acids) of phytoplankton abundance (Williams and Claustre,1991; Volkman et al., 1998; Bergé and Barnathan, 2005) indicatedthat food availability increased at the end of fall. Estimation of chloro-phyll a levels in waters outside the lagoon from analysis of satellite im-ages of water support that the highest food availability was observedfrom November to April/May with a particular bloom in Novemberthat matched the peak observed for PC2. Some of the pigments foundin C. corteziensis tissues correlated positively with chlorophyll a valuesreported in the water outside the lagoon, as was the case for phe-ophorbide a (r=0.73; Pb0.01) and pheophytin a (r=0.64; Pb0.05),both metabolic by-products of chlorophyll a (Aneeshkumar andSujatha, 2012), supporting the influence of oceanicwaters inside the la-goon, probably by the dynamics of sea currents and tides. Absorptionand storage of chlorophyll degradation products (i.e. pheophorbide,pheophythin, pyropheophythin, etc.) has been described in bivalves(Ansell, 1974; Gelder and Robinson, 1980) and pheophorbide a-likeproducts have been identified as primary indicators of digestive pro-cess in mussel Mytilus edulis (Hawkins et al., 1986). Pheophorbide ais the main product found in the digestive gland, stomach content,and feces of Placopecten magellanicus, followed by other productssuch as pheophythin a and pyropheophythin a (Robinson et al.,1989). Pheophorbide a and pheophitin a had increased levels fromNovember 2005 to April 2006, compared to previous months, and area good indicator of high food availability. The pigment that had a mostimportant loading on the PC2, was zeaxanthin, which is typical of cya-nobacteria (Aneeshkumar and Sujatha, 2012). Other pigments thatmay have been indicative of other kind of phytoplankton, i.e. fucoxan-thin and lutein, commonly used as fingerprints for diatoms, and greenalgae (Williams and Claustre, 1991;Wright et al., 1991), also presentedthis patternwith particular variation indicating that different algae par-ticipate in the initial bloom and subsequent abundance till the end ofthe period analyzed (Table 3).

The phytoplanctonic cycle at Laguna de Ceuta was described byIbarguen-Zamudio (2006) and consisted of a higher abundance of dia-toms (70% of all species) found during most of the year, with a bloomof dinoflagellates, particularly of Prorocentrum mexicanum, starting atthe end of fall. Some fatty acids are useful trophic biomarkers (Volkmanet al., 1998; Dalsgaard et al., 2003; Bergé and Barnathan, 2005; Pernetet al., 2012). During the highest abundance of phytoplankton starting inNovember, an increase in the proportion of 18:4n−3 in neutral lipids inNovember was observed, suggesting that dinoflagellates, that typicallycontains 18:4n−3 (Leblond and Chapman, 2000; Kharlamenko et al.,2001), contribute importantly to the phytoplankton bloom. Althoughthe content of 18:5n−3 of P. mexicanum is similar or even higher thanthe content f 18:4n−3 (Mansour et al., 1999; Gray et al., 2009), we did

Table 4Individual sterol proportions (% of total sterols) and concentrations of total free and esterified sterols in female tissues of Crassostrea corteziensis sampled from April 2005 to April 2006 in Laguna de Ceuta, Sinaloa, Mexico.

2005 2006

April May June July September October November December January February March April

Free fractionCholesterol 26.9±5.0cd 23.1±1.5cd 27.1±2.9cd 28.7±4.7bc 38.5±4.1ab 37.9±1.8ab 39.8±0.9a 31.9±1.9b 21.7±2.0cd 28.9±7.1bc 17.3±1.9d 28.1±6.0c

Brassicasterol 9.2±1.0bc 10.6±0.6bc 11.3±1.0b 10.0±1.4bc 11.0±0.7b 12.9±0.5a 13.0±0.3a 12.3±0.6ab 11.4±0.9b 7.5±0.8cd 8.1±0.8c 6.3±0.7d

Isofucosterol 4.5±1.0e 5.4±0.6d 8.5±1.5bc 6.9±1.7c 9.1±1.6b 9.7±1.1ab 10.9±1.4a 10.4±0.8ab 6.4±0.7cd 4.4±0.5e 4.5±0.7e 4.5±0.7e

Campesterol 3.6±0.5ab 3.3±0.2b 2.2±0.1bc 1.9±0.2c 2.2±0.1bc 2.5±0.1bc 2.8±0.1bc 3.7±0.2ab 4.0±0.3a 3.7±0.4ab 3.6±0.4ab 3.3±0.4ab

24‐Metylenecholesterol 4.8±0.7bc 4.6±0.6bc 4.3±0.4c 3.5±0.5cd 4.9±0.4b 5.8±0.2ab 6.0±0.1a 5.8±0.2ab 3.2±0.3d 2.4±0.3ed 2.5±0.3ed 2.3±0.2e

Stigmasterol 2.4±0.2b 2.6±0.2b 2.9±0.3ab 2.3±0.3b 2.5±0.2b 2.8±0.1b 3.9±0.1a 4.2±0.2a 2.4±0.2b 1.9±0.2b 1.8±0.2b 1.8±0.2c

Ergosterol 3.3±0.5ab 2.8±0.3bc 2.8±0.3bc 2.4±0.2c 3.1±0.5b 3.4±0.2a 3.4±0.2a 3.3±0.3ab 2.3±0.1cd 2.4±0.5c 2.1±0.3d 2.5±0.3c

β‐sitosterol+fucosterol 3.1±0.3c 4.1±0.4bc 4.8±0.3ab 4.1±0.4bc 3.7±0.3bc 4.6±0.3b 6.1±0.1a 4.5±0.2b 3.8±0.2bc 2.7±0.3d 3.3±0.2c 2.7±0.3d

t‐dehydrocholesterol 2.5±0.2c 3.1±0.2bc 3.5±0.4bc 2.9±0.4bc 4.0±0.3b 4.9±0.2a 5.2±0.1a 4.0±0.2ab 3.0±0.3bc 2.3±0.3d 2.4±0.2d 2.3±0.2d

c‐dehydrocholesterol 0.4±0.0d 0.6±0.0c 0.9±0.1ab 0.6±0.1c 0.9±0.1b 1.1±0.1a 0.9±0.1b 0.7±0.1bc 0.6±0.1c 0.5±0.1c 0.5±0.1cd 0.6±0.0c

Demosterol 1.6±0.2a 1.4±0.1b 0.8±0.0bc 0.7±0.1c 0.8±0.1c 1.0±0.1b 1.0±0.1b 1.4±0.2b 1.5±0.2a 1.6±0.2a 1.5±0.2a 1.4±0.2ab

Total (mg g−1) 7.5±1.5b 6.7±1.2b 10.1±1.5ab 13.1±2.3ab 19.8±4.5a 12.6±3.5ab 13.1±1.4ab 12.5±2.3ab 6.1±0.4b 7.9±2.0ab 4.8±0.6b 6.6±1.1b

Esterified fractionCholesterol 10.7±1.7bc 12.2±1.2b 8.8±1.5c 9.7±1.1bc 7.0±1.6cd 4.3±1.4d 2.0±0.4e 6.7±1.0cd 10.2±0.9bc 14.1±1.8b 14.4±1.7ab 15.7±1.5a

Brassicasterol 4.5±0.6cd 4.6±0.5c 3.4±0.7ed 3.6±0.5d 2.1±0.7e 1.3±0.5ef 0.4±0.1f 1.9±0.3e 4.8±0.5bc 6.2±1.0b 6.3±0.7a 6.3±0.7ab

Isofucosterol 4.3±0.4bc 6.6±0.9a 4.0±0.7bc 4.9±0.8bc 2.9±0.9c 1.8±0.6d 0.7±0.2e 1.8±0.3cd 5.1±0.6b 5.3±0.9ab 6.3±0.9a 4.6±1.0bc

Campesterol 2.3±0.4c 2.3±0.2bc 1.3±0.2cd 1.2±0.3d 0.7±0.2e 0.6±0.2f 0.4±0.0f 0.9±0.1ed 2.2±0.4c 2.9±0.4ab 3.1±0.4a 2.7±0.5b

24‐Metylenecholesterol 2.2±0.3a 2.2±0.2ab 1.5±0.3c 1.3±0.2c 1.1±0.2c 0.8±0.2cd 0.4±0.0d 1.0±0.3c 1.8±0.4bc 1.8±0.3b 1.7±0.3c 1.6±0.3c

Stigmasterol 3.1±0.4bc 3.4±0.5bc 2.7±0.4c 2.7±0.5c 2.1±0.7c 1.6±0.5cd 0.6±0.0d 1.6±0.2d 3.8±0.4b 3.9±0.6ab 3.6±0.5bc 4.4±0.8a

Ergosterol 1.0±0.2b 2.1±0.2a 1.1±0.2b 1.5±0.2b 0.8±0.1c 0.9±0.2b 0.9±0.2bc 1.4±0.4b 1.2±0.3b 1.7±0.2b 1.6±0.2b 2.0±0.3ab

β‐sitosterol+fucosterol 1.5±0.3c 1.8±0.2bc 1.2±0.3d 1.3±0.2cd 0.9±0.3ed 0.6±0.2e 0.2±0.0f 0.7±0.2ed 1.6±0.1bc 2.1±0.3b 2.6±0.3a 2.4±0.2ab

t‐dehydrocholesterol 1.5±0.1b 1.6±0.2b 1.2±0.2bc 1.4±0.2bc 1.0±0.2c 0.8±0.2cd 0.5±0.0d 1.0±0.2c 1.5±0.1b 1.9±0.1ab 2.2±0.3a 1.9±0.4ab

c‐dehydrocholesterol 0.6±0.1b 0.7±0.1b 0.6±0.1b 0.6±0.1b 0.5±0.1bc 0.4±0.1bc 0.3±0.0c 0.5±0.1b 0.6±0.0b 0.9±0.1ab 1.0±0.2a 0.8±0.2ab

Demosterol 0.8±0.1b 0.8±0.1ab 0.4±0.1cd 0.3±0.1ed 0.3±0.1e 0.3±0.1ed 0.6±0.2c 0.4±0.1d 0.7±0.1bc 1.0±0.1a 0.9±0.1a 1.0±0.2a

Total (mg g−1) 3.2±0.2c 3.9±0.4c 3.5±0.7c 4.6±0.3ab 4.2±1.5bc 1.8±0.5cd 1.0±0.2d 2.8±0.6c 3.8±0.5c 4.9±0.3ab 4.5±0.6b 5.3±0.9ab

Results are the mean±S.E. See Table 3 for statistical analysis.

178M.A.H

urtadoet

al./Com

parativeBiochem

istryand

Physiology,PartB163

(2012)172

–183

Table 5Individual fatty acid proportion (%) of neutral lipid fraction in Crassostrea corteziensis female tissues sampled from April 2005 to April 2006 in Laguna de Ceuta, Sinaloa, Mexico.

2005 2006

Apr May Jun Jul Sep Oct Nov Dec Jan Feb Mar Apr

14:0 1.9±0.1a 2.0±0.1a 1.6±0.1ab 1.5±0.2ab 1.4±0.1ab 1.3±0.2bc 0.9±0.1c 1.6±0.1ab 1.8±0.1ab 1.8±0.1ab 1.7±0.1ab 1.7±0.1ab

16:0 9.6±0.4 10.3±0.4 10.5±0.3 10.7±0.5 9.6±0.8 9.7±0.8 9.1±0.5 8.9±0.4 9.1±0.3 9.3±0.2 9.2±0.4 9.6±0.417:0 0.9±0.1ab 1.0±0.0ab 1.1±0.0a 1.1±0.0a 1.1±0.0a 1.1±0.0a 0.7±0.0b 0.8±0.0b 0.9±0.0ab 0.9±0.0ab 0.9±0.0ab 0.9±0.0ab

18:0 2.7±0.1bc 2.7±0.0bc 3.4±0.2ab 3.5±0.2ab 3.7±0.1a 3.8±0.1a 3.2±0.1abc 2.8±0.1bc 2.5±0.1c 2.8±0.1bc 2.7±0.1bc 2.9±0.1bc

16:1n−9 1.7±0.2 1.0±0.2 1.1±0.1 1.1±0.1 1.2±0.1 1.2±0.1 1.3±0.1 1.2±0.1 1.2±0.1 0.9±0.2 1.0±0.1 0.9±0.116:1n−7 1.9±0.1ab 2.1±0.1a 1.5±0.1abc 1.7±0.2abc 1.5±0.2abc 1.5±0.2abc 1.0±0.1c 1.2±0.1bc 1.7±0.1abc 1.9±0.1abc 1.8±0.1abc 1.7±0.1abc

18:1n−9 1.5±0.1b 1.8±0.1ab 2.1±0.1ab 2.1±0.1ab 1.9±0.1ab 2.1±0.2ab 2.5±0.2a 1.9±0.1ab 1.6±0.1b 1.6±0.1b 1.5±0.1b 1.6±0.0b

18:1n−7 3.0±0.2 3.1±0.2 2.6±0.1 2.6±0.2 2.6±0.3 2.4±0.3 2.2±0.1 2.3±0.1 2.7±0.1 2.9±0.0 2.8±0.2 2.8±0.120:1n−7 1.5±0.1 1.6±0.1 1.4±0.1 1.4±0.1 1.6±0.1 1.6±0.1 1.6±0.1 1.5±0.1 1.5±0.1 1.5±0.1 1.7±0.1 1.6±0.118:2n−6 1.1±0.1b 1.4±0.1b 1.5±0.1ab 1.5±0.1b 1.2±0.1b 1.1±0.1b 2.1±0.2a 1.6±0.1ab 1.3±0.1b 1.3±0.1b 1.3±0.1b 1.3±0.0b

18:3n−3 1.6±0.1b 1.7±0.1b 1.9±0.1b 1.6±0.1b 1.3±0.2b 1.1±0.2b 3.1±0.2a 1.6±0.1b 1.7±0.1b 1.6±0.1b 1.6±0.1b 1.5±0.1b

18:4n−3 2.1±0.1bc 2.1±0.1bc 1.9±0.1bcd 1.6±0.2cd 1.2±0.2d 1.2±0.2d 3.7±0.4a 2.2±0.1bc 2.9±0.1b 2.7±0.1b 2.7±0.2b 2.5±0.2bc

18:5n−3 1.0±0.0d 1.2±0.1d 1.8±0.2bc 2.2±0.3ab 2.7±0.3a 2.3±0.2ab 1.4±0.1cd 1.0±0.1d 0.8±0.0d 0.8±0.0d 0.8±0.0d 1.1±0.1d

20:4n−6 2.4±0.1cde 2.7±0.1bcd 3.0±0.1abc 3.3±0.3ab 3.6±0.3a 3.5±0.2a 2.6±0.2bcd 2.1±0.1de 1.7±0.1e 1.9±0.1e 1.9±0.1e 1.9±0.1e

20:5n−3 15.0±0.4a 13.8±0.6ab 11.0±0.6bc 10.7±0.8bc 9.4±0.9c 9.0±0.8c 10.9±0.2bc 13.3±0.5ab 13.6±0.2ab 15.2±0.3a 14.2±0.5a 13.4±0.7ab

22:4n−6 1.0±0.0 1.1±0.1 1.5±0.1 1.5±0.1 1.4±0.1 1.4±0.1 1.1±0.1 1.3±0.3 1.2±0.0 1.1±0.0 1.0±0.0 0.9±0.022:5n−3 2.2±0.1 2.0±0.1 2.1±0.1 2.2±0.1 2.4±0.1 2.3±0.2 2.2±0.1 2.5±0.2 1.9±0.0 2.0±0.1 2.0±0.1 1.9±0.122:6n−3 24.9±0.8bc 25.2±0.4ab 26.1±0.4ab 25.5±0.9ab 23.2±0.5bc 23.9±0.4bc 21.6±0.5c 25.9±0.3ab 28.7±0.3a 25.6±0.6ab 25.8±0.2ab 26.7±0.7ab

22:2i NMI 1.0±0.0b 1.0±0.1b 1.3±0.1ab 1.3±0.2ab 1.5±0.2ab 1.7±0.2a 1.6±0.1ab 1.5±0.0ab 1.0±0.0b 0.9±0.0b 1.0±0.1b 1.0±0.1b

22:2j NMI 7.4±0.5 7.1±0.4 6.3±0.4 6.2±0.7 9.1±1.0 9.8±0.9 9.3±0.6 7.4±0.2 5.8±0.3 5.9±0.5 6.8±0.5 6.8±0.3∑SAT. 15.8±0.6 16.8±0.6 17.4±0.4 17.6±0.6 16.5±0.9 16.6±1.2 14.5±0.6 14.7±0.4 15.0±0.4 15.4±0.3 15.2±0.6 15.7±0.6∑MUFA 13.8±0.4 12.6±0.4 12.1±0.2 12.2±0.4 12.7±0.7 12.5±0.4 12.7±0.3 11.7±0.2 11.9±0.3 11.8±0.5 11.8±0.3 11.4±0.2∑PUFA 55.9±0.4ab 55.5±0.4ab 55.6±0.5ab 54.8±0.6b 51.6±0.5c 51.2±0.4c 53.9±0.3bc 56.4±0.7ab 58.3±0.5a 56.8±0.4ab 56.0±0.5ab 55.4±0.3ab

∑Branched 6.8±0.4 7.6±0.6 8.0±0.6 9.0±0.8 9.6±0.7 9.2±0.9 8.4±0.5 9.4±0.9 8.4±0.6 9.7±0.3 9.8±0.9 10.1±0.7∑DMA 0.8±0.0 0.8±0.0 0.8±0.0 0.7±0.0 0.7±0.0 0.7±0.0 1.3±0.1 0.8±0.1 0.9±0.1 1.1±0.1 1.0±0.0 0.9±0.0∑NMI 9.1±0.5ab 8.8±0.5ab 8.2±0.5ab 8.1±0.8ab 11.3±1.1ab 12.2±1.0a 11.4±0.6ab 9.5±0.3ab 7.4±0.4b 7.4±0.5b 8.4±0.6ab 8.5±0.3ab

NMI = Non methylene-interrupted fatty acids; DMA = dimethylacetal. Results are the mean±S.E. See Table 3 for statistical analysis.

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not observe a consistent pattern of 18:5n−3 that would confirm aparticular bloom of this species. Nevertheless, 18:5n−3 in oysterwould not necessarily reflect its high content in microalgae for severalreasons such as retro-conversion to 18:4n−3 or use as intermediatein the synthesis of 22:6n−3, as previously suggested for other dino-flagellates species (Leblond and Chapman, 2000). Sterols are alsoused for food imprint of ingesting phytoplankton (Idler andWiseman, 1972; Volkman et al., 1998; Giner and Wikfors, 2011).Campesterol and 24‐methylenecholesterol are the major sterol(62%) in the diatoms (Yamaguchi et al., 1986), whereasbrassicasterol, stigmasterol, and β-sitosterol are characteristic main-ly of flagellates (Soudant et al., 1998; Mohammady, 2004). In accor-dance, the increased proportion of free brassicasterol (October toDecember), stigmasterol (November and December), and β-sitosterol(November), as potential signatures of flagellates, suggests their presencein this period.

A negative load of the proportion of the 20:4n−6 in phospholipidswas found in PC2, although there is probably little relation betweenfood availability and low level of this fatty acid. A high concentrationof 20:4n−6 is found in phosphatidylinositol (PI), from where it isused as a precursor of prostaglandin production (Deridovich andReunova, 1993; Rowley et al., 2005), which promotes spawning inmollusks (Ono et al., 1982; Martínez et al., 2000). The female gonadand larvae of P. maximus were also found to have a PI enriched withthe 20:4n−6 FA (Soudant et al., 1996). On the other hand, Pazos etal. (1996) found a negative correlation of the 20:4n−6 FA in phos-pholipids with the condition index during the reproductive cycle ofC. gigas. Indeed, the proportion of the 20:4n−6 FA in phospholipidswas positively correlated with temperature (r=0.75, Pb0.05), withhigher values during the end of the spawning season and at thehighest temperature (31 °C). But contrary to above studies, variationsof 20:4n−6 both in neutral and polar lipids of C. cortenziensisappeared not or only weakly linked to gonad development. Highlevels of the 20:4n−6 FA in polar lipids has also been related to stress(Delaporte et al., 2006) in C. gigas, and particularly during stressfulconditions of high temperature in C. virginica (Pernet et al., 2007). Itmay be speculated that increase of 20:4n−6 is associated to temper-ature related stress underwent by C. cortenziensis during summer.

The PC1 was composed of positive loadings of total carbohydratesand lipids, the ratio of 16:1n−7/16:0 and the sum of NMI in the neu-tral lipids (negative loading), the proportion of 20:5n−3 in neutrallipids, and the proportion of esterified sterols. The values of the PC1were high in winter and spring prior to gonad development, and de-creases during summer, reaching a minimum in autumn and startingto increase again in November (Fig. 1A). Annual variation in PC1 wasopposite to the temperature cycle and, from November–Decemberfollowed the increase in food availability (PC2) indicating an accumu-lation of reserves when temperatures decrease and just after the firstphytoplankton bloom. Several variables contributing to PC1, such ascarbohydrates, lipids and esterified sterols further point that theyconstitute a process of storage that starts during the resting reproduc-tive phase, as it will be discussed below.

The increase of esterified sterols measured from December couldbe associated with the phytoplankton bloom and the storage for thenext reproductive period. The most abundant sterol (both as freeand esterified forms) in tissues of females of C. corteziensis was cho-lesterol, as has been reported in other species of oysters (Teshima etal., 1980; Berenberg and Patterson, 1981; Gordon and Collins, 1982;Soudant et al., 1996; 1998). Bivalve mollusks have a limited abilityto synthesize cholesterol or convert it from phytosterols (Teshimaand Patterson, 1981; Holden and Patterson, 1991). The levels of ester-ified sterols were also clearly linked to PC1, namely to accumulation ofreserves but only partially to gonad development (from spring to fall).Such pattern differs some those observed in temperate specieswhere the esterified sterols increase during gametogenesis in femalegonads and are consumed during the larval development of P.

maximus (Soudant et al., 1996, 1998), and during embryogenesis ofC. gigas (Soudant et al., 2000).

Several fatty acids contribute positively or negatively to PC1. The20:5n−3 FA is accumulated in membrane, but it is also an energy sourceduring embryogenesis (Soudant et al., 1996) that could explain its accu-mulation in neutral lipids prior to gonad development. In contrast, the22:6n−3 FA is not generally used as an energy source, because it con-served in cellularmembranes for functional purposes (Hulbert, 2007). Al-though it has been reported that the content of 20:5n−3was higher than22:6n−3 in neutral lipids and similar in phospholipids during the repro-ductive cycle of C. gigas (Pazos et al., 1996), we found a higher content of22:6n−3 than 20:5n−3 in both fractions for C. corteziensis.

The ratio of 16:1n−7/16:0 is considered as a diatom biomarker. Itscontribution to PC1 rather than to PC2,which is associated to aNovemberbloom, is given by diatoms presence in Bahia Ceuta during most of theyear (Ibarguen-Zamudio, 2006). In contrast, the PC1 was also composedof the sum of the total NMIs in neutral lipids, but with a negative loading.TheNMIs are fatty acids that can be synthesized bymollusks (Kraffe et al.,2010) and they are commonly found in filter-feeders (Paradis andAckman, 1977; Copeman and Parrish, 2003). An increase in synthesis ofthe NMIs has been suggested to be associatedwith low levels of essentialfatty acids during maturation and reproduction in several bivalve mol-lusks (Paradis and Ackman, 1977; Soudant et al., 1999; Palacios et al.,2005). As shown by the PC1, there was a decrease of the NMIs whenthere was an accumulation of the 20:5n−3 or other reserves in tissues,supporting the idea that these fatty acids are synthesized during theshortage of the HUFAs, especially the 20:5n−3.

The third principal component (PC3), reflecting gonad development,was characterized by the accumulation of vitellin (Vn), and the increasein gonad coverage area (CGA). The role of vitellin in oyster reproductionas an important intra-oocyte reserve was established in C. gigas (Suzukiet al., 1992) and C. corteziensis (Arcos et al., 2009). Differential variationof CGA, oocyte diameter and Vn in relation to reproductive stages basedon histological analysis, suggest that the size of oocyte and gonad in-creased mainly between previtellogenic and vitellogenic stage, whereasVn concentration increased continuously from the previtellogenoic topostvitellogenic stages (Arcos et al., 2009). However these subtle differ-ences do not fully explain why low values of Vn and high values CGAwere observed in the present study in April 2005. Thereafter, with in-creasing temperatures, both variables presented practically the sameseasonal pattern with high values in May followed by a decrease inJune 2005, which confirms a first spawning period in June characterizedhistologically by Rodríguez-Jaramillo et al. (2008) at the same location.Then, Vn, and to a lower extent CGA, had a similar, although not so pro-nounced, increase during the second maturation (summer), concomi-tantly with the highest values of temperature, and then decreasedagain by December parallel to the decrease in temperature and the in-crease in food availability. A short resting reproductive period was ob-served from December to March and gonad development was againobserved from April with increasing temperatures. The lack of a full sea-sonal correspondence between food availability (PC2) and gonad devel-opment (PC3) points out that the reproductive strategy seemed to beconservative as reserves accumulated during fall and winter, aremaintained in spring and then decreased by the first spawning in Juneto finally reach the minimum values during further rematuration andspawning in summer. Therefore allocation of energy is directed to gonads(increase in CGA) mainly in spring during April and May, when reservesare still high in oysters, either in gonads or other tissues. These reserves,particularly carbohydrates, were gradually depleted from the firstspawning in June till the phytoplankton bloom in November, a periodinwhich rematuration and spawning occurred. This decrease in carbohy-drate levels suggested they are used as an energy source or for synthesisof somenonessential fatty acids to sustain vitellogenesis or spermatogen-esis, as shown for C. gigas (Matus de la Parra et al., 2005). The further in-crease of carbohydrates at the resting phase of the reproductive cycle isalso in accordance with results obtained for C. gigas and C. virginica,

Table 6Individual fatty acid proportion (%) from polar lipids (mainly phospholipids) in Crassostrea corteziensis female tissues of sampled from April 2005 to April 2006 in Laguna de Ceuta, Sinaloa, Mexico.

2005 2006

Apr May Jun Jul Sep Oct Nov Dec Jan Feb Mar Apr

14:0 0.3±0.1 0.2±0.0 0.3±0.0 0.2±0.0 0.4±0.1 0.3±0.0 0.4±0.0 0.4±0.1 0.3±0.0 0.2±0.0 0.2±0.0 0.2±0.116:0 3.1±0.8ab 2.0±0.4b 2.9±0.2ab 2.1±0.4b 4.1±0.8ab 3.7±0.9ab 6.7±1.1a 3.4±0.8ab 2.0±0.3b 1.7±0.3b 1.9±0.3b 1.7±0.5b

17:0 0.4±0.1ab 0.2±0.1b 0.4±0.0ab 0.2±0.0b 0.5±0.1ab 0.4±0.1ab 0.7±0.1a 0.4±0.1ab 0.2±0.0b 0.2±0.0b 0.2±0.0b 0.2±0.0b

18:0 1.4±0.4ab 0.7±0.2b 1.3±0.1ab 0.9±0.2ab 1.9±0.4ab 1.5±0.3ab 2.7±0.5a 1.4±0.3ab 0.7±0.1b 0.7±0.1b 0.8±0.1ab 0.7±0.2b

16:1n−9 1.9±0.2 1.2±0.2 1.2±0.1 1.3±0.1 1.3±0.1 1.4±0.1 1.3±0.1 1.3±0.1 1.3±0.1 1.0±0.3 1.1±0.1 1.1±0.116:1n−7 2.2±0.2ab 2.5±0.2a 1.7±0.1abc 2.0±0.2abc 1.7±0.2abc 1.7±0.2abc 1.1±0.1c 1.3±0.1bc 1.9±0.1abc 2.1±0.2ab 2.1±0.1abc 2.0±0.1abc

18:1n−9 1.7±0.2b 2.1±0.1ab 2.4±0.1ab 2.4±0.1ab 2.1±0.2ab 2.4±0.2ab 2.6±0.1a 2.1±0.1ab 1.8±0.1ab 1.8±0.1ab 1.7±0.1ab 1.8±0.0ab

18:1n−7 3.4±0.3 3.6±0.3 3.0±0.2 3.0±0.3 2.9±0.4 2.8±0.4 2.3±0.2 2.5±0.2 3.1±0.1 3.4±0.1 3.2±0.2 3.2±0.220:1n−7 1.7±0.2 1.9±0.1 1.6±0.1 1.6±0.1 1.8±0.1 1.8±0.1 1.7±0.1 1.6±0.1 1.6±0.1 1.7±0.1 1.9±0.1 1.8±0.118:2n−6 1.3±0.1b 1.6±0.1ab 1.7±0.1ab 1.8±0.1ab 1.3±0.2b 1.3±0.2b 2.2±0.1a 1.7±0.1ab 1.5±0.1ab 1.5±0.1ab 1.5±0.1ab 1.5±0.0ab

18:3n−3 1.8±0.1b 2.0±0.1b 2.2±0.2b 1.9±0.1b 1.5±0.2b 1.3±0.2b 3.2±0.2a 1.8±0.1b 1.9±0.1b 1.9±0.1b 1.8±0.1b 1.7±0.1b

18:4n−3 2.4±0.1abc 2.5±0.2abc 2.2±0.2bc 1.9±0.2bc 1.3±0.2c 1.4±0.3c 3.8±0.3a 2.4±0.1abc 3.3±0.2ab 3.0±0.1ab 3.0±0.2ab 2.9±0.3ab

18:5n−3 1.1±0.1cd 1.4±0.1bcd 2.1±0.2abcd 2.5±0.3abc 2.9±0.3a 2.6±0.2ab 1.5±0.1bcd 1.1±0.1cd 0.9±0.0d 1.0±0.1cd 0.9±0.0d 1.3±0.1cd

20:4n−6 2.8±0.2bcd 3.1±0.1abcd 3.5±0.1abc 3.8±0.3ab 4.0±0.3a 3.9±0.3a 2.8±0.2bcd 2.4±0.2cd 2.0±0.1d 2.2±0.1d 2.2±0.1d 2.1±0.1d

20:5n−3 16.9±0.8a 16.0±0.8a 12.6±0.7bc 12.5±1.0bc 10.6±1.2c 10.3±1.1c 11.4±0.4c 14.7±0.7ab 15.4±0.4ab 17.4±0.3a 16.2±0.8a 15.4±1.0ab

22:4n−6 1.1±0.0 1.3±0.1 1.7±0.1 1.7±0.1 1.5±0.0 1.6±0.1 1.2±0.2 1.5±0.4 1.3±0.0 1.2±0.0 1.1±0.0 1.1±0.022:5n−3 2.4±0.1 2.3±0.1 2.4±0.1 2.5±0.1 2.6±0.1 2.6±0.1 2.3±0.1 2.7±0.2 2.1±0.0 2.3±0.1 2.3±0.1 2.2±0.122:6n−3 27.9±0.8bcd 29.2±0.3bc 30.0±0.5ab 29.7±1.0ab 25.7±0.6d 26.8±0.5cd 22.5±0.5e 28.5±0.6bcd 32.5±0.4a 29.3±0.7bc 29.3±0.3bc 30.6±0.5ab

22:2i NMI 1.1±0.0b 1.1±0.1b 1.5±0.1ab 1.5±0.2ab 1.7±0.1ab 1.9±0.1a 1.7±0.1ab 1.7±0.1ab 1.2±0.1b 1.1±0.1b 1.1±0.1b 1.2±0.1b

22:2j NMI 8.2±0.5bc 8.3±0.5bc 7.2±0.5bc 7.2±0.8bc 10.0±0.9bc 10.9±0.7a 9.7±0.7bc 8.1±0.3bc 6.6±0.3b 6.8±0.6bc 7.7±0.5bc 7.8±0.4bc

∑SAT. 5.6±1.5ab 3.6±0.7ab 5.3±0.3ab 4.0±0.7ab 7.5±1.5ab 6.5±1.4ab 11.1±1.8a 6.0±1.3ab 3.6±0.4ab 3.1±0.5b 3.6±0.5ab 3.1±0.8b

∑MUFA 15.4±0.3 14.6±0.4 13.9±0.3 14.2±0.6 14.1±0.8 14.1±0.7 13.2±0.2 12.8±0.1 13.5±0.3 13.5±0.6 13.5±0.5 13.1±0.3∑PUFA 62.7±1.5abc 64.4±0.7ab 63.8±0.8ab 63.8±1.1ab 57.3±1.7bc 57.6±1.8bc 56.1±0.9c 62.2±1.6abc 66.1±1.0a 65.0±0.4ab 63.6±1.1abc 63.7±0.9ab

∑Branched 7.6±0.3 8.8±0.7 9.2±0.6 10.4±0.8 10.5±0.7 10.2±0.7 8.8±0.6 10.3±1.0 9.5±0.7 11.1±0.4 11.1±1.0 11.6±0.7∑DMA 0.9±0.0bc 0.9±0.0bc 0.9±0.0bc 0.8±0.0c 0.8±0.0c 0.8±0.0c 1.3±0.1a 0.9±0.1bc 1.1±0.1ab 1.3±0.1a 1.2±0.0ab 1.1±0.0ab

∑NMI 10.2±0.6ab 10.2±0.6ab 9.4±0.6ab 9.4±0.9ab 12.5±1.0ab 13.5±0.8a 11.9±0.8ab 10.5±0.4ab 8.4±0.4b 8.5±0.6b 9.6±0.6ab 9.7±0.4ab

NMI = Nonmethylene-interrupted fatty acids; DMA = dimethylacetal. Results are the mean±S.E. See Table 3 for statistical analysis.

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where an accumulation of glycogen was observed at the end of fall andwinter (Deslous-Paoli and Héral, 1988).

Temperate species such as C. gigas and C. virginica have a conserva-tive strategy, where glycogen reserves stored in the vesicular connec-tive tissue (Berthelin et al., 2000) during autumn and part of winterare used together with available food during the spring phytoplanktonbloom to sustain the gametogenic process (Deslous-Paoli and Héral1988; Matus de la Parra et al., 2005) that usually concludes in a singlespawning in summer (Heffernan et al., 1989; Lango-Reynoso et al.,2006). Although the reproductive period of C. cortenziensis is occurringover a longer period, the typical pattern of a conservative strategy wasobserved with the increase in the accumulation of reserves at the endof the period of main reproductive activity when temperature is de-creasing and food availability increased with phytoplanctonic blooms.The factor analysis presented in this work was adequate to describethis major picture with a particular identification of the variation ofmain factors influencing reproduction throughout the year of this spe-cies at this location.

Acknowledgments

We are grateful to J. Guevara (Ostrícola Guevara) for oysterdonation. Special thanks to J.L. Ramírez, C. Rodríguez-Jaramillo,M. Reza, F. Hernández-Sandoval, M. Manzano-Sarabia, E. Meza, O. Arjona,G. González, C. Becerra, and F. Pérez for their technical assistance and toDr. Ellis Glazier for editing the English language text. This research wassupported by grants from SAGARPA 2003-02-035 to A.M. Ibarra andSEP-CONACYT2006‐24333 to E. Palacios, and a fellowship fromCONACYTto M.A. Hurtado.

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