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Fatty acids of densely packed embryos of Carcinus maenas reveal homogeneous maternal provisioning and no within-brood variation at hatching.

Abstract. Embryonic development of decapod crustaceans relies on yolk reserves supplied to offspring through maternal provisioning. Unequal partitioning of nutritional reserves during oogenesis, as well as fluctuating environmental conditions during incubation, can be sources of within-brood variability. Ultimately, this potential variability may promote the occurrence of newly hatched larvae with differing yolk reserves and an unequal ability to endure starvation and/or suboptimal feeding during their early pelagic life. The present study evaluated maternal provisioning by analyzing fatty acid (FA) profiles in newly extruded embryos of Carcinus maenas. Also assessed were the dynamics of such provisioning during embryogenesis, such as embryo location within the regions of the brooding chamber (left external, left internal, right external, and right internal). The FA profiles surveyed revealed a uniform transfer of maternal reserves from the female to the entire mass of embryos, and homogeneous embryonic development within the brooding chamber. Although C. maenas produces a densely packed mass of embryos that are unevenly distributed within its brooding chamber, this factor is not a source of within-brood variability during incubation. This finding contrasts with data already recorded for larger-sized brachyuran crabs, and suggests that the maternal behavior of C. maenas promotes homogeneous lipid catabolism during embryogenesis.

Introduction

Decapod crustaceans have become popular model organisms with which to address important topics in marine ecology, such as those related to parental care (Fernandez et al., 2000). Females of most of these species (except for the Dendrobranchiata) brood their embryos in their abdomen for variable periods of time, generating a favorable environment for their offspring--at least until hatching. This form of parental care is known to present significant energetic costs due to the active brooding behavior of the ovigerous females (Fernandez et al., 2000; Fernandez and Brante, 2003).

Embryonic development in decapod crustaceans is lecithotrophic; thus, the quantitative and qualitative transfer of nutrients during oogenesis, from the female to what will later be the yolk reserves of the developing embryos, plays a key role in embryogenesis (Rosa et al., 2007). Maternal provisioning to developing embryos has been extensively investigated in crustaceans, namely, through biochemical analysis of female gonads (e.g., Rosa and Nunes, 2002, 2003; Smith et al., 2004) and newly extruded embryos (e.g., Morais et al., 2002; Rosa et al., 2003, 2005; Calado et al., 2005; Wu et al., 2007; Li et al., 2012). Nonetheless, to date, no study has ever attempted to determine if maternal provisioning in brachyuran crabs is a source of offspring variability, especially in species that brood large numbers of embryos. In other words, do females provide comparable yolk reserves to all embryos within a brood?

Another potential source of offspring variability, already highlighted for brachyuran crabs, concerns the supply of oxygen to developing embryos during their incubation in the brooding chamber (Fernandez et al., 2003). Indeed, embryos located at the periphery of the brooding chamber are likely to be more easily oxygenated than those in the inner regions (Fernandez et al., 2003; Fernandez and Brante, 2003). Brachyuran crabs commonly display densely packed embryos, but, unlike other benthic marine invertebrates (e.g., opisthobranchs), are not covered with protective gel. This gel enhances the passive provision of oxygen to embryos located in the inner regions of the brood (Strathmann and Strathmann, 1995; Lee and Strathmann, 1998). In this way, while suitable levels of oxygenation may be difficult to achieve in inner embryos of densely packed broods, female brachyuran crabs have evolved active brooding behaviors (e.g., abdominal flapping) that assure a suitable supply of oxygen to the entire brood (Baeza and Fernandez, 2002).

Oxygen limitation may delay early ontogeny (Hartnoll and Paul, 1982; Cohen and Strathmann, 1996; Brante et al., 2003; Fernandez et al., 2003); hypoxia-induced metabolic suppression decreases the potential for embryo development (Zhou et al., 2001; Hochachka and Somero, 2002; Rosa et al., 2013; Alter et al., 2015). Thus, if embryos located in the internal regions do not receive the same, or similar, levels of oxygen as those embryos in the external regions, their energetic reserves will be catabolized at a slower rate (Chaffee and Strathmann, 1984; Fernandez et al., 2003). Under this scenario, significant shifts are expected to occur in the biochemical profile of internal and external embryos of decapods during the incubation period.

In the present study, we used the fatty acid (FA) profile of embryos of Carcinus maenas (Linnaeus, 1758) as a biochemical proxy to test the following null hypotheses: 1) whether maternal provisioning during oogenesis in C. maenas was homogeneous, and the whole mass of newly extruded embryos displayed a similar FA profile; and 2) that the location of developing embryos within the brooding chamber of females does not influence their FA profile by the end of embryogenesis (prior to hatching). Since numerous studies have already addressed the biochemical dynamics of FA during embryogenesis in decapod crustaceans (e.g., Rosa et al., 2005, 2007; Li et al., 2012), shifts in the pool of FAs present in early- and late-stage embryos are not discussed.

Materials and Methods

Sampling

Thirty ovigerous females of Carcinus maenas were collected, using trawl nets, between March and April 2012, in Canal de Mira, Ria de Aveiro (40[degrees] 37' 17" N, 8[degrees] 44' 56" W), a coastal lagoon on the northwestern coast of Portugal. Females were transported to the laboratory and sorted according to their carapace width (CW), measured between the first pair of lateral spines, and the development stage of their embryos. Developing embryos were classified by the following criteria (Rosa et al., 2007): early-stage (newly extruded embryos), uniform yolk and absence of cleavage and eyes; or late-stage (embryos ready to hatch in < 48 h), almost no yolk present and embryo fully developed. From the 30 ovigerous females collected, 10 specimens of a similar size (average CW of 44.1 [+ or -] 0.7 mm) were selected, 5 brooding embryos in the early stage and 5 brooding embryos in the late stage. To identify their locations, the brooding chamber of each female was first divided into two sides, the left (L) and right (R); then each side was divided into two regions, the external (E; periphery of the egg mass) and internal (I; inner region of the egg mass), resulting in a total of four areas: left external (LE); left internal (LI); right external (RE); and right internal (RI). All embryos from each area were carefully removed with forceps. A total of 40 embryo samples (4 areas within the brooding chamber X 2 embryonic stages X 5 females = 40) were collected for analysis. Immediately after collection, 30 embryos from each sample were haphazardly selected for measurement, using a stereomicroscope; their volume was determined using formula V: V = (4/3) [pi] [r.sup.3], for spheroid embryos. All embryo samples were freeze-dried and stored at -32 [degrees]C for later biochemical analysis.

Fatty acid analysis

Total lipid extracts were obtained using the Bligh and Dyer (1959) method. A subsample of 15 mg of freeze-dried embryos was selected from the total egg mass present in each of the four regions (LE, LI, RE, and RI) of each individual female (4 regions X 5 females X 2 embryonic stages = 40). Each subsample was resuspended in 1 ml of ultrapure water; 3.75 ml of methanol: chloroform 2:1 (v/v) was added to the suspension, which was vortexed and incubated on ice for 30 min. An additional volume of 1.25 ml of chloroform was added, along with 1.25 ml of ultrapure water. Following vigorous vortexing, samples were centrifuged at 1000 rpm for 5 min at room temperature to obtain a two-phase solution: an aqueous top phase and an organic bottom phase, from which the lipids were retrieved. Lipid extracts were dried with a nitrogen flow and stored at -32 [degrees]C for posterior analysis.

To quantify the total amount of phospholipids (PL), a phosphorus assay was performed according to Bartlett and Lewis (1970). To quantify the total PL extract, 5% of the sample volume was used, then dried with a nitrogen flow. Perchloric acid (70%) was added to the samples, which were then incubated for 1 h at 180 [degrees]C. After incubation, 3.3 ml of water, 0.5 ml of ammonium molybdate (2.5% m/v), and 0.5 ml of ascorbic acid (10% m/v) were added to each sample, followed by incubation for 10 min at 100 [degrees]C in a water bath. Standard solutions from 0.1 to 3.0 [micro]g of phosphate underwent the same treatment as the samples. Absorbance of the mixtures was measured at 800 nm, at room temperature, in a microplate ultraviolet-visible (UV-vis) spectrophotometer.

Fatty acids were analyzed by gas chromatography-mass spectrometry (GC-MS) after transesterification of the embryos' total lipid extracts (30 [micro]g of total PL). Fatty acid methyl esters (FAMEs) were prepared using a methanolic solution of potassium hydroxide (2 mol [1.sup.-1]) according to the method described previously by Aued-Pimentel et al. (2004). The FAMEs were resuspended in 40 [micro]m of hexane; 2 [micro]m of this solution was used for GC-MS analysis on an Agilent Technologies 6890N Network (Santa Clara, CA), equipped with a DB-1 column 30 m in length, 0.25 mm internal diameter, and 0.1 [micro]m film thickness (J&W Scientific, Folsom, CA). The GC-MS was connected to an Agilent 5973 Network Mass Selective Detector (Agilent Technologies) operating with an electron impact mode at 70 eV and scanning at a range of 40-500 m/z in a 1-second cycle in full-scan mode acquisition. The oven temperature was programmed from an initial temperature of 40 [degrees]C, remaining at this temperature for 0.5 min, followed by a linear increase to 220 [degrees]C at 20 [degrees]C per min, a linear increase of 2 [degrees]C per min to 240 [degrees]C, then 5 [degrees]C per min until reaching 250 [degrees]C. The injector was set at 220 [degrees]C and the detector, at 230 [degrees]C. Helium was used as the carrier gas at a flow rate of 1.7 ml/min. Identification of the FAMEs was performed by comparing the retention time and mass spectrum of each FAME relative to 34 mixed FAME standards (C6-C24, Supelco 37 Component FAMEs Mix), and confirmed by comparison with the Wiley chemical database and the spectral library, the "AOCS Lipid Library" (AOCS, 2012).

Data analysis

After checking for assumptions (using the Shapiro-Wilk and Levene's tests to determine normality and homogeneity of variance, respectively), we tested for significant differences in the volume of the early- and late-stage embryos, using a nested ANOVA with two factors: side of the brooding chamber where the embryo was located (with two levels: left and right), and region within the brood (with two levels: external and internal) nested in the side of the brooding chamber. Two-way ANOVAs were performed using STATISTICA v8 (StatSoft Inc., Tulsa, OK).

The relative content of each fatty acid was expressed as the percentage of the total pool of FAs. For statistical analysis, only FAs representing more than 1 % of the total pool of FAs were considered. For a better understanding of our results, it is important to know that we considered only the classes of FAs containing the most abundant FAs, namely, saturated FA (SFA), monounsaturated FA (MUFA), polyunsaturated FA (PUFA), highly unsaturated FA (HUFA), epoxy FA (EpFA), and branched FA (BrFA). While PUFAs are commonly defined as all FAs with [greater than or equal to] two double bonds, in the present study we distinguished between PUFA (FAs with two or three double bonds) and HUFA (FAs with [greater than or equal to] four double bonds).

Multivariate statistical analyses were performed to determine significant differences in 1) the fatty acid profile of early-stage embryos; 2) FA class profile of early-stage embryos; 3) FA profile of late-stage embryos; and 4) FA class profile of late-stage embryos, from the four different areas of the brooding chamber (LE, LI, RE, and RI). Prior to statistical analysis, and in order to down-weight the contributions of quantitatively dominant FA, the raw data matrix was log(x + 1)-transformed. Immediately afterward, a new matrix was assembled using the Bray-Curtis similarity coefficient.

Permutational multivariate analyses of variance (PERMANOVA) were used to compare FA composition and FA class composition of the four different areas of the brooding chamber (LE, LI, RE, and RI) (Kelly and Scheibling, 2012; Anderson and Walsh, 2013). PERMANOVAs were performed for the early- and late-stage embryos, using two factors: side of the brooding chamber (with two levels: left and right) and region of the brooding chamber (with two levels: external and internal) nested in the side of the brooding chamber. To visualize inter-individual differences between samples, Principal Coordinate Analyses (PCO) were performed for the early- and late-stage embryos. The contribution of the FAs to inter-individual differences was plotted in the PCO of each embryonic stage. All multivariate statistical tests were performed with Primer 6.1 software with PERMANOVA add-on (Primer-E Ltd., Plymouth, UK).

Results

While embryo volume of Carduus maenas doubled during incubation, no significant differences in average volume were detected between the early- and late-stage embryos brooded in each region of the brooding chamber (LE, LI, RE, and RI) ([F.sub.1,596] = 0.003, P = 0.957; [F.sub.1,596] = 0.002, P = 0.912; V =0.013 [+ or -] 0.001 m[m.sup.3] and V = 0.026 [+ or -] 0.002 [mm.sup.3], for early- and late-stage embryos, respectively).

The most relevant fatty acids recorded from each area of the brooding chamber, and for the entire mass of embryos, are summarized in Tables 1 and 2 (embryos in the early and late stages, respectively). For a detailed list of all FAs identified, see supplementary tables (SM_l.doc, http://www.biolbull.org/content/supplemental). The 5 most abundant FAs, representing 50% of the total pool of FAs recorded, were the same in embryos from both the early and late stages (Fig. 1), namely, 16:0 (palmitic acid; PA), 16:1n-7 (palmitoleic acid), 18:1n-9 cis (oleic acid), 20:5n-3 (eicosapentaenoic acid; EPA), and 22:6n-3 (docosahexaenoic acid; DHA).

In the early-stage embryos, the most abundant fatty acid class was the monounsaturated fatty acids, representing around 40% of the total pool of FAs (22.82% [+ or -] 1.92% for SFA, 40.09% [+ or -] 3.97% for MUFA, and 22.77% [+ or -] 3.34% for HUFA) (Table 1). However, in the late-stage embryos the contribution of MUFA decreased, reaching values more like those of SFA and HUFA (27.60% [+ or -] 2.87% for SFA, 32.44% [+ or -] 2.50% for MUFA, and 29.14% [+ or -] 3.92% for HUFA) (Table 2).

PERMANOVA analysis of embryos in the early stage showed no significant effect of factor side or region on the FA profiles (side: P = 0.729; region: P = 0.991) or FA classes (side: P = 0.674; region: P = 0.892). Furthermore, the same analysis of embryos in the late stage showed no significant effect of side or region on the FA profiles (side: P = 0.932; region: P = 0.961) or FA classes (side: P = 1; region: P = 0.972).

Principal Coordinate Analyses revealed low variability between the different areas of the brooding chamber in the early-stage (Fig. 2a) and late-stage embryos (Fig. 2b). Nevertheless, PCOs showed the natural variability of sampled females and the contribution of FAs to these inter-individual differences.

Discussion

The results reported in the present study do not allow us to reject our first null hypothesis: that maternal provisioning during oogenesis in Carcinus maenas is homogeneous, and the whole mass of newly extruded embryos displays a similar fatty acid profile. This finding does not agree with Leal et al. (2013), who recorded significant differences in the FA profile of newly extruded embryos (early-stage) across the brooding chamber of the European clawed lobster Homams gammarus. We emphasize that our work is only the third study to address this topic in decapods. As the study by Pochelon et al. (2011) focused solely on an intermediate stage of embryonic development (neither early or late), results from the present study are comparable only to those from Leal et al. (2013). Therefore, it may be speculative to advance any solid argument about why within-brood variation was not noted in the newly extruded embryos of C. maenas. Nevertheless, we must stress that C. maenas and H. gammarus belong to different infraorders, Brachyura and Astacidae, respectively (De Grave et al., 2009). It is possible that the metabolic pathways involved in lipid accumulation during oogenesis in these two species may somehow differ.

Maternal provisioning is influenced by maternal nutritional status (Tuck et al., 1997; Racotta et al., 2003), environmental conditions, feeding regimes (Brillon et al., 2005; Calado et al., 2010), and factors related to the biology of the species (e.g., time between molts or subsequent broods) (Vensimo et al., 2011). During ovarian maturation, as much as 60% of the lipid content of female decapods may be transferred to their oocytes (Herring, 1973). The source of these lipids is still unclear; lipids may be mobilized from reserves stored in the hepatopancreas (Harrison, 1990), or derived directly from the ingestion of food (Clarke, 1982).

Although ovarian maturation is a continuous process in decapod crustaceans (e.g., Rotllant et al., 2005; Seegert et al, 2014), differences between the Leal et al (2013) study and our own results may have originated from the predominance of one pathway in the allocation of reserves for each decapod species. In this way, different lipidomic pathways during ovarian maturation might justify a dissimilar response in within-brood provisioning between species. Lipids originating in the hepatopancreas are not fully depleted during ovarian maturation (Tuck et al., 1997; Rosa and Nunes, 2003; Smith et al., 2004), and are more homogeneous than those lipids derived from dietary resources. Thus, species showing a prevalence for resource allocation pathways from hepatopancreas-originated lipid reserves are likely more apt to display homogeneous embryonic provisioning. On the other hand, as previous studies in lobsters and shrimps have already demonstrated (Cahu et al., 1995; Smith et al., 2004; Calado et al., 2010), female diet significantly affects fatty acid profiles shown by produced embryos. Therefore, species favoring this lipidomic pathway will likely display higher variability in embryonic provisioning. Despite the remarkable diversity of dietary items consumed by an opportunistic feeder such as C. maenas (Baeta et al., 2006; Chaves et al., 2010), in this study it was clearly shown that maternal provisioning is not a source of offspring variability, and that the incorporation of lipid reserves in developing oocytes must occur in a synchronous and uniform way during gonadal maturation.

Our data also do not allow us to reject our second null hypothesis, affirming that the location of developing embryos within the brooding chamber does not influence their fatty acid profile by the end of embryogenesis (prior to hatching). Previous studies by Baeza and Fernandez (2002) and Fernandez et al (2002, 2003) reported dramatic differences in oxygen availability between the center and the periphery of the brooding chamber of two large-sized brachyuran crabs (Cancer setosus and Homalaspis plana). Indeed, these differences are even more accentuated during early embryonic development (Fernandez and Brante, 2003). Such differences in oxygen levels within the brooding chamber invariably lead to varying metabolic rates (Alter et al., 2015), thus increasing energy demand and saturated fatty acid oxidation (Leal et al., 2013). Overall, as oxygen levels influence embryonic development (Hartnoll and Paul, 1982), internal embryos should present a delay when compared with external embryos (Fernandez et al., 2003). In our study, FA analysis showed synchronous embryonic development in the four brooding areas surveyed. In Carcinus maenas, oxygen conditions in the embryo mass are known to change drastically between early and late embryonic development (Fernandez and Brante, 2003). The absence of an asynchronous trend in developing embryos suggests that C. maenas exerts an efficient oxygen supply on the embryonic mass being incubated, through the well-known maternal behavioral patterns of abdominal flapping and pleopod movements (Baeza and Fernandez, 2002; Brante etal, 2003; Fernandez and Brante, 2003; Silva et al., 2007), which promote intense water movement (Fernandez etal, 2006).

Although female ventilation must be the main factor responsible for homogeneous embryonic development, other factors also may have contributed--although likely to a lesser extent--to such synchronous embryonic development. The increase in embryo volume during embryogenesis enlarges the embryonic surface area for gas exchange (Chaffee and Strathmann, 1984), and facilitates the passive supply of oxygen into the mass of developing embryos (Fernandez et al., 2006). However, the presence of branched, cyclopropyl, and odd-numbered fatty acids reflects the presence of a stable population of bacteria on developing embryos (Dalsgaard etal, 2003). The role of the bacterial communities present in developing embryos is still largely unknown. The presence of bacterial films covering developing embryos of marine invertebrates has been commonly associated with negative effects, such as decreased gaseous exchange, reduced embryonic development, and even death (Fisher, 1976; Biermann et al., 1992; Przeslawski and Benkendorff, 2005; Silva et al., 2007). Nevertheless, symbiotic bacteria may also prevent infection by pathogenic fungi on the surface of crustacean embryos (Gil-Turnes et al., 1989; Gil-Turnes and Fenical, 1992). In this regard, the presence of a structurally stable bacterial population during the embryogenesis of C. maenas (as revealed by the consistent levels of branched, cyclopropyl, and odd-numbered FAs) could impair the proliferation of deleterious microorganisms and fungi, thus exerting a protective function and indirectly favoring oxygen diffusion (Cohen and Strathmann, 1996; Cronin and Seymour, 2000; Peters et al., 2012).

Fernandez et al. (2003) also suggested that asynchronous development could imply asynchronous hatching. Although ovigerous females of C. maenas release their larvae during two or more main events (Zeng and [N.sub.a]ylor, 1997), according to our results these extended hatching events are probably not a consequence of asynchronous embryonic development. The rhythms of larval release are likely associated with tidal cycles in order to facilitate the export of these larvae from estuarine waters into the ocean (Queiroga et al., 1997; Anger, 2001).

In conclusion, the present study demonstrated that maternal investment in Carcinus maenas was uniformly distributed among newly extruded embryos, and that, in spite of a densely packed mass of embryos, those

incubated in the internal and external regions of the brood displayed similar biochemical profiles by the end of embryogenesis. Future studies should try to confirm this apparent efficiency shown by ovigerous females of C. maenas in providing suitable levels of oxygen to developing embryos, by coupling the experimental approaches described by Baeza and Fernandez (2002) and Fernandez et al (2002, 2003) with lipidomic analyses. This approach will be important to evaluate whether C. maenas is "an exception to the rule" within the Brachyura. By using species that are similar in size to C. maenas, and larger and smaller sympatric crab species, researchers may determine whether adult body size is the key variable ruling the efficiency of these brooding behaviors. It may also be possible to investigate whether this efficiency was shaped along the natural history of the Brachyura, and if it is somehow reflected in their phylogeny.

Acknowledgments

F. Rey, A. S. P. Moreira, and F. Ricardo were supported by Ph.D. scholarships (SFRH/BD/62594/2009, SFRH/BD/80553/2011, and SFRH/BD/ 84263/2012, respectively), and funded by the Fundacao para a Ciencia e Tecnologia (FCT) (QREN-POPH-Tipe 4.1--Advanced training, subsidized by the European Social Fund and national funds MEC). The present study was funded by FEDER through COMPETE, Programa Operacional Factures de Competitividade, and by [N.sub.a]tional funding through FCT, within the research project NO RESET PTDC/BIA-BIC/116871/2010.

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FELISA REY (1), ANA S. P. MOREIRA (2), FERNANDO RICARDO (1), MANUEL A. COIMBRA (2), M. ROSARIO M. DOMINGUES (2), PEDRO DOMINGUES (2), RUI ROSA (3), HENRIQUE QUEIROGA (1), AND RICARDO CALADO (1,*)

(1) Departamento de Biologia & CESAM, and (2) QOPNA, Departamento de Quimica, Universidade de Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal; and (3) MARE--Marine and Environmental Sciences Centre, Laboratorio Maritime da Guia, Faculdade de Ciencias da Universidade de Eisboa, Avenida Nossa Senhora do Cabo 939, 2750-374 Cascais, Portugal

Received 29 October 2015; accepted 29 February 2016.

(*) To whom correspondence should be addressed. E-mail: rjcalado@hotmail.com

Abbreviations: BrFA, branched fatty acid: CW, carapace width; EpFA, epoxy fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; GC-MS, gas chromatography-mass spectrometry; HUFA. highly unsaturated fatty acid; LE, left external region of brooding chamber; LI. left internal region of brooding chamber; MUFA, monounsaturated fatty acid; PL, phospholipids; PUFA, polyunsaturated fatty acid; RE, right external region of brooding chamber; RI, right internal region of brooding chamber; SFA, saturated fatty acid.

Table 1

Fatty acid (FA) profile (expressed as percentages of total pool of FAs)
of early-stage embryos of the green crab Carcinus maenas from four
different regions within the brooding chamber (left external (LE), left
internal (LI), right external (RE), and right internal (Rl)), and
average values ([+ or -] standard deviation) of all embryo masses

                          Left                 Left
Fatty acid             external (%)         internal (%)

14:0               0.96 [+ or -] 0.22   0.84 [+ or -] 0.08
16:0              15.94 [+ or -] 0.53  15.05 [+ or -] 0.78
18:0               4.16 [+ or -] 0.63   3.96 [+ or -] 0.33
[SIGMA] SFA (a)   23.09 [+ or -] 0.99  21.82 [+ or -] 0.56
16:1n-7           15.87 [+ or -] 2.65  15.01 [+ or -] 2.39
18:1n-9c          11.21 [+ or -] 1.29  10.95 [+ or -] 1.60
18:1n-7            4.75 [+ or -] 0.54   4.65 [+ or -] 0.77
20:1n-9            2.14 [+ or -] 1.04   2.19 [+ or -] 1.15
20:1n-7            3.26 [+ or -] 0.97   3.33 [+ or -] 1.04
[SIGMA] MUFA (b)  40.17 [+ or -] 4.77  39.71 [+ or -] 4.30
18:2n-6            0.84 [+ or -] 0.31   0.81 [+ or -] 0.27
18:3n-3            1.13 [+ or -] 0.14   1.12 [+ or -] 0.18
[SIGMA] PUFA (c)   6.08 [+ or -] 1.92   6.63 [+ or -] 1.67
20:4n-6            2.05 [+ or -] 0.29   2.13 [+ or -] 0.30
20:5n-3            8.46 [+ or -] 1.88   8.64 [+ or -] 1.72
22:5n-3            2.10 [+ or -] 0.18   2.32 [+ or -] 0.49
22:6n-3            8.34 [+ or -] 3.38   8.79 [+ or -] 3.18
[SIGMA] HUFA (d)  22.66 [+ or -] 3.60  23.80 [+ or -] 3.05
BrFA 1             1.95 [+ or -] 0.32   1.86 [+ or -] 0.20
BrFA 2             1.57 [+ or -] 0.17   1.50 [+ or -] 0.16
[SIGMA] BrFA (e)   5.68 [+ or -] 0.52   5.29 [+ or -] 0.35
EpFA 1             1.52 [+ or -] 0.41   1.81 [+ or -] 0.39
[SIGMA] EpFA (f)   1.74 [+ or -] 0.37   2.04 [+ or -] 0.35

                        Right                 Right
Fatty acid            external (%)         internal (%)

14:0               1.05 [+ or -] 0.11   1.04 [+ or -] 0.22
16:0              15.66 [+ or -] 1.72  16.53 [+ or -] 1.71
18:0               4.07 [+ or -] 0.71   4.03 [+ or -] 0.49
[SIGMA] SFA (a)   22.84 [+ or -] 2.50  23.76 [+ or -] 2.77
16:1n-7           15.79 [+ or -] 2.36  16.46 [+ or -] 2.51
18:1n-9c          11.06 [+ or -] 1.69  10.89 [+ or -] 1.82
18:1n-7            4.50 [+ or -] 0.60   4.95 [+ or -] 0.58
20:1n-9            2.21 [+ or -] 1.21   2.08 [+ or -] 1.06
20:1n-7            3.25 [+ or -] 1.09   3.13 [+ or -] 1.00
[SIGMA] MUFA (b)  40.12 [+ or -] 3.94  40.69 [+ or -] 4.19
18:2n-6            0.67 [+ or -] 0.32   0.85 [+ or -] 0.38
18:3n-3            1.19 [+ or -] 0.16   1.17 [+ or -] 0.17
[SIGMA] PUFA (c)   6.41 [+ or -] 1.24   6.01 [+ or -] 1.47
20:4n-6            2.80 [+ or -] 1.47   2.22 [+ or -] 0.48
20:5n-3            8.02 [+ or -] 2.10   8.22 [+ or -] 1.43
22:5n-3            2.07 [+ or -] 0.22   1.86 [+ or -] 0.24
22:6n-3            8.15 [+ or -] 3.30   7.96 [+ or -] 3.81
[SIGMA] HUFA (d)  22.72 [+ or -] 3.33  21.79 [+ or -] 4.14
BrFA 1             1.89 [+ or -] 0.22   1.97 [+ or -] 0.23
BrFA 2             1.54 [+ or -] 0.16   1.62 [+ or -] 0.24
[SIGMA] BrFA (e)   5.47 [+ or -] 0.84   5.58 [+ or -] 1.32
EpFA 1             1.58 [+ or -] 0.4    1.43 [+ or -] 0.50
[SIGMA] EpFA (f)   1.82 [+ or -] 0.42   1.66 [+ or -] 0.46

                      All embryo
Fatty acid            masses (%)

14:0               0.97 [+ or -] 0.18
16:0              15.75 [+ or -] 1.31
18:0               4.04 [+ or -] 0.51
[SIGMA] SFA (a)   22.82 [+ or -] 1.92
16:1n-7           15.77 [+ or -] 2.34
18:1n-9c          11.00 [+ or -] 1.48
18:1n-7            4.69 [+ or -] 0.60
20:1n-9            2.15 [+ or -] 1.03
20:1n-7            3.24 [+ or -] 0.94
[SIGMA] MUFA (b)  40.09 [+ or -] 3.97
18:2n-6            0.78 [+ or -] 0.30
18:3n-3            1.15 [+ or -] 0.16
[SIGMA] PUFA (c)   6.35 [+ or -] 1.48
20:4n-6            2.30 [+ or -] 0.79
20:5n-3            8.38 [+ or -] 1.67
22:5n-3            2.09 [+ or -] 0.33
22:6n-3            8.30 [+ or -] 3.16
[SIGMA] HUFA (d)  22.77 [+ or -] 3.34
BrFA 1             1.92 [+ or -] 0.23
BrFA 2             1.56 [+ or -] 0.18
[SIGMA] BrFA (e)   5.53 [+ or -] 0.80
EpFA 1             1.60 [+ or -] 0.42
[SIGMA] EpFA (f)   1.83 [+ or -] 0.40

Values are averages ([+ or -] standard deviation) of embryos from five
different females (n = 5).
(a) SFA (saturated fatty acid): 12:0, 14:0, 15:0, 16:0, 17:0, 18:0,
19:0, 20:0, 22:0.
(b) MUFA (monounsaturated fatty acid): 14:1n-5, 15:1n-1, 16:1n-7,
16:1n-5, 7-methyl-hexadec-6-enoate, 17:1n-9, 17:1n-8, 18:1n-9c,
18:1n-9t, 18:1n-7, 18:1n-5, 19:1n-9, 19:1n-8, 20:1n-9, 20:1n-7,
22:1n-11, 22:1n-9,
(c) PUFA (polyunsaturated fatty acid): 18:2n-6, 18:3n-3, 18:2n-3,
19:2n-7, 20:2n-9, 20:2n-7, 20:2n-6, 20:3n-4, 22:2n-9, 22:3n-6,
(d) HUFA (highly unsaturated fatty acid): 20:4n-6, 20:5n-3, 21:5n-3,
21:6, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3.
(e) BrFA (branched fatty acid): 4,8,12-trimethyl-tridecanoate,
9-methyl-tetradecanoate, 12-methyl-tetradecanoate (anteiso),
13-methyl-tetradecanoate (iso), 14-methyl-pentadecanoate (iso),
10-methyl-hexadecanoate, 14-methyl-hexadecanoate (anteiso),
15-methyl-hexadecanoate (iso), 15-methyl-heptadecanoate (anteiso),
16-mefhyl-heptadecanoate (iso), 16-methyl-octadecanoate (anteiso),
17-methyl-octadecanoate (iso).
(f) EpFA (epoxy fatty acid): 10,13-epoxy-l
1,12-dimethyl-octadecadienoate,
12,15-epoxy-13,14-dimethyl-eicosadienoate. BrFA 1,
14-methyl-hexadecanoate (anteiso); BrFA 2, 15-methyl-hexadecanoate
(iso); EpFA 1, 12,15-epoxy-13,14-dimethyl-eicosadienoate.

Table 2

Fatty acid (FA) profile (expressed as percentages of total pool of FAs)
of late-stage embryos of the green crab Carcinus maenas from four
different regions within the brooding chamber (left external (LE), left
internal (LI), right external (RE), and right internal (RI)), and
average values ([+ or -] standard deviation) of all embryo masses

                          Left                Left
Fatty acid             external (%)        internal (%)

14:0               0.48 [+ or -] 0.26   0.75 [+ or -] 0.42
16:0              17.82 [+ or -] 3.26  19.09 [+ or -] 2.80
18:0               6.04 [+ or -] 0.43   6.34 [+ or -] 0.41
[SIGMA] SFA (a)   26.44 [+ or -] 3.39  28.55 [+ or -] 2.87
16:1n-7            9.20 [+ or -] 1.34  10.06 [+ or -] 3.47
18:1n-9c          12.07 [+ or -] 1.49  11.28 [+ or -] 1.20
18: 1n-7           5.71 [+ or -] 0.91   5.35 [+ or -] 0.73
20:1n-9            1.23 [+ or -] 0.53   1.20 [+ or -] 0.45
20:1n-7            2.32 [+ or -] 0.89   2.06 [+ or -] 0.52
[SIGMA] MUFA (b)  32.19 [+ or -] 1.45  32.10 [+ or -] 4.29
18:2n-6            0.96 [+ or -] 0.43   0.77 [+ or -] 0.35
18:3n-3            0.72 [+ or -] 0.24   0.75 [+ or -] 0.13
[SIGMA]PUFA (c)    4.54 [+ or -] 1.28   4.19 [+ or -] 0.90
20:4n-6            2.52 [+ or -] 0.92   2.85 [+ or -] 1.28
20:5n-3           15.45 [+ or -] 1.96  14.48 [+ or -] 2.83
22:5n-3            1.32 [+ or -] 0.68   1.23 [+ or -] 0.31
22:6n-3            9.81 [+ or -] 2.36   8.78 [+ or -] 2.60
[SIGMA] HUFA (d)  30.04 [+ or -] 2.08  28.44 [+ or -] 6.43
BrFA 1             1.72 [+ or -] 0.31   1.63 [+ or -] 0.20
BrFA 2             1.58 [+ or -] 0.26   1.54 [+ or -] 0.17
[SIGMA] BrFA (c)   5.44 [+ or -] 0.51   5.50 [+ or -] 0.82
EpFA 1             0.99 [+ or -] 0.42   0.75 [+ or -] 0.16
[SIGMA] EpFA (f)   1.13 [+ or -] 0.39   0.95 [+ or -] 0.25

                        Right                Right
Fatty acid           external (%)         internal (%)

14:0               0.55 [+ or -] 0.26   0.37 [+ or -] 0.18
16:0              18.96 [+ or -] 3.05  17.93 [+ or -] 1.94
18:0               6.16 [+ or -] 0.48   6.34 [+ or -] 0.52
[SIGMA] SFA (a)   27.74 [+ or -] 3.59  26.66 [+ or -] 2.08
16:1n-7            9.81 [+ or -] 1.04   8.75 [+ or -] 1.78
18:1n-9c          11.86 [+ or -] 0.60  12.37 [+ or -] 1.61
18: 1n-7           5.91 [+ or -] 0.69   5.94 [+ or -] 0.83
20:1n-9            1.28 [+ or -] 0.56   1.21 [+ or -] 0.55
20:1n-7            2.25 [+ or -] 0.86   2.27 [+ or -] 0.81
[SIGMA] MUFA (b)  32.84 [+ or -] 1.35  32.13 [+ or -] 2.60
18:2n-6            1.00 [+ or -] 0.87   1.00 [+ or -] 0.80
18:3n-3            0.69 [+ or -] 0.18   0.60 [+ or -] 0.12
[SIGMA]PUFA (c)    4.15 [+ or -] 1.29   4.28 [+ or -] 1.02
20:4n-6            2.47 [+ or -] 0.42   2.28 [+ or -] 1.25
20:5n-3           15.24 [+ or -] 1.54  16.20 [+ or -] 2.21
22:5n-3            1.14 [+ or -] 0.51   1.24 [+ or -] 0.38
22:6n-3            8.82 [+ or -] 3.23   9.63 [+ or -] 1.46
[SIGMA] HUFA (d)  28.73 [+ or -] 3.59  30.29 [+ or -] 3.43
BrFA 1             1.63 [+ or -] 0.39   1.61 [+ or -] 0.34
BrFA 2             1.49 [+ or -] 0.49   1.64 [+ or -] 0.35
[SIGMA] BrFA (c)   5.24 [+ or -] 1.21   5.48 [+ or -] 0.98
EpFA 1             0.85 [+ or -] 0.34   0.90 [+ or -] 0.19
[SIGMA] EpFA (f)   1.10 [+ or -] 0.37   0.96 [+ or -] 0.17

                      All embryo
Fatty acid            masses (%)

14:0               0.53 [+ or -] 0.31
16:0              18.74 [+ or -] 2.62
18:0               6.23 [+ or -] 0.44
[SIGMA] SFA (a)   27.60 [+ or -] 2.87
16:1n-7            9.53 [+ or -] 2.01
18:1n-9c          11.97 [+ or -] 1.27
18: 1n-7           5.75 [+ or -] 0.77
20:1n-9            1.22 [+ or -] 0.48
20:1n-7            2.21 [+ or -] 0.72
[SIGMA] MUFA (b)  32.44 [+ or -] 2.50
18:2n-6            0.93 [+ or -] 0.61
18:3n-3            0.68 [+ or -] 0.17
[SIGMA]PUFA (c)    4.25 [+ or -] 1.05
20:4n-6            2.52 [+ or -] 0.97
20:5n-3           15.29 [+ or -] 2.10
22:5n-3            1.19 [+ or -] 0.45
22:6n-3            9.15 [+ or -] 2.32
[SIGMA] HUFA (d)  29.14 [+ or -] 3.92
BrFA 1             1.66 [+ or -] 0.30
BrFA 2             1.54 [+ or -] 0.32
[SIGMA] BrFA (c)   5.34 [+ or -] 0.86
EpFA 1             0.85 [+ or -] 0.28
[SIGMA] EpFA (f)   1.01 [+ or -] 0.29

Values are averages ([+ or -] standard deviation) of embryos from five
different females (n = 5).
(a) SFA (saturated fatty acid): 12:0. 14:0, 15:0, 16:0, 17:0, 18:0,
19:0, 20:0, 22:0.
(b) MUFA (monounsaturated fatty acid): 14:1n-5, 15:1n-1, 16:1n-7,
16:1n-5, 7-methyl-hexadec-6-enoate, 17:1n-9, 17:1n-8, 18:1n-9c,
18:1n-9r, 18:1n-7, l8:1n-5, 19:1n-9, 19:1n-8, 20:1n-9, 20:1n-7,
22:1n-11, 22:1n-9.
(c) PUFA (polyunsaturated fatty acid): 18:2n-6, 18:3n-3, 18:2n-3,
19:2n-7, 20:2n-9, 20:2n-7, 20:2n-6, 20:3n-4, 22:2n-9, 22:3n-6.
(d) HUFA (highly unsaturated fatty acid): 20:4n-6, 20:5n-3, 21:5n-3,
21:6, 22:4n-6, 22:5n-6, 22:5n-3, 22:6/1-3.
(e) BrFA (branched fatty acid): 4,8,12-trimethyl-tridecanoate,
9-methyl-tetradecanoate, 12-methyl-tetradecanoate (anteiso),
13-methyl-tetradecanoate (iso), 14-methyl-pentadecanoate (iso),
10-methyl-hexadecanoate, 14-methyl-hexadecanoate (anteiso),
15-methyl-hexadecanoate (iso), 15-methyl-heptadecanoate (anteiso),
16-methyl-heptadecanoate (iso), 16-mefhyl-octadecanoate (anteiso),
17-methyl-octadecanoate (iso).
(f) EpFA (epoxy fatty acid): 10,13-epoxy-l
1,12-dimethyl-octadecadienoate, 12,15-epoxy-13,
14-dimethyl-eicosadienoate. BrFA 1, 14-methyl-hexadecanoate (anteiso);
BrFA 2. 15-methyl-hexadecanoate (iso); EpFA 1, 12,15-epoxy-13,
14-dimethyl-eicosadienoate.
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Author:Rey, Felisa; Moreira, Ana S.P.; Ricardo, Fernando; Coimbra, Manuel A.; Domingues, M. Rosario M.; Dom
Publication:The Biological Bulletin
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Date:Apr 1, 2016
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