Mar Biol (2007) 151:935–947

DOI 10.1007/s00227-006-0535-6

RESEARCH ARTICLE

Embryogenesis of decapod crustaceans with diVerent life history traits, feeding ecologies and habitats: a fatty acid approach

R. Rosa · R. Calado · L. Narciso · M. L. Nunes

Received: 4 April 2006 / Accepted: 23 October 2006 / Published online: 6 December 2006© Springer-Verlag 2006

Abstract Variations in embryo size and fatty acid(FA) dynamics during embryogenesis were evaluatedin deep-sea pandalids and portunid swimming crabsfrom the Portuguese continental margin and MadeiraIsland slope and compared with previous data on neri-tic and deep-sea lobsters and shrimps (collectedbetween February 2001 and March 2004). Inter-speciWcvariations in embryo size seem to be dictated primarilyby phylogeny rather than by diVerences in reproduc-tive or early life history traits. FA reserves were signiW-cantly correlated with embryo size (P < 0.001).Principal component analysis revealed diVerencesamong three groups (1—neritic caridean shrimps, 2—deep-sea pandalids of the genus Plesionika, and lob-sters, 3—portunid crabs and the deep-sea pandalid

Chlorotocus crassicornis, Costa 1871). Group 1 wasclearly separated by PC1 mainly due to the higher per-centage of essential C18 (linoleic and linolenic acids)and C20 (namely eicosapentaenoic) polyunsaturatedFA (speciWc markers of primary producers). PC2 sepa-rated Group 2 from Group 3 due to diVerences in thepercentage of several saturated FA (including odd-numbered FA—bacterial markers) and C18 monoun-saturated FA (namely 18:1n ¡ 9, a general marker ofcarnivory). Therefore, these diVerences among groupsseem to result from distinctions in diet and ecologicalniche. Intra-speciWc diVerences in FA compositionbetween western and southern Plesionika martia mar-tia (A. Milne-Edwards, 1883) populations may reXecthigher water temperatures on the south sub-tropicalcoast. Lobster embryonic development was moredemanding of lipid energy than that of the other deca-pod species, which may reXect an evolutionary trend indecapod taxa related to an increasing degree of lecitho-trophy. However, a lower FA catabolism can be inter-preted as an enhanced independence of the newlyhatched larvae from external energy sources. HigherFA content at hatching and, as a consequence, agreater independence from the external environmentshould increase the chances of larval survival.

Introduction

An important step in the evolution of many multicellu-lar organisms was the development of the oviparouslife strategy, where embryogenesis occurs indepen-dently of the maternal body and, consequently, growthand energy provision are dependent on endogenousyolk reserves (e.g. lipovitellins and lipid droplets in

Communicated by J.P. Grassle, New Brunswick.

Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00227-006-0535-6 and is accessible for authorized users.

R. Rosa (&)Biological Sciences Center, University of Rhode Island, 100 Flagg Road, Kingston, RI 02881, USAe-mail: rrosa@etal.uri.edu

R. Calado · L. NarcisoDepartamento de Biologia Animal, Faculdade de Ciências da, Universidade de Lisboa, Laboratório Marítimo da Guia, Estrada do Guincho, Forte N.S. da Guia, 2750-642 Cascais, Portugal

R. Rosa · M. L. NunesDepartamento de Inovação Tecnológica e Valorização dos Produtos da Pesca, IPIMAR, Avenida de Brasília, 1449-006 Lisboa, Portugal

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936 Mar Biol (2007) 151:935–947

crustaceans; Lee 1991; Lee et al. 2006). During this lec-ithotrophic phase of ontogeny (prior to exogenousfeeding), larvae must obtain all the nutrients requiredfor homeostasis and development from the eggs. Theprecise sequence of bio-macromolecule consumption[amino acids, lipids and fatty acids (FAs), and carbohy-drates] varies both qualitatively and quantitativelyamong species (Sargent et al. 1989). Endogenousreserves must be used gradually in accordance with theneeds of speciWc cells as imposed by the genetic pro-gram of the embryo.

The quantity and quality of embryo FA reserves areimportant parameters that impact larval quality and sur-vival, and the rate of FA utilization is a useful determi-nant of the nutritional requirements of crustaceanlarvae. Females may transfer as much as 60% of theirlipid reserves to their eggs (Herring 1973), suggestingminimal lipid metabolism independent of egg provision(Rosa and Nunes 2002, 2003a, b). Lipids are used by thedeveloping embryo both as substrates for energy metab-olism (namely acylglycerols but also polar lipids; Sargent1995) and as structural components in membrane bio-genesis (namely phospholipids and cholesterol; Rosaet al. 2003, 2005). Crustaceans are well known for theirenvironmental plasticity and eclecticism, with speciesadapted to a great variety of environmental conditions.The preferential utilization of neutral and/or polar lipidsas energy sources is species-dependent and may reXectenvironmental adaptations (Narciso 1999).

Embryo size can be regarded as an estimate of energycontent since the lipid and FA contents are generallycorrelated with embryo volume and with the time inter-val between spawning and hatching or larval Wrst feeding(Rainuzzo et al. 1997). However, embryo energy con-tent can also be density-dependent (Anger et al. 2002),which may aVect direct inter-speciWc comparisons.Embryo size increases with latitude and decreases withaverage water temperature (Thatje et al. 2004). Initialembryo size also varies intra-speciWcally between latitu-dinally or geographically separated populations (Lardiesand Wehrtmann 2001), between diVerent reproductiveseasons (Boddeke 1982), and on a year-to-year basis inthe same locality (Kattner et al. 1994).

In the present study, we compare embryo size, watercontent and FA dynamics during embryogenesis ofthree deep-sea pandalid shrimps (Decapoda, Caridea,Pandalidae) and two portunid swimming crabs (Deca-poda, Brachyura, Portunidae). Chlorotocus crassicor-nis is an epibenthic (organism that lives on the bottom)pandalid shrimp that occurs in the Eastern CentralAtlantic and Mediterranean Sea between 75 and 600 mof depth. Plesionika martia martia is a nektobenthic(pelagic organism in close association with the bottom)

pandalid distributed throughout the Atlantic and Med-iterranean Sea at depths between 200 and 1,200 m(Udekem d’Acoz 1999). Plesionika narval is a pandalidspecies of wide distribution at low latitudes, from thesouth-western Iberian Peninsula to Angola (Gonzálezet al. 1997). Adults are nektobenthic but can be alsofound in shallow-water, aphotic, marine caves (Bisco-ito 1993). Macropipus tuberculatus is a polybiinid port-unid crab that occurs from Western Norway and theFaeroe Islands to Morocco (Zariquiey Alvarez 1968).It is an epibenthos feeder that can be found on varioussoft bottoms between 50 and 2,760 m depth. Henslow’sswimming crab, Polybius henslowii, is a benthic port-unid crab with pelagic phases (depth range 0–1,250 m)distributed from the British Isles to Morocco, includingthe Azores and Canarias Islands and Western Mediter-ranean Sea (Della Croce 1961).

Though there are many comparative studies on crus-tacean reproductive traits (e.g. Wenner and Kuris1991; Anger and Moreira 1998), there are only fewconcerning chemical composition of eggs (e.g. Angeret al. 2002). Therefore, the results obtained for thepresent Wve species are compared with our previouslypublished data on neritic and deep-sea shrimps andlobsters. The eVect of phylogeny, environmental condi-tions, diVerent habitats, and distinct early life historieson FA composition and metabolism of decapod crusta-cean embryos is discussed.

Materials and methods

Sampling

Ovigerous females of C. crassicornis (Costa, 1871), P.martia martia (A. Milne-Edwards, 1883), and M. tuber-culatus (Roux, 1830) were collected in September andOctober 2003 on the upper slope oV the south Portu-guese coast (400–600 m in Sagres area) aboard the com-mercial crustacean trawler “Costa Sul” (Table 1). P.henslowii (Leach, 1820) females were collected in March2004 by commercial Wshing vessels in Peniche (westerncoast of Portugal; Fig. 1) and the egg-bearing females ofP. narval (Fabricius, 1787) were sampled from April toMay 2003 on the Madeira Island slope (near Funchal;Fig. 1) with shrimp traps (at 600–700 m depth) on boarda small trap-Wshing vessel of the “Museu Municipal doFunchal—História Natural.” The embryo mass wasremoved from the females and embryos were classiWedaccording to the following criteria (modiWed from Katt-ner et al. 1994): stage 1—uniform yolk and no embry-onic development visible; stage 2—eyes clearly visiblewith ½ yolk consumed; stage 3—almost no yolk present

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and embryo fully developed. To determine embryo vol-ume, 30 embryos were taken from each female (ninefemales, three per embryonic stage) and length andwidth were measured under a stereomicroscope (Olym-pus®, model SZ6045TR) with a calibrated micrometereyepiece. To perform the biochemical analyses andwater content determinations, diVerent embryo batches(10–20 similar sized females at the same stage of devel-opment) were pooled. Pooled embryo samples werestored in liquid nitrogen.

Embryo volume and water content

Embryo volume was calculated using the formulae: (1)V = 1/6(�W2L) (V = volume, W = width, L = length)

for oblate spheroid embryos (Turner and Lawrence1979) of Astacidea and Caridea; and (2) V = 4/3(�r3)for spheroid embryos of Brachyura. Water content wasdetermined in triplicate by measuring the diVerencebetween wet and dry weight of the embryo samples.The freeze-drying procedure was performed in aSavant VP100®.

Fatty acid analysis

The determination of FA proWle was based on theexperimental procedure of Lepage and Roy (1986)modiWed by Cohen et al. (1988). The FA methyl esterswere analysed in a CP 3800 Varian gas chromatographequipped with an auto-sampler and Wtted with a Xame

Table 1 Sampling area and period, depth range (m), DMA (m), life strategy, habitat and main feeding guild of decapod crustaceansfrom the Portuguese margin and Madeira Island slope

B benthic, Eb endobenthic, Nb nektobenthic, P pelagic, N neritic or coastal, D demersal, Int intertidal, Inf infralitoral, DMA depth ofmaximum abundancea Data from Morais et al. (2002)b According to Orav-Kotta (2004)c Data from Calado et al. (2005)d According to Calado and Narciso (2000)e Present studyf According to Cartes et al. (2002)g Data from Rosa et al. (2005)h According to Cristo and Cartes (1998)i Data from Rosa et al. (2003)

Species (abbreviations) Sampling area

Period DMA (m) Life strategy and habitat

Main feeding guild

CarideaPalaemonidaePalaemon elegansa (P. ele) Cascais February–June 2001 0–5 Nb (Int) Mesograzer

(mesoherbivore)b

Palaemon serratusa (P. ser) Cascais February–June 2001 5–40 (20–25) Nb (Inf) Mesograzer (mesoherbivore)b

HippolytidaeLysmata seticaudatac (L. set) Cascais August–September 2001 0–60 (15–30) Nb (Int–Inf) Mesograzerd

PandalidaeChlorotocus crassicornise (C. cra)

Sagres September–October 2003 75–600 (100–400) Nb (D) Infaunal feederf

Plesionika martia martiaa (P. mar—west)

Cascais February–June 2001 200–1,200 (200–700) Nb (D) Macroplankton feederf

P. martia martiae (P. mar—south)

Sagres September–October 2003 200–1,200 (200–700) Nb (D) Macroplankton feederf

Plesionika narvale (P. nar) Funchal April–May 2003 4–900 (200–400) Nb (N and D) Macroplankton feederf

AstacideaNephropidaeHomarus gammarusg (H. gam)

Cascais February–June 2001 0–150 (0–50) B–Eb (N and D) Macroplankton feederh

Nephrops norvegicusi (N. nor)

Cascais February–June 2001 100–800 (300–600) B–Eb (D) Macroplankton feederh

BrachyuraPortunidaeMacropipus tuberculatuse (M. tub)

Sagres September–October 2003 50–2,760 (100–500) B–P (N and D) Epibenthos feederf

Polybius henslowiie (P. hen) Peniche March 2004 0–1,250 (0–600) B–P (N and D) Epibenthos feederf

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ionization detector. The separation was carried outwith helium as the carrier gas in a DB-Wax polyethyl-ene glycol column (30 m £ 0.25 mm id) programmedto start at 180°C for 5 min, heating at 4°C per minutefor 10 min, and holding at 220°C for 25 min, with adetector at 250°C. A split injector (100:1) at 250°C wasused. FA methyl esters were identiWed by comparisonof their retention time with those of chromatographicSigma standards. Peak areas were determined usingthe Varian software and the FA 23:0 was used as aninternal standard.

Lipid energy consumption

Fatty acid utilization is expressed as percent ofdecrease, i.e. the diVerence between the mass per FAat the start (stage 1) and end of embryonic develop-ment (stage 3). To quantify the lipid energy consump-tion during embryogenesis the Rubner’s coeYcient(9.5 kcal g¡1 for lipids; Winberg 1971) was used with

FA data. Since total FA content represents at least80% of the total lipid content, the lipid catabolic ratewas sub-estimated.

Statistical analysis

Two-way ANOVAs were used to test the eVects of spe-cies and embryonic stages on embryo volume, watercontent, and FA composition following validation ofthe normal distribution of the data. All post hoc pair-wise comparisons were done using Tukey’s multiplecomparison tests (Statistica 6.0). DiVerences were con-sidered signiWcant at P < 0.05. The relationshipsbetween embryo size and FA content and embryo sizeand water content increase were also investigated usingcorrelation analyses (non-parametric Spearman corre-lation coeYcients). Principal component analysis(PCA) was performed on the correlation matrix of theFA variables. The values used were percentages oftotal FA values in order to remove the eVect of concen-tration (and embryo size) that would otherwise be themajor controlling factor in the subsequent analysis.Prior to analysis, the proportion data were transformedwith the formula: log%/(100 ¡ %) to ensure homosce-dasticity (McCullagh and Nelder 1989).

Results

Embryo volume and water content

Embryo volume increased signiWcantly during embry-onic development of decapod crustaceans (P < 0.05;Fig. 2). The smallest increase was observed in thedeep-sea pandalid shrimp C. crassicornis (45%) andthe highest in palaemonid shrimp Palaemon serratus(188%). Among all species, the smallest embryo vol-ume occurred in the portunid crabs (P < 0.05; 0.03–0.05 mm3 in stage 3) and the highest in the lobstersHomarus gammarus (6.1 mm3) and Nephrops norvegi-cus (1.9 mm3). The water content also increased signiW-cantly with development stage (P < 0.05; Fig. 3). Thesmallest increase was observed in the pandalid shrimps(<18%) and portunid crabs (<17%) and the highest inthe lobster H. gammarus (45%) and palaemonidshrimps (40–41%). However, there was no positivecorrelation between the embryo volume increase andwater content increase (r2 = 0.26, P > 0.05).

Fatty acids

Embryo FA reserves were signiWcantly correlated withembryo size (r2 = 0.68, P < 0.001). The quantitatively

Fig. 1 Map of the Portuguese continental and Madeira Islandcoasts with sampling areas (rectangle marks) of: 1—Polybiushenslowii (present study); 2—Palaemon elegans, Palaemon serra-tus (adapted from Morais et al. 2002), and Lysmata seticaudata(adapted from Calado et al. 2005); 3—Plesionika martia martia(adapted from Morais et al. 2002), Nephrops norvegicus (adaptedfrom Rosa et al. 2003), and Homarus gammarus (adapted fromRosa et al. 2005); 4—Cholorotus crassicornis, P. martia martia,and Macropipus tuberculatus (present study); 5—Plesionikanarval (present study)

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most important saturated fatty acids (SFA) in the crus-tacean embryos were palmitic (16:0) and stearic (18:0)acids. The other SFA detected were 14:0, 15:0, 17:0,19:0, 20:0, and 22:0.

Palmitic acid content showed signiWcant diVerencesthroughout embryogenesis of pandalids and portunidcrabs (P < 0.05), primarily in stage 1 (Tables 2, 3). Thehighest values of this FA were attained by the formergroup. In comparison with previously published data(Supplementary online material), all caridean shrimpsshowed intermediate values [26.6–31.4 �g mg¡1 dryweight (dw)], lobsters had the highest contents (48.3–49.7 �g mg¡1 dw) and the portunid crabs had the lowest(21.7–25.0 �g mg¡1 dw; P < 0.05). In relation to 18:0 con-tent in stage 1, the inter-speciWc diVerences (P < 0.05)were not so clear between groups. The inter-speciWc dis-similarities of the saturated fraction content (SFA) werea reXection of its major FAs (lobsters > carideanshrimps > portunid crabs). In terms of percentage of

total FA some interesting observations can be made(Table 4 and Supplementary online material). Oneresult that stands out is the stability or increase that isobserved in the proportion of SFA throughout embry-onic development, as opposed to a signiWcant decrease(P < 0.05) when expressed on a dry weight basis.

The major monounsaturated fatty acids (MUFA)were palmitoleic (16:1n ¡ 7), oleic (18:1n ¡ 9), and vac-cenic (18:1n ¡ 7) acids. Other MUFA detected include17:1n ¡ 8, 20:1n ¡ 9, 20:1n ¡ 7, 22:1n ¡ 11, and22:1n ¡ 9. Although there were diVerences in stage 116:1n ¡ 7 (P < 0.05) within pandalids and portunid crabs(Tables 2, 3), there was no clear distinction between car-idean and portunid groups, and the highest concentra-tions were found in the lobsters (>22 �g mg¡1 dw instage 1; supplementary online material). The majorMUFA, 18:1n ¡ 9, was similar in both portunids(»22 �g mg¡1 dw) but there were signiWcant diVerencesamong the pandalids (P < 0.05) and among all caridean

Fig. 2 Embryo volume (mm3) of decapod crustacean embry-os at diVerent stages of embry-onic development. Values are means § SD (n = 90). White bars—new data, grey bars—published data. For species abbreviations see Table 1

0,0

0,1

0,2

0,3

0,4

P. ele P. ser L. set C. cra P. mar

(west)

P. mar

(south)

P. narv H. gam N. nor P. hen M. tub

ovggE

lue

mm

m(

3

)

Stage I Stage II Stage III

0

2

4

6

8

H. gam N. nor

Fig. 3 Water content (% of wet weight) of decapod crustacean embryos at diVerent stages of embryonic development. White bars—new data, grey bars—published data. Values are means § SD (n = 3). For species abbreviations see Table 1

40

50

60

70

80

90

P. ele P. ser L. set C. cra P. mar(west)

P. mar(south)

P. narv H. gam N. nor P. hen M. tub

Wat

ocre

ntne

t(%

ww

)

Stage I Stage II Stage III

Swim. crabs LobstersCaridean shrimps

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shrimps (ranging from 20 to 45 �g mg¡1 dw; Table 2 andSupplementary online material; P < 0.05). The lobstershad concentrations >69 �g mg¡1 dw. A fairly similar ten-dency for 18:1n ¡ 7 was found (Tables 2, 3 and Supple-mentary online material). Unlike the SFA results, allspecies showed a decreasing trend in the relative per-centage of total MUFA throughout embryogenesis(Table 4 and supplementary online material).

The most prevalent polyunsaturated fatty acids(PUFA) were the linoleic (18:2n ¡ 6), linolenic(18:3n ¡ 3), arachidonic (ARA, 20:4n ¡ 6), eicosapen-taenoic (EPA, 20:5n ¡ 3), and docosahexaenoic

(DHA, 22:6n ¡ 3) acids. Additional PUFA detectedinclude 16:4n ¡ 3, 18:3n ¡ 6, 18:4n ¡ 3, 20:2n ¡ 6,20:3n ¡ 3, 20:4n ¡ 3, 22:4n ¡ 6, 22:5n ¡ 6, and22:5n ¡ 3.

Quantities of 18:2n ¡ 6 ranged between 2 and3 �g mg¡1 dw in the pandalids and portunids (with theexception of P. narval; Tables 2, 3) and was signiW-cantly lower (P < 0.05) than those in the other caride-ans previously studied (Supplementary onlinematerial). Similar inter-speciWc diVerences wereobserved in 18:3n ¡ 3, 20:4n ¡ 6, and 20:5n ¡ 3. Quan-tities of 22:6n ¡ 3 were signiWcant diVerent within and

Table 2 Embryo FA composition (�g mg¡1 dw) of deep-sea pandalids Chlorotocus crassicornis, Plesionika martia martia, and Plesion-ika narval at diVerent stages of embryonic development

Values are means of triplicate samples § SD

Fas C. crassicornis P. martia martia (south) P. narval

1 2 3 1 2 3 1 2 3

14:0 4.8 § 0.0 3.8 § 0.0 2.9 § 0.0 3.1 § 0.0 2.6 § 0.0 1.9 § 0.0 3.1 § 0.0 2.3 § 0.0 1.1 § 0.015:0 1.5 § 0.0 1.2 § 0.0 0.9 § 0.0 0.8 § 0.0 0.7 § 0.0 0.6 § 0.0 1.2 § 0.0 0.8 § 0.0 0.5 § 0.016:0 30.2 § 0.1 27.0 § 0.4 22.4 § 0.1 28.3 § 0.1 23.8 § 0.1 19.8 § 0.0 31.4 § 0.3 24.0 § 0.0 15.6 § 0.217:0 1.2 § 0.0 1.1 § 0.0 1.0 § 0.0 0.3 § 0.0 0.2 § 0.0 0.1 § 0.0 1.4 § 0.0 1.0 § 0.0 0.7 § 0.018:0 6.0 § 0.0 6.0 § 0.1 5.1 § 0.0 7.0 § 0.0 5.5 § 0.0 4.7 § 0.0 10.8 § 0.1 8.8 § 0.0 6.6 § 0.119:0 0.4 § 0.0 0.4 § 0.0 0.3 § 0.0 0.2 § 0.0 0.2 § 0.0 0.1 § 0.0 0.2 § 0.0 0.2 § 0.0 0.1 § 0.020:0 0.9 § 0.0 0.4 § 0.0 0.3 § 0.0 0.7 § 0.0 0.6 § 0.0 0.3 § 0.0 0.5 § 0.0 0.4 § 0.0 0.4 § 0.022:0 0.7 § 0.0 0.6 § 0.0 0.3 § 0.0 0.4 § 0.0 0.4 § 0.0 0.2 § 0.0 0.3 § 0.0 0.4 § 0.0 0.1 § 0.0�Saturated 45.7 § 0.2 40.5 § 0.7 33.2 § 0.2 40.9 § 0.2 34.0 § 0.1 27.7 § 0.1 48.9 § 0.5 38.0 § 0.0 25.3 § 0.3

Iso 14:0 0.3 § 0.0 0.2 § 0.0 0.4 § 0.0 0.2 § 0.0 0.1 § 0.0 0.1 § 0.0 0.2 § 0.0 0.1 § 0.0 0.1 § 0.0Anteiso 14:0 0.1 § 0.0 0.1 § 0.0 0.1 § 0.0 0.1 § 0.0 0.1 § 0.0 0.0 § 0.0 0.1 § 0.0 0.1 § 0.0 0.0 § 0.0Iso 16:0 0.8 § 0.0 0.7 § 0.0 0.5 § 0.0 0.7 § 0.0 0.5 § 0.0 0.3 § 0.0 0.6 § 0.0 0.4 § 0.0 0.2 § 0.0Anteiso 16:0 0.3 § 0.0 0.2 § 0.0 0.1 § 0.0 0.3 § 0.0 0.2 § 0.0 0.2 § 0.0 0.4 § 0.0 0.3 § 0.0 0.2 § 0.0�Branched 1.6 § 0.0 1.2 § 0.0 1.1 § 0.0 1.3 § 0.0 0.9 § 0.0 0.7 § 0.0 1.3 § 0.0 0.9 § 0.0 0.5 § 0.0

16:1n ¡ 7 6.7 § 0.0 5.4 § 0.1 3.7 § 0.0 15.4 § 0.1 13.1 § 0.0 9.5 § 0.0 8.8 § 0.0 6.1 § 0.0 2.8 § 0.017:1n ¡ 8 0.6 § 0.0 0.8 § 0.0 0.7 § 0.0 1.3 § 0.0 1.1 § 0.0 0.8 § 0.0 0.8 § 0.0 0.6 § 0.0 0.5 § 0.018:1n ¡ 9 23.7 § 0.1 19.6 § 0.1 11.2 § 0.1 44.6 § 0.2 35.1 § 0.2 26.4 § 0.0 43.0 § 0.5 34.7 § 0.1 21.5 § 0.218:1n ¡ 7 4.5 § 0.0 3.9 § 0.1 3.3 § 0.0 11.9 § 0.0 10.3 § 0.0 8.4 § 0.0 7.3 § 0.0 5.5 § 0.0 4.4 § 0.020:1n ¡ 9 1.7 § 0.0 1.4 § 0.0 0.8 § 0.0 2.3 § 0.0 1.7 § 0.0 1.1 § 0.0 0.9 § 0.0 0.7 § 0.0 0.4 § 0.020:1n ¡ 7 0.7 § 0.0 0.5 § 0.0 0.3 § 0.0 0.6 § 0.0 0.6 § 0.0 0.3 § 0.0 0.7 § 0.0 0.6 § 0.0 0.4 § 0.022:1n ¡ 11 1.0 § 0.0 0.8 § 0.0 0.5 § 0.0 0.4 § 0.0 0.4 § 0.0 0.2 § 0.0 0.2 § 0.0 0.2 § 0.0 0.3 § 0.022:1n ¡ 9 0.3 § 0.0 0.3 § 0.0 0.1 § 0.0 0.4 § 0.0 0.3 § 0.0 0.1 § 0.0 0.1 § 0.0 0.2 § 0.0 0.1 § 0.0�Monounsaturated 39.2 § 0.1 32.6 § 0.2 20.5 § 0.2 77.0 § 0.4 62.7 § 0.2 46.8 § 0.1 61.8 § 0.5 48.6 § 0.1 30.4 § 0.3

16:3n ¡ 3 1.3 § 0.0 1.0 § 0.0 0.7 § 0.0 0.8 § 0.0 0.7 § 0.0 0.5 § 0.0 1.2 § 0.0 0.9 § 0.0 0.5 § 0.016:4n ¡ 3 0.1 § 0.0 0.2 § 0.0 0.2 § 0.0 0.9 § 0.0 0.8 § 0.0 0.7 § 0.0 1.2 § 0.0 1.2 § 0.0 0.9 § 0.018:2n ¡ 6 2.7 § 0.1 2.2 § 0.0 1.8 § 0.0 2.6 § 0.0 2.1 § 0.0 1.5 § 0.0 11.7 § 0.3 10.2 § 0.1 6.9 § 0.118:3n ¡ 6 0.4 § 0.0 0.3 § 0.0 0.3 § 0.0 0.0 § 0.0 0.0 § 0.0 0.1 § 0.0 0.1 § 0.0 0.2 § 0.0 0.1 § 0.018:3n ¡ 3 1.1 § 0.0 2.0 § 0.0 1.7 § 0.0 1.4 § 0.0 1.1 § 0.0 0.8 § 0.0 0.9 § 0.0 0.7 § 0.0 0.5 § 0.018:4n ¡ 3 0.8 § 0.0 1.8 § 0.0 1.4 § 0.0 0.8 § 0.0 0.6 § 0.0 0.3 § 0.0 0.9 § 0.0 0.8 § 0.0 0.4 § 0.020:2n ¡ 6 0.6 § 0.0 0.6 § 0.0 0.5 § 0.0 0.9 § 0.0 0.6 § 0.0 0.4 § 0.0 0.7 § 0.0 0.6 § 0.0 0.6 § 0.020:4n ¡ 6 2.4 § 0.0 2.8 § 0.0 0.4 § 0.0 2.9 § 0.0 2.5 § 0.0 2.1 § 0.0 2.5 § 0.0 1.9 § 0.0 1.8 § 0.020:3n ¡ 3 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.020:4n ¡ 3 0.5 § 0.0 0.8 § 0.0 0.7 § 0.0 0.8 § 0.0 0.6 § 0.0 0.4 § 0.0 0.3 § 0.0 0.1 § 0.0 0.1 § 0.020:5n ¡ 3 8.9 § 0.0 5.3 § 0.0 2.2 § 0.1 14.5 § 0.2 12.9 § 0.1 11.4 § 0.0 8.3 § 0.0 5.4 § 0.0 4.4 § 0.122:4n ¡ 6 2.4 § 0.0 3.1 § 0.1 2.4 § 0.0 0.4 § 0.0 0.3 § 0.0 0.2 § 0.0 0.1 § 0.0 0.0 § 0.0 0.0 § 0.022:5n ¡ 6 0.6 § 0.0 0.7 § 0.0 0.4 § 0.0 0.6 § 0.0 0.4 § 0.0 0.4 § 0.0 0.6 § 0.0 0.3 § 0.0 0.2 § 0.022:5n ¡ 3 0.5 § 0.0 0.7 § 0.0 0.2 § 0.0 0.7 § 0.0 0.6 § 0.0 0.5 § 0.0 0.9 § 0.0 0.4 § 0.0 0.2 § 0.022:6n ¡ 3 15.1 § 0.0 10.0 § 0.1 7.5 § 0.1 20.5 § 0.3 16.8 § 0.1 13.9 § 0.0 10.7 § 0.1 7.4 § 0.0 6.2 § 0.1�Polyunsaturated 36.8 § 0.1 32.3 § 0.4 21.0 § 0.1 48.0 § 0.7 40.2 § 0.2 33.4 § 0.1 40.1 § 0.2 30.2 § 0.1 22.9 § 0.2�(n ¡ 3) 28.2 § 0.1 21.7 § 0.2 14.6 § 0.1 40.4 § 0.6 34.0 § 0.2 28.5 § 0.1 24.2 § 0.0 16.9 § 0.1 13.2 § 0.1�(n ¡ 6) 8.2 § 0.0 10.2 § 0.2 6.1 § 0.0 7.4 § 0.1 6.0 § 0.0 4.8 § 0.0 15.6 § 0.3 13.2 § 0.1 9.6 § 0.1�Total FA 122.9 § 0.4 106.3 § 1.3 75.7 § 0.4 166.9 § 1.3 137.6 § 0.2 108.5 § 0.2 151.8 § 1.2 117.6 § 0.2 79.0 § 0.8

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Mar Biol (2007) 151:935–947 941

between all decapod groups (P < 0.05) with the highestvalues recorded in the lobsters and the swimming crabP. henslowii (>30 �g mg¡1 dw in stage 1; Tables 2, 3and Supplementary online material).

Total PUFA concentrations varied signiWcantly withinand between the decapod groups (P < 0.05). Similar toSFA, PUFA proportions were maintained or increasedthroughout embryogenesis (Table 4 and Supplementaryonline material). MUFA were used preferentially forenergetic purposes (Fig. 4), with the exception of theneritic shrimps (Palaemon and Lysmata) and the port-unid P. henslowii. These species had a greater contribu-tion of PUFA to the overall lipid catabolism becausePUFA was the major FA fraction in their embryo proWle,

representing >42% of total lipids in stage 1 (MUFA rep-resented <33%). In contrast, the lobsters, pandalids andM. tuberculatus contained <34% of PUFA and >40%MUFA with the exception of C. crassicornis.

To compare the FA proWle (with transformed per-centage values) of all crustacean species in stage 1, aPCA was done with 25 FAs as variables (Fig. 5). TheWrst principal component (PC) explained 25.0% ofthe variance and the second PC explained 19.0% of thevariance. Since the third PC explained 15.4% of thevariance but did not add additional information andthe other PCs had eigenvalues <2.0 (explaining only asmall proportion of the variance), they were not inves-tigated further.

Table 3 Embryo FA compo-sition (�g mg¡1 dw) of portun-id swimming crabs Polybius henslowii and Macropipus tu-berculatus at diVerent stages of embryonic development

Fas P. henslowii M. tuberculatus

1 2 3 1 3

14:0 3.7 § 0.0 3.1 § 0.1 1.7 § 0.0 1.9 § 0.0 1.1 § 0.015:0 1.1 § 0.0 1.2 § 0.0 0.8 § 0.0 0.7 § 0.0 0.4 § 0.016:0 25.0 § 0.1 22.1 § 0.3 14.4 § 0.1 21.7 § 0.2 14.6 § 0.117:0 1.3 § 0.0 1.2 § 0.0 1.0 § 0.0 1.5 § 0.0 0.7 § 0.018:0 8.1 § 0.0 7.1 § 0.1 5.0 § 0.0 6.4 § 0.0 4.2 § 0.019:0 0.2 § 0.0 0.2 § 0.0 0.2 § 0.0 0.4 § 0.0 0.2 § 0.020:0 0.8 § 0.0 0.2 § 0.0 0.4 § 0.0 0.5 § 0.0 0.8 § 0.022:0 0.2 § 0.0 0.2 § 0.0 0.1 § 0.0 0.2 § 0.0 0.1 § 0.0� Saturated 40.3 § 0.1 35.3 § 0.5 23.6 § 0.1 33.3 § 0.3 22.0 § 0.1

Iso 14:0 0.2 § 0.0 0.2 § 0.0 0.2 § 0.0 0.2 § 0.0 0.1 § 0.0Anteiso 14:0 0.1 § 0.0 0.1 § 0.0 0.1 § 0.0 0.1 § 0.0 0.0 § 0.0Iso 16:0 0.7 § 0.0 0.8 § 0.0 0.6 § 0.0 0.7 § 0.0 0.4 § 0.0Anteiso 16:0 0.5 § 0.0 0.5 § 0.0 0.4 § 0.0 0.5 § 0.0 0.3 § 0.0�Branched 1.5 § 0.0 1.6 § 0.0 1.2 § 0.0 1.4 § 0.0 0.8 § 0.0

16:1n ¡ 7 9.8 § 0.1 9.0 § 0.1 4.6 § 0.0 15.9 § 0.2 9.8 § 0.117:1n ¡ 8 0.6 § 0.0 0.8 § 0.0 0.6 § 0.0 0.5 § 0.0 0.3 § 0.018:1n ¡ 9 21.9 § 0.0 20.8 § 0.3 12.3 § 0.1 22.0 § 0.1 14.6 § 0.118:1n ¡ 7 5.7 § 0.0 6.2 § 0.1 4.1 § 0.0 5.0 § 0.1 4.1 § 0.020:1n ¡ 9 2.1 § 0.0 0.4 § 0.0 1.0 § 0.0 1.6 § 0.0 1.5 § 0.020:1n ¡ 7 1.3 § 0.0 0.7 § 0.0 0.6 § 0.0 2.4 § 0.0 0.4 § 0.022:1n ¡ 11 0.5 § 0.0 0.3 § 0.0 0.1 § 0.0 1.2 § 0.0 0.7 § 0.022:1n ¡ 9 0.3 § 0.0 0.3 § 0.0 0.2 § 0.0 0.5 § 0.0 0.3 § 0.0�Monounsaturated 42.2 § 0.1 38.5 § 0.6 23.4 § 0.2 49.1 § 0.4 31.7 § 0.1

16:3n ¡ 3 1.6 § 0.0 1.2 § 0.0 0.7 § 0.0 1.6 § 0.0 0.8 § 0.016:4n ¡ 3 1.2 § 0.0 1.3 § 0.0 0.8 § 0.0 1.9 § 0.0 0.9 § 0.018:2n ¡ 6 2.2 § 0.0 1.8 § 0.0 1.1 § 0.0 2.6 § 0.0 1.2 § 0.018:3n ¡ 6 0.2 § 0.0 0.3 § 0.0 0.1 § 0.0 0.2 § 0.0 0.1 § 0.018:3n ¡ 3 0.8 § 0.0 0.9 § 0.0 0.4 § 0.0 1.1 § 0.0 0.5 § 0.018:4n ¡ 3 1.4 § 0.0 1.1 § 0.0 0.5 § 0.0 0.7 § 0.0 0.3 § 0.020:2n ¡ 6 1.1 § 0.0 1.2 § 0.0 1.4 § 0.0 0.8 § 0.0 0.7 § 0.020:4n ¡ 6 3.2 § 0.0 2.8 § 0.0 1.8 § 0.0 1.6 § 0.1 2.3 § 0.020:3n ¡ 3 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.0 0.0 § 0.020:4n ¡ 3 1.1 § 0.0 1.0 § 0.0 0.5 § 0.0 0.5 § 0.0 0.3 § 0.020:5n ¡ 3 20.8 § 0.1 19.4 § 0.3 13.7 § 0.1 12.8 § 0.1 9.2 § 0.122:4n ¡ 6 0.6 § 0.0 0.5 § 0.0 0.3 § 0.0 0.4 § 0.0 0.3 § 0.022:5n ¡ 6 0.9 § 0.0 0.8 § 0.0 0.4 § 0.0 0.5 § 0.0 0.4 § 0.022:5n ¡ 3 2.2 § 0.0 1.5 § 0.0 0.9 § 0.0 1.6 § 0.0 1.1 § 0.022:6n ¡ 3 34.4 § 0.2 33.0 § 0.4 20.5 § 0.3 17.2 § 0.1 14.3 § 0.1�Polyunsaturated 71.9 § 0.4 67.0 § 0.9 43.2 § 0.4 43.9 § 0.0 32.6 § 0.2�(n ¡ 3) 63.5 § 0.4 59.4 § 0.0 37.9 § 0.4 37.5 § 0.0 27.4 § 0.1�(n ¡ 6) 8.2 § 0.0 7.4 § 0.0 5.2 § 0.0 6.1 § 0.1 4.9 § 0.0�Total FA 155.7 § 0.6 142.2 § 2.0 91.3 § 0.8 127.4 § 1.1 86.9 § 0.4

Values are means of triplicate samples § SD

123

942 Mar Biol (2007) 151:935–947

Three groups were distinguished with the calculatedPCs, namely the neritic caridean species (Group 1), thedeep-sea pandalids of the genus Plesionika and lob-sters (Group 2), and the portunid crabs and the panda-lid C. crassicornis (Group 3).

The PC loading plot (Fig. 5b) revealed that C18PUFA, 20:5n ¡ 3, 20:4n ¡ 6, 22:5n ¡ 3, as well as22:6n ¡ 3, 20:1n ¡ 9, and 22:1n ¡ 1 were the mostimportant FA for group diVerentiation along PC1,while the saturated 14:0, 15:0, 16:0, 17:0, 20:0, and 22:0as well as C18 MUFA (18:1n ¡ 9 and 18:1n ¡ 7) werethe most important along PC2. The same analysis was

done using stage 3 data, but although Group 1 wasclearly separated from the others, there was no distinc-tion between Groups 2 and 3. FA trophic markersEPA/DHA and 18:1n ¡ 7/18:1n ¡ 9 ratios and percent-age of 18:1n ¡ 9 were used to determine species’degree of carnivory and the sum of odd-numbered FA(namely 15:0 and 17:0) was used as a marker of bacte-rial production (Fig. 6). EPA/DHA and 18:1n ¡ 7/18:1n ¡ 9 ratios were signiWcantly higher in Group 1(P < 0.05; Fig. 6a). Group 2 showed the highest per-centages of 18:1n ¡ 9 and the lowest of odd-numberedFA (P < 0.05; Fig. 6b).

Table 4 Relative FA composition (% total FAs) of embryos of Chlorotocus crassicornis, Plesionika martia martia (south) and Plesion-ika narval, Polybius henslowii and Macropipus tuberculatus at diVerent stages of embryonic development (only the quantitatively mostimportant FAs are represented)

FAs C. crassicornis P. martia martia (south) P. narval P. henslowii M. tuberculatus

1 2 3 1 2 3 1 2 3 1 2 3 1 3

16:0 24.56 25.41 29.63 16.95 17.31 18.25 20.68 20.44 19.79 16.07 15.57 15.80 17.07 16.7618:0 4.88 5.68 6.74 4.19 3.98 4.29 7.09 7.51 8.39 5.17 5.00 5.49 5.02 4.80�Saturated 37.17 38.10 43.92 24.49 24.69 25.56 32.20 32.32 31.99 25.89 24.83 25.87 26.15 25.30

16:1n ¡ 7 5.47 5.04 4.94 9.25 9.55 8.76 5.81 5.19 3.57 6.29 6.31 5.00 12.52 11.2418:1n ¡ 9 19.28 18.39 14.77 26.73 25.52 24.30 28.36 29.48 27.19 14.07 14.62 13.50 17.29 16.8418:1n ¡ 7 3.65 3.68 4.30 7.15 7.50 7.79 4.81 4.72 5.59 3.68 4.33 4.47 3.95 4.71�Monounsaturated 31.94 30.68 27.14 46.12 45.57 43.11 40.69 41.33 38.51 27.12 27.07 25.65 38.56 36.47

18:2n ¡ 6 2.19 2.07 2.35 1.56 1.54 1.39 7.68 8.70 8.72 1.42 1.28 1.25 2.05 1.3518:3n ¡ 3 0.90 1.90 2.29 0.83 0.83 0.71 0.59 0.56 0.66 0.53 0.66 0.43 0.87 0.5920:4n ¡ 6 1.95 2.66 0.52 1.72 1.80 1.97 1.62 1.59 2.27 2.05 1.98 1.96 1.25 2.6320:5n ¡ 3 7.22 4.97 2.86 8.67 9.37 10.48 5.44 4.63 5.61 13.38 13.61 15.04 10.08 10.5722:6n ¡ 3 12.26 9.40 9.91 12.30 12.17 12.85 7.07 6.27 7.91 22.06 23.22 22.46 13.46 16.46�Polyunsaturated 29.92 30.41 27.79 28.78 29.20 30.82 26.41 25.71 29.04 46.17 47.10 47.37 34.45 37.49

Fig. 4 Lipid energy consumption (kJ g¡1 dw) during the embry-onic development (from the early to the later stage) of decapodcrustaceans. White bars—new data, grey bars—published data(note: since total FA content represents utmost 80% of total lipid

content, the lipid catabolic rate is sub-estimated). Values of incu-bation periods (weeks) and the percentage of utilization of PUFAduring embryogenesis (values placed inside and above columns)are also given. For species abbreviations see Table 1

0

2

4

6

8

10

P. ele P. ser L. set C. cra P. mar(west)

P. mar(south)

P. narv H. gam N. nor P. hen M. tub

Ene

rgy

cosn

muitpo

Jk(n

g-1

dw

SFA MUFA PUFA

26

40

53

68

4330

2543

5952

28

24-44 16Incubation (weeks): 2-4

Swim. crabs LobstersCaridean shrimps

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Mar Biol (2007) 151:935–947 943

Discussion

Embryo volume increase during embryogenesis (from10% in P. martia martia to 45% in H. gammarus) wasattributed to the osmotic intake of water, which seemsto be a response to high internal osmolality of theembryos. Embryos are osmotically protected byembryo membranes, but some time before hatchingmembrane permeability increases, resulting in wateruptake and an increase in wet weight (Charmantierand Charmantier-Daures 2001). Additionally, water isalso a by-product of respiration and, consequently, theretention of metabolic water is also likely.

Embryo size diVered substantially between Astaci-dea (lobsters) and the other infra-orders (Caridea andBrachyura). Lobsters produce bigger embryos, have areduced number of larval stages (3–4 zoeal stages) and

long development times (6–11 months) (Farmer 1975).In contrast, the brachyuran crabs M. tuberculatus andP. henslowii have small embryos, Wve zoeal stages plusmegalopa and their embryogenesis lasts 4 months(Zariquiey Alvarez 1968). The caridean shrimps hadintermediate sizes, with the largest embryos beingobserved in the neritic palaemonids. Palaemon spp.shrimps have 7–9 zoeal stages (dos Santos 1999), Lys-mata seticaudata has 9 zoeae and 1 megalopa (Caladoet al. 2004), and the pandalid shrimps have 11 zoealstages (Barnich 1996). All of these caridean specieshave development times of 2–4 weeks from spawningto hatching. Although there are evident diVerences inlarval morphogenesis (number of instars), all decapodspecies analysed show the same mode of larval nutri-tion and place of development, namely planktotrophic(as opposed to lecithotrophic) and planktonic (inopposition to demersal and epibenthic; more details inthe section below). Therefore, the inter-speciWc diVer-ences in egg size may be inXuenced more by phylogenythan by diVerences in reproductive or early life historytraits. In terms of decapod phylogeny, recent Wndingssuggest: (1) within Pleocyemata, the natant lineages(including Caridea) have an early origin, around420 mya; (2) the placement of Brachyura as the basalreptant lineage, i.e. the earliest diverging group withinthe reptant clade (clade that originated 385 mya),which is also supported by fossil records conWrming thelong evolutionary history of Brachyura; and (3) Astaci-dea is monophyletic and originated 325 mya (see Fig. 2in Porter et al. 2005). Based on these phylogenetic rela-tionships and divergence times, we can speculate thatthe bigger egg sizes of Astacidea (lobsters) may be anevolutionary trend in decapod taxa that is related to anincreasing degree of lecithotrophy, which is consistentwith a general decrease in the number and variabilityof larval stages (Anger and Harms 1990). In support ofthis trend, the evolutionarily recent group of lithodidcrabs (Anomala) seems to have developed a completeendotrophic larval development, i.e a non-feedingmode of development from hatching to metamorphosisof the late megalopa (Kattner et al. 2003).

Increased egg size with large energy reserves andsmall clutch sizes (i.e. reduced fecundity) has been con-sidered as a latitudinal pattern (from the equatortowards the poles) in reproductive traits in carideanshrimps (Anger et al. 2004). In polar ecosystems, largeyolky eggs are indicative of higher endotrophy in lar-vae and of their reduced dependence on planktonicfood sources (Kattner et al. 2003). Prolonged broodingperiods and extended hatching rhythms are also lifehistory traits expected in these regions (Thatje et al.2003, 2005). These energy saving reproductive strate-

Fig. 5 Principal component analysis based on the FA composi-tions of decapod crustacean embryos in the early stage of embry-onic development. a PC plot. b Loading plot of FAs and theircontribution to the spread along PC1 and PC2. For species abbre-viations see Table 1

123

944 Mar Biol (2007) 151:935–947

gies in response to cold and food limitation are alsoobserved in deep-sea environments (Morley et al.2006), where there is also an overall scarcity of avail-able food resources (Labropoulou and Kostikas 1999)and a relatively stable physical environment (Gage andTyler 1991). Deep-sea species tend to have a smallernumber of large embryos to counteract the decrease inlarval survival probabilities with increasing depth(King and Butler 1985). However, it has been morediYcult to demonstrate the “evolutionary temperatureadaptation” (see Clarke 2003) in the deep sea than inhigher latitudes. Company and Sardà (1998) looked atthe reproductive patterns of Wve deep-water speciesfrom the genus Plesionika and no signiWcant relation-ship was found between egg size and depth distribu-tion. Furthermore, Herring (1974) demonstrated thatabbreviated development in several oceanic species ofCaridea was not related to depth distribution.

With the present caridean shrimps, egg size did notseem to be directly related to depth or to the major abi-otic (temperature) and biotic (food availability) factorsassociated with it. The largest embryos were observedin the shallow-water palaemonids and the smallest inthe deep-sea pandalid species in the genus Plesionika(C. crassicornis had similar stage 3 embryo sizes as Pal-aemon elegans). However, it is worth noting that thedeep-sea Plesionika spp. do not have demersal, lecitho-trophic larval development; instead the larvae migrateand develop in or just below the euphotic zone, wherethere is a rich supply of food, phytoplankton and sus-

pended matter (i.e. planktotrophic larval development;Omori 1974). The greater investment per embryo inthe neritic palaemonid species (i.e. increased embryosize and higher FA content) may be a response to thehighly variable environmental conditions that plankto-trophic larvae face. They hatch in habitats character-ized by strong currents and wave action and,consequently, may be adapted to a broader range ofenvironmental conditions than the larvae of deep-seaand pelagic species.

Three groups were distinguished in the PCA,namely the neritic caridean species (Group 1), thedeep-sea pandalids from the genus Plesionika and lob-ster (Group 2), and the portunid crabs and the panda-lid C. crassicornis (Group 3). The diVerentiation alongPC1 was mainly due to the C18 and C20 PUFA andthese diVerences may result from diVerences in dietand ecological niche. Palaemon spp. and L. seticaudata(Group 1, from intertidal and infralitoral zones) areomnivorous species feeding mainly on macroalgae (e.g.Laminaria spp. and Rhodophyceae), and to a lesserextent on moss, detritus, and small arthropods (Lag-ardère 1971; Calado and Narciso 2000; Orav-Kotta2004), while Plesionika are active predators of macro-plankton (namely euphausiids; Cartes et al. 2002;Cartes 1993), lobsters prey mainly on polychaetes,decapod crustaceans and Wsh (Cristo and Cartes 1998),swimming crabs are epibenthos feeders, and C. crassi-cornis is an infaunal feeder (Cartes et al. 2002). There-fore, due to these trophic diVerences it is not surprising

Fig. 6 Inter-speciWc diVer-ences in FA trophic markers during the early stage of embryonic development. a EPA/DHA and 18:1n ¡ 7/18:1n ¡ 9 ratios, and b 18:1n ¡ 9 and percentage of odd-numbered FAs

0

1

2

3

P. ele P. ser L. set P. mart(west)

P. mar(south)

P. narv H. gam N. nor P. hen M. tub C. cra

b

5

10

15

20

25

30

81:1

n-(

9%

)

18:1n-9 Odd-numbered

ΣO

dd-n

ub

mdere

FA

(%)

0

1

2

3

4

5

EP

/A

HD

A

a

0

Group 1

0,2

0,4

0,6

0,8

18:1

n-/7

81:1

n-9

EPA/DHA 18:1n-7/18:1n-9

Group 3 Group 2

123

Mar Biol (2007) 151:935–947 945

to see Group 1 with higher percentages of essential C18(linoleic acid, 18:2n ¡ 6 and linolenic acid, 18:3n ¡ 3)and C20 PUFA (namely EPA)—fatty acid trophicmarkers (FATM) of macroalgae. Moreover, EPA/DHA and 18:1n ¡ 7/18:1n ¡ 9 ratios (Fig. 6a), i.e.FATM used to determined the degree of carnivory(Auel et al. 2002; Scott et al. 2002), were signiWcantlyhigher in Group 1 than in the other groups (P < 0.05).Higher values suggest lower trophic levels due to thefact that DHA is highly conserved throughout the foodchain and because 18:1n ¡ 9 is the major FA in marineanimals and a general marker of carnivory (Fig. 6b; seeDalsgaard et al. 2003).

The distinction between Group 2 (genus Plesionikaand lobsters) and Group 3 (portunids and C. crassicor-nis) was mainly due to diVerences in the percentage ofC18 MUFA, namely 18:1n ¡ 7 and 18:1n ¡ 9 and sev-eral SFA, including the odd-numbered 15:0 and 17:0.These dissimilarities (Fig. 6b) can result from theoccurrence or absence of detritivorous/scavengerbehaviours. Odd-numbered FAs are known to belargely biosynthesized by marine heterotrophic bacte-ria, which are particularly abundant in marine sedi-ments (Volkman et al. 1998).

Fatty acid proWles are thought to reXect habitat tem-perature, with colder and/or deeper water speciesshowing a higher degree of unsaturation in their FAproWle (Narciso 1999). The Xuidity of biological mem-branes is dependent on the unsaturation of FAs inmembrane lipids (Los and Murata 2004). BecausePUFA have lower melting points than saturated FAs,they maintain the Xuidity of membrane phospholipidsat low temperatures (Munro and Thomas 2004). Theresults presented here (Table 4 and Supplementaryonline material) do not support the trend of increasedFA unsaturation with increased depth/decreasedtemperature, possibly owing to the limited depth andtemperature ranges tested.

Intra-speciWc diVerences in FA proWle wereobserved between the P. martia martia from the westand south coast of Portugal. The southern populationhad smaller embryo volumes and lower total FA con-tents than the western population, which may be a con-sequence of higher incubation temperatures in watersoV the south coast. The south continental slope of Por-tugal is dominated by a sub-tropical branch of EasternNorth Atlantic Central Water (Fiúza et al. 1998), andMediterranean waters originating in the outXow fromthe Strait of Gibraltar. The Mediterranean outXowruns below the 300-m isobath along the south coast(Ambar and Howe 1979). These Wndings are corrobo-rated by data from the sub-tropical pandalid of thesame genus from Madeira Island—P. narval, which had

similar embryo size to those from the southern (tem-perate) population of P. martia martia, but even lowerFA content. The larger embryos and higher FA con-tents found in the western population of P. martia mar-tia may provide suYcient endogenous reserves forlonger incubation. The southern temperate and thesub-tropical populations revealed higher energy con-sumption throughout embryogenesis than the westerntemperate population. In fact, embryo metabolic rateincreases with temperature, which decreases the dura-tion of embryonic and larval development andenhances the utilization rates of endogenous reserves(Evjemo et al. 2001; García-Guerrero et al. 2003).

During embryogenesis unsaturated fatty acids(UFA) were used up at a higher rate than SFA; withinthe UFA, MUFA (primarily 16:1n ¡ 7, 18:1n ¡ 9, and18:1n ¡ 7) were consumed to a greater extent thanPUFA (% of utilization is given in Fig. 4). Since SFAare non-essential and can be synthesized de novo orobtained by desaturation of MUFA and PUFA, theirlower rate of consumption may suggest either a selec-tive retention during embryonic development or par-tial utilization and replacement. The importance ofPUFA in crustacean ontogeny has been extensivelyinvestigated during the past several decades (Anger2001). They contribute to the functional maturation ofthe central nervous system (Bell and Dick 1990; Foxet al. 1994). Embryo concentration of ARA (derivativeof the precursor of the n ¡ 6 series of essential FAs—linoleic acid, 18:2n ¡ 6) and DHA (formed from theprecursor of the n ¡ 3 series, linolenic acid, 18:3n ¡ 3)depends, in part, on the intake of the appropriate pre-cursors in the diet.

The substantial decrease in the FA content duringembryogenesis is directly linked to neutral lipid catab-olism, namely of triacylglycerols (TAG), sterols, anddiacylglycerols (see lipid class results in lobsters; Rosaet al. 2003, 2005). The utilization of TAG duringembryonic development implies a release of free fattyacids, which besides energy production can also bediverted to growth (namely PUFA) by conversion andincorporation into polar lipids. Phospholipids areimportant as structural membrane components and asemulsifying agents in biological systems. They also playan active role in lipid transport in the hemolymph andFA absorption within the body (Teshima 1997).

Lobster embryonic development is much more lipidenergy demanding than that of the other decapod spe-cies studied. This may be also a reXection of the evolu-tionary trend (of increasing degree of lecithotrophy) indecapod taxa, as was discussed before. On the otherhand, a lower FA consumption can be interpreted as anenhanced independence of the newly hatched larva on

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external energy sources (Wehrtmann and Graeve1998). Higher FA content at hatching and, as a conse-quence, a greater independence from the external envi-ronment should increase the chances of larval survival.

Acknowledgments The Foundation for Science and Technol-ogy supported this study through a doctoral grant to the Wrst au-thor and also through the research project POCTI/BSE/43340/2001. Gratitude is due to Dr Ricardo Araújo and Dr Manuel Bi-scoito from the Estação de Biologia Marinha do Funchal—MuseuMunicipal do Funchal (Historia Natural) for their help obtainingthe Madeira specimens. The authors would also like to thank S.Morais, A. Rodrigues, C. Pires and T. Pimentel for their supportduring Weld and laboratory work and Dr Brad Seibel for criticallyreading and editing the English text. The experiments describedcomply with current Portuguese and EU laws.

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