Comparative Biochemistry and Physiology Part B 131(2002) 695–712

1096-4959/02/$ - see front matter� 2002 Elsevier Science Inc. All rights reserved.PII: S1096-4959Ž02.00042-8

Comparison of growth and lipid composition in the green abalone,Haliotis fulgens, provided specific macroalgal diets

Matthew M. Nelson *, David L. Leighton , Charles F. Phleger , Peter D. Nicholsa,b, a,c a d

Department of Biology, San Diego State University, San Diego, CA 92182, USAa

Department of Zoology, University of Tasmania, Hobart, Tasmania 7001, Australiab

Marine Bioculture, Leucadia, CA 92024, USAc

CSIRO Marine Research, Hobart, Tasmania 7001, Australiad

Received 26 January 2001; received in revised form 10 May 2001; accepted 19 May 2001

Abstract

Lipid composition of abalone was examined over a one-year interval. A feeding trial was designed to cover a fullreproductive cycle in young adult green abalone,Haliotis fulgens, consisting of five diet treatments: the macrophyticalgal phaeophyteEgregia menziesii, rhodophyteChondracanthus canaliculatus, chlorophyteUlva lobata, a composite ofthe three algae and a starvation control. The lipid class, fatty acid, sterol and 1-O-alkyl glyceryl ether profiles weredetermined for foot, hepatopancreasygonad tissues and larvae. The major fatty acids were 16:0, 18:0, 18:1(n-7)c, 18:1(n-9)c, 20:4(n-6), 20:5(n-3) and 22:5(n-3), as well as 14:0 for abalone fed brown and red algae. 4,8,12-Trimethyltridecanoicacid, derived from algae, was detected for the first time inH. fulgens (hepatopancreas complex, 1.2–13.9%; larvae,0.5% of total fatty acids). Diacylglyceryl ethers were present in larvae(0.6% of total lipid). The major 1-O-alkylglycerols were 16:0, 16:1 and 18:0. Additionally, 18:1(n-9) was a major component in hepatopancreasygonad and larvae.The major sterol was cholesterol(96–100% of total sterols). Highest growth rates were linked to temperature andoccurred in abalone fed the phaeophyteE. menziesii (43 mmØday , 56 mgØday yearly mean), an alga containing the–1 –1

highest levels of C polyunsaturated fatty acids and the highest ratio of 20:4(n-6) to 20:5(n-3). This study provides20

evidence of the influence of diet and temperature on seasonal changes in abalone lipid profiles, where diet is moststrongly related to body mass and temperature to shell length. The allocation of lipids to specific tissues in green abaloneclarifies their lipid metabolism. These results provide a basis for improving nutrition of abalone in mariculture throughformulation of artificial feeds.� 2002 Elsevier Science Inc. All rights reserved.

Keywords: Abalone; Diacylglyceryl ether; Fatty acid;Haliotis fulgens; Larvae; Lipid; Polyunsaturated; Sterol

1. Introduction

Elements that favor reproductive success maybe critical for species that reproduce by broadcastspawning. Knowledge of such factors may beespecially valuable for species approaching threat-ened status, as is the case with southern Californiaabalone. Abalone mariculture, whether oriented to

*Corresponding author. Tel.:q613-6232-5268; fax:q613-6232-5123.

E-mail address: mmnelson@utas.edu.au(M.M. Nelson).

supply of seed for fishery enhancement or theproduction of adults andyor juveniles for seafoodmarkets, depends on maintenance of broodstock inprime condition (Leighton, 1989). There isincreasing evidence that in abalone, specific die-tary lipids play important roles in gonadogenesis(Uki and Watanabe, 1992). Macroalgae, whichcomprise the diet of these herbivorous gastropods,have lipid profiles which differ between taxa, andvary both geographically and seasonally(Johns etal., 1979; Nelson et al., 2002). Postlarval abalone

696 M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

rely upon high protein and lipid-containingmicroalgae, and as juveniles undergo a dietarytransition to feeding on macroalgae. A number ofbiochemical dietary influences on abalone havebeen documented, as reviewed by Olley andThrower (1977), Fleming et al.(1996). Althoughall elements of diet are nutritionally important toabalone growth, including carbohydrate and pro-tein components, lipid content and class composi-tion may be especially vital to abalone nutrition.Lipids may be synthesized de novo by herbivores,but some have been demonstrated to be derivedonly from the diet and are thus considered essential(Uki et al., 1986b). Growth rates of the abaloneH. discus hannai have been correlated with classand quantity of dietary lipid(Uki et al., 1985,1986b). Since marine algae differ in their lipidcomposition, growth of herbivorous species maybe significantly affected by the species of macroal-gae consumed(Uki et al., 1986a).

Abalone tissues show seasonal changes in lipids,as well as other components. Many molluscsexhibit seasonal changes in proximate composi-tion, as in the limpetNacella (P.) macquariensis(Simpson, 1982), the oysterOstrea edulis (Ruizet al., 1992), and the scallopsP. yessoensis (Tak-ahashi and Mori, 1971) and P. maximus (Soudantet al., 1996), and changes in saturation of fattyacids, as has been shown in the scallopPlacopec-ten magellanicus (Napolitano and Ackman, 1993).Growth rate and gonad maturation inH. cracher-odii (Leighton and Boolootian, 1963; Webber,1970) and larval settlement success inH. rufescens(Slattery, 1992) were shown to vary seasonally.For abalone, information is needed to quantifylipid components in the diet and the effects ofdietary lipid on both somatic and gonadal growth.Knowledge regarding the dietary sources and inthe essentiality and roles of specific lipids in thesemarine molluscs is vital.

In this study, selectivity of lipid incorporationin tissues of the green abalone,Haliotis fulgensPhilippi, was examined. Specific attention wasgiven to arachidonic acid and eicosapentaenoicacid found in macroalgae upon which the adultscommonly feed and the possible effects of thesefatty acids on abalone growth. Evidence wassought to confirm that these eicosanoid precursorsare obtained from, and the quantities may beinfluenced by, the diet. We attempted, by a feedingexperiment, to gain evidence that the diet plays amajor role in growth and gonadal maturation by

influencing a shift of lipids between somatic andgonadal depots; the composition of the diet canaffect energy allocation.

2. Materials and methods

2.1. Aquaria

A feeding trial was conducted over a one yearperiod(October, 1997 to October, 1998) to exam-ine seasonal patterns in abalone growth and gonadmaturation. Shell length and total body masseswere measured monthly and tissue samples takenquarterly for lipid analyses. Seawater facilities atthe Marine Biology Division Experimental Aquar-ium, the Scripps Institution of Oceanography(SIO), La Jolla, CA, were utilized. Seawater sup-plied to laboratories is pumped from the SIO pierand filtered through(Tahitian) sand filters. Theaquaria consisted of sixteen 18.9 l clear or{nat-ural’ polyethylene pails, each covered with a plas-tic screen and with screened PVC pipe drainsplaced near the upper lip for exit of seawater.Mixing by aeration and flow facilitated faecesremoval while retaining abalone and algae. Theflow rate was 1.5 lØmin . Air stones were placed–1

in each container, and were replaced monthly.During monthly measurements, abalone wereremoved and aquaria were cleaned to removeserpulid polychaete worm tubes and biofilm.Remaining algal food was removed, weighed andfresh algae provided.

2.2. Experimental design

The year long feeding experiment consisted offive diet treatments, including three classes ofmacroalgae: the phaeophyteEgregia menziesii(Turner) Areschoug, the rhodophyteChondracan-thus canaliculatus (Harvey) Guiry wformerlyGigartina canaliculatata (Harvey)x and the chlo-rophyteUlva lobata (Ku tzing) Setchell and Gard-ner. In another treatment the abalone were fed amixture of the three algae. The final treatment wasa starvation control. Three replicate experimentalunits were used per treatment, which consisted offive abalone and the assigned diet in each aquari-um. The remaining container was assigned algaeonly, to test for any changes in mass due to growthor deterioration. Treatments were randomizedamong aquaria.

697M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

Female green abalone,H. fulgens (ns75), weresupplied by Marine Bioculture, Leucadia, CA, inSeptember 1997. Animals were approaching firstsexual maturity with mean maximum length of55.5 mm(42.2–68.7 mm) and mean mass of 20.0g (8.4–41.6 g). The abalone had been culturedfrom gametes spawned by wild broodstock andwere considered of normal genetic heterogeneity.Individual abalone were labeled and coated withclear epoxy and affixed to the center of the shell.Abalone were fed a mixed algal diet ad libitumfor a six-week acclimation period prior to com-mencement of controlled feeding. One abalone peraquarium(3Øtreatment ) was sacrificed quarterly–1

for lipid analyses, and several abalone excessiveto the experimental design were maintained as aprecautionary measure. The temperature of thewater flowing through aquaria was recorded week-ly, ranging from 17 to 218C (Fig. 2b). Normaltemperature data are recorded at SIO pier near theseawater system intake.

2.3. Morphometrics

Maximum shell length(mm) was measuredmonthly using vernier callipers. Mass(g) wasmeasured monthly with abalone blotted to removeexcess water. Soft body mass and shell mass wererecorded for abalone sacrificed quarterly. Shellswere cleaned of epizootic growth. To performperiodic measurements, water was removed fromthe aquaria, which caused abalone to relax theirhold for easy removal. Ford–Walford plots(Ford,1933; Walford, 1946) were utilized to visualizegrowth by plotting initial vs. final measurements.Growth rates for shell length(GR : mmØday )–1

L

and in body mass(GR : mgØday ) were calcu-–1M

lated with an equation adapted from Uki et al.(1986b). Specific growth rates were calculatedwith the equation:

–1 –1w xSGR(%Øday )s100 ln(L )-ln(L ) Ødays1 0

Such equations compensate for differences ininitial size (Mai et al., 1995, 1996).

2.4. Macroalgal collection

Algae were collected weekly.E. menziesii(freshly detached; blades excluding pneumato-cysts) was collected from Tourmaline Beach, andU. lobata from Perez Cove and Dana Landing´Marinas in Mission Bay, San Diego, CA.C.

canaliculatus was collected from Shore Line Park,Santa Barbara, CA. Specimens were selected forhigh quality (live, healthy plants); freshness, fullcolor, with neither obvious deterioration nor epi-phytes. After rinsing and agitation under flowingseawater for 10 min(to assist the removal ofepiphytes), algae were blotted on paper towels toremove surface moisture and weighed.

2.5. Abalone larvae

Two batches of larvae(10 000 total) were pro-duced by Marine Bioculture from two separatespawnings ofH. fulgens. Larvae were placed as 2ml of packed larvae in 10 ml vials. All were earlystage veliger larvae, approximately 250mm inlength.

2.6. Lipid analyses

Foot (columellar muscle) tissue, hepatopan-creasygonad tissue and larvae of the abalone wereanalyzed, as well as the algae specified above(Nelson et al., 2002). Samples were dissected,frozen in a –708C freezer and lyophilized at theV. Vacquier laboratory, SIO. Samples were trans-ported frozen(dry ice) by air to CSIRO MarineResearch, Australia, and stored at –708C untilanalysis with methods used as outlined in Nelsonet al. (2002). Samples were homogenized, rehy-drated and quantitatively extracted overnight usinga modified Bligh and Dyer(1959) one-phaseMeOH–CHCl –H O extraction. Individual lipid3 2

classes were quantified using a TLC-FID analyzer;fatty acids, sterols and 1-O-alkyl glycerols werequantified with a gas chromatograph(GC) andconfirmed using a GC-mass spectrometer. Lowgonad mass necessitated that gonad and hepato-pancreas were analyzed together. To gain the vol-ume of tissue required and reduce the number ofsamples, replicate numbers 2 and 3 were pooledinto one sample for analysis.

3. Results

3.1. Morphometrics

The Ford–Walford plot of shell length illustratesthat abalone fedE. menziesii by far showed thegreatest change in mean shell length(q16.4 mm;Fig. 1), followed by abalone on the compositealgae treatment(q6.1 mm). Abalone fedC. can-

698 M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

Fig. 1. Ford–Walford plot of mean shell length ofH. fulgensfed different algal diets for one year. Presented as mean"S.E.

aliculatus and U. lobata showed a similar changein shell length(q1.1 and 0.8 mm, respectively),and starved abalone showed no shell growth. Meanbody mass data also revealed that abalone fedE.menziesii had the greatest positive effect ongrowth, with a 21.3 g gain in mass. Animals fedthe composite algal diet showed a change(q7.4g) that was 1y3 that of those fedE. menziesii,which reflected the amount of brown alga given.Starved abalone had decreased mass(–5.8 g), asdid those fedU. lobata (–3.3 g). There was asmall change in body mass in those fedC. canal-iculatus (–0.4 g).

Abalone fedE. menziesii had the greatest growthrates(GR) which averaged 42.8mmØday in shell–1

length and 55.6 mgØday in body mass over the–1

entire year. Other diets provided lower averagegrowth: composite diet(15.9 mmØday , 19.4–1

mgØday ), C. canaliculatus (2.9 mmØday , –1.1–1 –1

mgØday ) and U. lobata (2.1 mmØday , –8.6–1 –1

mgØday ). The starved abalone had negative–1

growth rates. All treatments showed bimodal GRwith growth depression in May(Fig. 2a, Fig. 3a),for corresponding minimums during mid-April forlength and late April for mass. Even at the firsttime interval (Oct.–Nov.), E. menziesii diet hadthe most pronounced positive effect on GR(44.0mmØday ). The GR (length) was highest during–1

L

the first peak(January, 69.1mmØday ) with a–1

lower second peak occurring in July(64.7mmØday , Fig. 2a). The first peak of the GR–1

M

(mass) plot in animals fedE. menziesii was inMarch (50.2 mgØday ), and the second larger–1

peak was in July(119.7 mgØday ; Fig. 3a). The–1

corresponding peaks in mean specific growth rates(%Øday ) were 0.118 and 0.101 for length–1

(SGR ) and 0.223 and 0.399 for mass(SGR ).L M

The bimodal peaks in mass growth rates wereevident for all diets compared to shell lengthgrowth rates.

Percentage soft body mass(% of total mass) ofabalone is the mass of tissue compared to the massof the entire individual(tissue and shell). Nodifferences were apparent between seasons or treat-ments (53.2–66.1%), except the starvation treat-ment, which approached 50% in spring. Percentagewater content(% of total mass) in abalone tissuesincreased in spring and decreased into autumn,and values were similar among tissues and betweentreatments(72.2–82.9%). There was one excep-tion; the starvation treatment had the highest watercontent(92% in spring).

3.2. Lipid content

The lipid content(mgØg dry mass; Table 1)–1

differed between treatments. Lipid in the footremained low over the year(15–34 mgØg ), was–1

high in the hepatopancreasygonad(HG) (36–131mgØg ) and showed temporal variation. Lipid–1

cumulatively increased in the HG of animals fedE. menziesii from winter (75 mgØg ) to autumn–1

(131 mgØg ), and slightly decreased in the foot–1

(27–17 mgØg , winter–autumn). Lipid content in–1

HG of animals fed C. canaliculatus remainedconstant over the year(;39 mgØg ) and the foot–1

lipid was also constant(;30 mgØg ), although a–1

slight decrease occurred in autumn(22 mgØg ).–1

For abalone fedU. lobata, HG lipids were high inwinter (129 mgØg ), dropped to 37 mgØg in–1 –1

spring, and increased to 48 mgØg by autumn.–1

The amount of lipid in foot of abalone fedU.lobata did not change(;26 mgØg ). On average,–1

HG of animals fed the composite diet decreasedfrom winter (84.9 mgØg ) to summer (48.5–1

mgØg ), and increased again to 75 mgØg by–1 –1

autumn. Lipid in foot of animals with the compos-ite diet gradually decreased from winter(27.9mgØg ) to autumn(15.4 mgØg ).–1 –1

699M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

Fig. 2. Intra-annual comparison of(a) the mean growth rate of shell length ofH. fulgens fed different algal diets to(Presented asmean"S.E.) (b) the ambient temperature of aquarium water for 1997–1998 and Scripps Institution of Oceanography(SIO) pier bottom(5 m) water for 1995–1996.

3.3. Lipid classes

Polar lipid (PL) comprised a major lipid classin all samples(38–93% of total lipid; Table 1),although triacylglycerol(TAG) was a major com-ponent(up to 56%) in some HG samples. TAGwas highest in animals fedE. menziesii andincreased over the year(from 27 to 56% of total

lipid). TAG was next highest in the compositealgal diet (5–32%), followed by generally lowerabundance in the remaining treatments(0.4–10.6%), with the exception of theU. lobatadiet in winter(53%). The starvation treatment hadequal amounts of TAG in HG and foot tissues(10.6%). Sterol(ST) were generally higher in foot(7.4–26.7%) than in HG (2.6–12.7%), and free

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Fig. 3. Intra-annual comparison of(a) the mean growth rate of mass ofH. fulgens fed different algal diets to(b) the sum of arachidonicacid wAA, 20:4(n-6)x and eicosapentaenoic acidwEPA, 20:5(n-3)x in gonadyhepatopancreas tissues ofH. fulgens and (c) the sum ofAA and EPA in the macroalgae fed toH. fulgens. Presented as mean"S.E.

701M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

Table 1Percentage lipid class composition ofHaliotis fulgens tissuesa

Tissue Wax DAGE TAG Free fatty Sterols Polar Lipid: mg gy1

treatment Ester acid lipid dry mass

FootE. menziesiiWinter – – – 0.7"0.5 10.9"1.9 88.4"1.4 27.4"4.9Spring – – – 0.5"0.1 18.0"0.6 81.6"0.5 28.7"0.4Summer – – – 0.5"0.1 17.4"0.4 82.2"0.4 19.8"2.8Autumn – – – – 7.5"1.1 92.5"1.1 17.3"4.8C. canaliculatusWinter – – – – 13.7"7.0 86.3"7.0 30.5"1.6Spring – – – 1.1"0.5 17.7"0.4 81.2"0.1 29.3"3.9Summer – – – 0.4"0.2 17.6"1.1 82.0"1.3 30.8"4.8Autumn – – – – 7.4"0.2 92.6"0.2 21.8"0.5U. lobataWinter – – – – 16.1"0.2 83.9"0.2 26.2"13.3Spring – – – 0.4"0.1 16.9"0.3 82.6"0.2 27.8"0.4Summer – – – 0.3"0.1 18.2"0.1 81.5"0.0 24.6"1.8Autumn – – – – 8.2"1.7 91.8"1.7 24.2"2.3CompositeWinter – – – 0.4"0.5 13.5"3.2 86.2"2.7 27.9"0.1Spring – – – 0.4"0.1 17.5"1.1 82.1"1.2 26.9"0.3Summer – – – 0.3"0.0 16.3"0.2 83.4"0.2 24.8"2.6Autumnb – – – – 7.8"2.3 92.2"2.3 15.4"1.3StarvedWinter – – – – 8.9"0.5 91.1"0.5 33.7"3.4Spring 1.3"0.2 – 10.6"10.3 2.3"0.4 26.7"1.0 59.1"9.1 31.1"2.1

HepatopancreasyGonadE. menziesiiWinter 2.9"0.1 – 26.7"1.9 10.8"0.6 5.5"0.6 54.1"2.0 74.7"5.1Spring 4.3"0.0 – 38.7"4.2 10.7"0.6 6.3"2.7 40.1"1.0 76.6"0.6Summer 0.6"0.3 0.2"0.1 45.0"3.5 10.4"4.8 5.4"2.4 38.3"3.2 91.3"4.6Autumn 0.3"0.0 – 55.8"2.8 2.9"1.1 2.6"0.2 38.4"1.6 130.5"15.0C. canaliculatusWinter 0.8"1.2 – 3.8"5.4 2.6"0.9 10.2"1.9 82.5"9.4 36.3"14.4Spring 0.4"0.1 – 5.8"1.7 6.2"0.9 5.7"0.9 81.8"1.6 42.3"10.4Summer – – 0.9"1.2 4.2"2.4 8.4"2.9 86.6"4.1 39.3"13.5Autumn 0.1"0.0 – 0.5"0.4 5.5"0.3 7.9"6.3 86.0"5.6 39.8"8.2U. lobataWinter 1.5"1.1 – 53.3"0.6 7.3"4.1 4,2"2.5 33.6"7.0 128.6"30.1Spring – – 1.1"0.8 9.5"2.0 7.4"0.4 82.0"3.2 37.0"1.2Summer 1.4"2.0 – 3.9"5.5 8.4"0.2 7.5"1.9 78.9"9.5 45.2"7.3Autumn 0.7"0.8 – 0.4"0.0 6.3"5.3 8.0"0.7 84.6"3.8 48.0"3.8CompositeWinter 4.0"4.0 0.2"0.3 18.9"26.7 8.0"1.3 7.6"0.6 61.4"31.7 84.9"27.3Spring 2.1"0.1 – 28.2"8.5 7.0"2.2 5.3"2.4 57.3"13.0 60.3"9.1Summer 1.6"1.0 – 5.0"2.7 9.2"2.4 8.8"1.9 75.4"0.6 48.5"11.4Autumn 0.5"0.0 – 32.1"30.6 9.1"6.9 5.1"2.2 53.2"21.5 75.1"32.0StarvedWinter – – 7.6"8.4 11.5"5.9 11.2"3.5 69.7"10.9 39.0"10.9Spring 1.6"0.1 – 10.6"2.7 9.6"3.7 12.7"2.2 65.6"4.4 42.2"23.2Larvae 0.9"0.4 0.6"0.2 71.2"1.3 2.6"0.6 2.5"0.7 22.1"2.0 23.7"16.2

Presented as mean"S.D.,ns2a

ns3; (y), below detection; DAGE, diacylglyceryl ether; TAG, triacylglycerol.b

702 M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

fatty acids (FFA) were present in all samples(0.3–11.5%). Wax ester(WE) was found only inHG (0.1–4.3%), and diacylglyceryl ethers(DAGE) were only detected in a few samples(0.2%). In abalone larvae, lipid was dominated byTAG (71% of total lipids), followed by PL(22%).ST and FFA were minor(2.5–2.6%) and WE andDAGE were-1% (Table 1). The amount of lipidwas 24 mgØg dry mass.–1

3.4. Fatty acids

Palmitic acid (16:0) was the major fatty acid(FA) in all samples(21–33% of total FA; Tables2 and 3). The second most abundant FA werecis-vaccenic acidw18:1(n-7)c, 6–15%x, arachidonicacid wAA, 20:4(n-6), 4–15%x, oleic acidw18:1(n-9)c, 1–13%x, myristic acid(14:0, 1–9%), eicosa-pentaenoic acidwEPA, 20:5(n-3)x (3–8%) and 22:2non-methylene interrupted(NMI) diunsaturatedFA (2–6%). Docosapentaenoic acidwDPA, 22:5(n-3)x was present in all samples, with percentageshigher in foot (6–12%) than in HG (2–8%).Docosahexaenoic acidwDHA, 22:6(n-3)x was aminor FA (-1%). Only the HG contained 4,8,12-trimethyltridecanoic acid(4,8,12-TMTD), whichwas more abundant in animals fedU. lobata(1.2–13.9%) than in other treatments(1.0–7.3%).Total FA (mgØg dry mass) in HG varied with–1

diet: E. menziesii (50–101 mgØg ), C. canalicu-–1

latus (17–22 mgØg ), U. lobata (19–95 mgØg ),–1 –1

composite algal diet(27–49 mgØg ) and starved–1

animals(16–30 mgØg ; Table 3). Total FA in the–1

foot was lower than in HG throughout the year(7–19 mgØg dry mass) in all abalone(Table 2).–1

The sum of AA and EPA(mgØg dry mass) in–1

HG showed a seasonal pattern, ranging from 3 to9 mgØg in all abalone (Table 3, Fig. 3b).–1

Amounts in foot were lower(1–3 mgØg ; Table–1

2). The ratio of AAyEPA also varied throughoutthe year, from 1–3 in HG and 2–4 in foot of allanimals(Tables 2 and 3).

In larvae, major FA included palmitic acid(16:0,30% of total FA), with equal amounts of 18:1(n-9)c and 18:1(n-7)c (14%), followed by 16:1(n-7)c (10%; Table 4). The AAyEPA ratio was low(1.3), since abundances of AA and EPA weresimilar (4 and 3%, respectively). DPA was low(2%) and DHA was not detected. The proportionsof total SFAyMUFAyPUFA were 3:3:1. BranchedFA were not abundant.

3.5. Sterols and phytol

Cholesterol was the major ST in adult abalone(97–98% of total ST; Table 5), with a number ofminor components detected: dehydrocholesterolwtr(trace,-0.05%)–1%x, desmosterol(tr), 24-meth-ylenecholesterol(tr–1%) and fucosterol(tr–1%).The only stanol detected was cholestanol(1–2%).Phytol (trans-3,7,11,15-tetramethylhexadec-2-en-1-ol), the isoprenoid side-chain of chlorophyll,was detected in the non-saponifiable lipid fraction.HG was the only tissue that contained phytol(66.4mgØg dry mass), with a low ratio of phytol to–1

ST of 0.02. Representative data are shown for thespring E. menziesii treatment. In larvae, the onlyintegrated ST was cholesterol, although traceamounts of other ST were present.

3.6. 1-O-Alkyl glyceryl ethers

In HG of abalone fedE. menziesii, analyzedfrom spring samples, the major 1-O-alkyl glycerylethers(GE) were 16:0(37% of total GE), 18:1(n-9) (36%), and 18:0(20%), as well as a smallamount of 16:1(8%), and other trace GE(Table6). Trace amounts(-0.05%) of 16:0, 18:1(n-9)and 18:0 were detected in the foot. In larvae, themajor GE were 16:0(43%) and 18:1(n-9) (37%),followed by 16:1 (9%), 18:0 (8%), 20:1 (4%)and several trace components.

4. Discussion

4.1. Lipid classes

The lipid content was consistently higher in HG(36–131 mgØg dry mass) than in foot from all–1

treatments(15–34 mgØg ; Table 1). Lipid content–1

in abalone tissues were generally higher than intheir macroalgal diets(2–30 mgØg ) (Nelson et–1

al., 2002), indicating that selective storage of lipidoccurs in HG. Viscera serve as lipid storage pools,and visceral lipid levels and growth in the abaloneH. tuberculata and H. discus hannai have beenpositively correlated(Mercer et al., 1993). Of noteis the increase of lipid in the HG of abalone fedE. menziesii, which increased from winter toautumn(75 to 131 mgØg ). The increase in lipid–1

in the foot of animals fedE. menziesii in winter issimilar to findings forH. discus, fed a phaeophytediet (Eisenia bicyclis and Ecklonia cava); lipidcontent in the foot muscle ofH. discus increased

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Table 2

Percentage fatty acid composition of the foot tissues ofHaliotis fulgensa

Fatty acid E. menziesii C. canaliculatus U. lobata Composite Starve

Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring

14:0 4.8"0.0 4.2"0.3 4.4"0.1 2.3"1.1 2.3"0.5 3.1"0.1 3.1"0.1 1.7"0.4 1.9"0.5 2.7"0.1 2.2"0.1 1.3"0.2 2.5"0.1 3.4"0.0 3.3"0.3 2.0"0.2 1.6"0.9 2.5"0.1

15:0 0.9"0.0 0.8"0.1 0.7"0.0 0.4"0.1 0.8"0.1 1.6"0.0 2.1"0.4 1.7"0.4 0.8"0.1 1.5"0.1 1.8"0.1 1.4"0.1 0.9"0.1 1.3"0.0 1.5"0.1 1.0"0.2 0.6"0.1 1.0"0.2

16:0 32.7"0.0 27.9"4.6 25.7"0.8 25.1"0.4 24.3"0.2 24.9"0.6 24.5"0.4 23.9"0.0 24.4"0.2 26.0"0.2 24.7"0.7 24.4"0.7 24.3"0.0 25.3"0.3 25.8"0.2 25.7"0.5 23.7"0.4 20.6"1.7

17:0 1.2"0.0 1.1"0.2 1.0"0.1 0.9"0.1 1.1"0.0 1.6"0.1 2.3"0.1 2.7"0.2 1.2"0.0 1.4"0.1 1.7"0.1 2.1"0.0 1.2"0.1 1.3"0.0 1.5"0.1 1.7"0.3 1.3"0.2 1.4"0.2

18:0 7.5"0.1 8.0"1.2 7.4"0.1 8.6"0.3 8.0"0.3 7.0"0.2 7.7"0.4 9.4"0.9 8.3"0.3 7.2"0.5 7.2"0.0 9.9"0.6 7.6"0.0 7.4"0.3 7.1"0.1 8.4"0.4 9.3"0.7 8.5"0.1

Sum SFA 47.2"0.2 42.0"6.5 39.2"1.1 37.3"2.1 36.3"1.1 38.2"1.1 39.8"1.4 39.3"2.0 36.6"1.2 38.8"1.1 37.6"1.0 39.1"1.6 36.5"0.3 38.7"0.7 39.3"0.8 38.8"1.6 36.5"2.3 34.0"2.3

16:1(n-7)c 1.0"0.1 0.8"0.1 0.7"0.0 0.8"0.0 0.7"0.1 0.8"0.2 0.5"0.0 0.7"0.0 0.7"0.1 0.8"0.0 0.7"0.0 0.7"0.1 0.6"0.0 0.9"0.1 0.7"0.1 0.8"0.1 0.8"0.2 1.3"0.2

18:1(n-9)c 6.7"0.0 7.4"1.3 5.9"0.0 6.6"0.3 6.6"0.0 7.1"0.5 6.1"0.2 6.6"0.4 6.3"0.4 5.5"0.4 4.9"0.4 6.3"1.1 5.9"0.5 6.2"0.4 5.9"0.0 6.1"0.2 7.1"0.6 9.8"0.9

18:1(n-7)c 7.7"0.2 7.6"2.2 7.6"0.3 8.7"0.4 7.4"0.1 7.0"0.2 7.1"0.4 8.2"0.1 7.1"0.4 7.9"0.1 7.7"0.2 8.0"0.4 7.0"0.0 7.4"0.0 7.0"0.5 8.0"0.4 7.4"0.4 6.1"0.6

20:1(n-9)c 3.2"0.0 3.7"0.4 3.9"0.1 4.3"0.3 3.6"0.1 3.0"0.3 3.5"0.1 3.7"0.0 3.5"0.1 3.3"0.0 3.9"0.2 3.7"0.0 3.8"0.1 3.1"0.2 3.5"0.1 4.1"0.1 4.0"0.4 3.9"0.4

20:1(n-7)c 0.2"0.0 0.3"0.0 0.3"0.0 0.4"0.0 0.3"0.0 0.2"0.0 0.3"0.1 0.4"0.0 0.3"0.0 0.2"0.0 0.2"0.0 0.6"0.3 0.3"0.0 0.3"0.0 0.2"0.0 0.4"0.0 0.5"0.0 2.1"0.7

Sum MUFA 18.7"0.3 19.8"4.1 18.5"0.5 20.8"1.0 18.6"0.3 18.2"1.2 17.6"0.8 19.6"0.6 18.0"1.0 17.8"0.6 17.5"0.8 19.2"1.9 17.6"0.7 17.9"0.7 17.3"0.7 19.3"0.8 19.8"1.6 23.3"2.8

18:2(n-6) 3.2"0.1 2.6"0.2 2.2"0.1 3.3"0.0 2.6"0.5 2.4"0.4 0.9"0.1 2.8"0.6 3.1"0.2 2.8"0.6 2.9"0.1 2.9"0.3 3.3"0.2 2.9"0.2 2.8"0.1 3.4"0.1 3.4"0.0 1.6"0.1

18:3(n-3) 1.9"0.1 1.4"0.1 1.5"0.1 1.7"0.2 1.2"0.1 0.7"0.1 1.3"0.1 0.5"0.1 2.0"0.1 3.4"0.3 3.6"0.9 2.6"0.8 1.9"0.2 2.2"0.1 1.9"0.1 2.6"0.1 1.4"0.2 0.6"0.1

20:4(n-6) 10.8"0.9 11.9"4.7 12.4"0.8 11.0"1.0 14.0"0.8 14.2"3.0 14.1"0.7 11.8"0.5 12.5"0.2 10.5"0.1 10.6"2.4 10.0"0.1 13.3"0.6 12.2"0.3 13.7"0.6 11.6"0.2 12.3"0.1 9.8"1.2

20:5(n-3) 4.4"0.3 3.1"0.9 5.0"0.1 5.5"0.4 4.8"0.1 3.6"0.6 5.0"0.3 6.7"0.5 5.1"0.4 5.8"0.0 5.3"1.0 5.9"0.1 5.6"0.1 5.3"0.4 4.3"0.5 5.9"0.5 5.4"0.6 5.5"0.8

22:4(n-6) 1.6"0.2 0.9"1.3 – 2.3"0.3 3.2"0.1 5.4"0.4 1.7"2.5 2.6"0.1 3.0"0.0 2.4"0.3 1.1"1.6 3.2"0.3 3.1"0.4 2.6"0.2 3.3"4.6 2.4"0.1 2.9"0.5 4.0"0.0

22:5(n-3) 5.7"0.7 8.7"6.0 11.8"0.4 7.6"0.1 9.9"0.3 8.6"1.2 10.9"2.8 7.9"0.3 10.3"0.3 9.5"0.8 12.2"2.1 8.3"0.1 9.2"1.2 9.2"0.9 9.0"3.7 7.1"0.2 8.9"0.5 10.8"0.1

22:2 NMI 3.2"0.1 5.5"0.0 5.3"0.1 5.1"0.0 5.4"0.1 5.4"0.6 5.4"0.6 4.6"0.3 5.2"0.1 4.7"0.2 4.9"0.4 4.5"0.1 5.2"0.3 4.8"0.3 4.9"0.1 4.4"0.2 5.1"0.2 6.1"0.0

Sum PUFA 30.7"2.4 34.2"13.1 38.3"1.6 36.5"2.1 41.1"1.9 40.3"6.2 39.3"7.2 36.9"2.5 41.3"1.3 39.1"2.2 40.6"8.4 37.3"1.8 41.6"2.9 39.3"2.4 39.9"9.7 37.5"1.5 39.4"2.0 38.3"2.2

Other 3.4 4.1 4.0 5.4 3.9 3.3 3.3 4.2 4.1 4.3 4.3 4.4 4.3 4.1 3.4 4.4 4.3 4.3

Total mg gy1 7.6"2.0 13.9"5.3 8.0"0.9 8.0"0.1 9.3"3.1 10.0"0.7 10.5"1.3 11.4"1.8 14.0"4.2 15.9"2.5 9.2"0.3 9.6"0.4 12.7"6.5 9.2"7.6 8.0"0.5 7.4"0.9 19.1"0.5 12.8"0.3

Ratio AAyEPA 2.48 3.77 2.47 1.99 2.95 3.90 2.86 1.76 2.45 1.81 1.98 1.68 2.38 2.29 3.16 1.98 2.25 1.79

Presented as mean"S.D., ns2; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; AA, arachidonic acidw20:4(n-6)x; EPA, eicosapentaenoic acidw20:5(n-3)x; Other includes alla

components-1% include: 16:1(n-5)c, 16:1(n-9)c, 17:1, i17:0, 4,8,12-TMTD, 18:1(n-5), 18:3(n-6), 18:4(n-3), 20:0, 20:2(n-6), 20:3(n-6), 20:4(n-3), 22:1, 22:3(n-6), 22:4(n-3), 22:6(n-3), C PUFA.24

704M

.M.

Nelson

etal.

/C

omparative

Biochem

istryand

Physiology

Part

B131

(2002)695–712

Table 3

Percentage fatty acid composition of the gonadyhepatopancreas tissues ofHaliotis fulgensa

Fatty acid E. menziesii C. canaliculatus U. lobata Composite Starve

Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring

14:0 6.7"0.8 6.9"0.6 8.9"0.5 8.7"0.9 4.1"0.9 3.7"0.6 3.0"0.1 4.7"1.5 8.1"0.4 1.8"0.0 2.0"0.3 1.8"0.6 5.8"2.7 5.5"0.4 4.3"0.0 6.5"2.0 4.3"1.3 2.4"0.4

15:0 0.6"0.0 0.4"0.0 0.5"0.0 0.3"0.0 1.6"0.6 1.5"0.1 2.3"0.3 1.9"0.1 0.4"0.1 1.4"0.0 1.6"0.2 1.5"0.1 0.9"0.5 0.7"0.0 1.4"0.3 0.9"0.3 1.1"0.3 1.1"0.2

16:0 21.7"1.2 24.3"0.9 26.8"1.5 28.6"3.5 25.2"1.2 23.0"1.6 24.0"0.3 24.4"3.9 29.6"0.5 21.1"1.7 21.9"0.2 20.8"0.3 26.2"4.2 22.2"0.5 24.4"2.6 23.9"2.8 24.2"3.7 20.5"0.2

17:0 0.5"0.0 0.4"0.1 0.4"0.0 0.3"0.0 1.4"0.5 1.8"0.1 2.7"0.1 2.2"0.0 0.3"0.1 1.6"0.0 1.9"0.3 2.2"0.8 0.8"0.5 0.7"0.0 1.3"0.2 0.7"0.4 1.2"0.3 1.8"0.5

18:0 4.0"0.2 3.5"0.3 4.3"0.1 3.3"0.3 4.5"0.8 4.3"0.1 5.3"0.5 5.0"0.5 3.2"0.2 4.6"0.1 4.5"0.3 5.6"1.2 3.8"1.2 3.2"0.2 4.7"0.3 3.9"0.7 5.1"0.5 6.0"0.5

Sum SFA 33.4"2.2 35.5"1.9 40.8"2.2 41.1"4.7 36.8"4.0 34.3"2.4 37.2"1.3 38.2"6.0 41.6"1.3 30.5"1.8 31.9"1.3 31.9"3.0 37.5"9.0 32.4"1.2 36.1"3.4 35.8"6.3 36.0"6.2 31.8"1.8

i17:0 0.2"0.0 0.3"0.0 0.2"0.1 0.0"0.1 0.5"0.0 0.4"0.0 0.7"0.2 0.9"0.2 0.1"0.0 0.6"0.0 1.2"0.7 0.8"0.2 0.2"0.1 0.3"0.0 0.4"0.1 0.3"0.2 0.5"0.1 1.1"0.6

4,8,12-TMTD 5.1"0.1 2.3"0.7 3.0"0.3 1.0"0.1 4.8"3.2 3.4"0.3 3.5"1.2 1.4"0.4 1.2"0.2 13.9"2.5 12.4"1.2 6.8"3.0 6.2"6.2 3.3"0.2 7.3"0.9 4.2"3.2 3.1"0.7 2.4"0.1

16:1(n-7)c 1.5"0.2 6.7"1.3 3.3"0.1 6.8"1.7 2.4"2.3 3.7"0.6 0.7"0.1 1.8"0.7 7.6"0.8 1.1"0.6 1.3"0.7 0.7"0.0 4.2"5.0 7.9"0.7 2.2"0.8 3.0"2.5 3.9"2.1 2.4"1.2

18:1(n-9)c 5.9"0.2 11.8"0.0 8.9"0.3 11.2"0.8 5.7"2.1 7.4"0.8 3.5"0.2 5.0"1.3 12.4"0.5 2.2"0.7 3.1"2.3 2.3"0.7 7.5"5.5 12.2"0.3 5.7"0.8 7.1"3.4 7.7"2.0 6.8"2.3

18:1(n-7)c 6.3"0.5 11.4"0.1 11.7"0.6 14.1"1.0 7.5"1.8 8.6"0.4 6.2"1.1 9.2"1.1 12.7"1.0 7.3"0.4 7.7"1.3 6.7"0.8 8.8"4.5 11.0"0.2 7.8"0.4 9.1"3.6 8.4"1.5 6.9"1.1

20:1(n-9)c 5.6"0.4 5.1"0.6 5.6"0.3 4.3"0.6 6.0"1.2 5.9"1.4 5.9"0.7 6.4"0.5 4.0"0.2 5.5"0.3 7.9"2.6 6.5"0.3 4.7"1.2 6.5"1.4 7.8"0.7 5.1"0.7 5.3"0.7 7.4"1.6

20:1(n-7)c 1.2"0.1 0.9"0.1 1.2"0.1 1.0"0.3 0.8"0.6 0.7"0.2 0.4"0.1 0.6"0.0 0.9"0.1 0.4"0.0 0.7"0.2 0.7"0.1 0.7"0.3 1.0"0.1 0.7"0.2 0.9"0.2 0.7"0.1 1.1"0.0

Sum MUFA 20.4"1.3 36.0"2.1 30.7"1.3 37.4"4.5 22.5"8.0 26.2"3.3 16.7"2.1 23.0"3.7 37.5"2.6 16.4"2.0 20.7"7.1 16.8"1.8 25.9"16.4 38.7"2.7 24.3"2.8 25.2"10.3 26.0"6.5 24.7"6.2

18:3(n-6) 2.1"0.2 0.3"0.0 0.7"0.2 0.4"0.2 0.1"0.1 0.2"0.0 – – 0.3"0.2 0.4"0.2 0.2"0.0 0.1"0.2 0.1"0.1 0.2"0.0 – – 0.1"0.1 –

18:4(n-3) 0.1"0.0 0.3"0.1 0.6"0.0 0.3"0.1 0.1"0.0 0.4"0.1 0.3"0.2 0.5"0.1 0.4"0.2 1.2"0.2 0.8"0.4 0.9"0.6 0.4"0.5 0.1"0.0 0.5"0.0 1.0"0.5 0.2"0.3 0.1"0.2

18:2(n-6) 3.7"0.4 1.4"0.1 2.3"0.3 1.5"0.3 1.9"0.4 0.8"0.2 1.7"0.9 2.6"0.1 1.8"0.5 3.4"0.2 2.8"0.2 3.1"1.0 2.1"1.4 1.3"0.0 2.4"0.2 3.0"1.4 1.6"0.4 1.0"0.7

18:3(n-3) 3.8"0.2 1.6"0.5 1.7"0.3 1.6"0.6 0.5"0.1 1.5"0.3 0.7"0.5 0.6"0.1 1.3"0.4 6.1"0.5 4.5"1.7 5.8"3.1 2.5"1.3 1.3"0.2 2.5"0.2 3.4"1.2 1.0"0.1 1.4"0.9

20:4(n-6) 11.1"1.5 8.4"0.8 5.1"0.8 4.0"0.9 11.8"2.4 11.5"1.1 14.6"1.9 9.1"2.0 3.6"0.7 5.7"0.2 6.5"0.4 8.7"3.5 8.8"4.1 6.9"0.9 8.8"0.2 8.4"2.6 12.4"3.5 14.2"1.0

20:5(n-3) 4.7"0.4 3.6"0.0 3.4"0.3 4.0"1.2 4.3"0.6 5.0"0.1 7.2"0.1 6.3"1.6 2.9"0.2 7.9"0.8 5.5"1.8 7.0"0.8 4.4"2.0 3.7"0.1 4.8"0.5 4.8"1.0 4.0"0.2 5.3"0.6

20:3(n-6) 2.3"0.2 0.7"0.0 1.7"0.3 1.0"0.5 0.5"0.1 0.4"0.0 0.5"0.2 1.0"0.2 0.4"0.0 0.5"0.1 0.4"0.0 0.5"0.3 0.6"0.2 0.5"0.0 0.6"0.0 1.5"0.1 0.3"0.0 0.4"0.2

20:4(n-3) 0.8"0.0 0.6"0.0 1.4"0.0 0.5"0.6 0.7"0.2 0.8"0.1 0.7"0.3 1.3"0.3 0.7"0.0 0.9"0.1 1.0"0.3 0.7"0.1 0.6"0.3 0.9"0.1 0.8"0.2 0.8"0.1 0.6"0.0 0.7"0.4

22:4(n-6) 0.9"0.2 1.0"0.2 0.5"0.0 0.4"0.2 1.9"0.1 1.7"0.2 1.5"0.2 1.0"0.4 0.8"0.2 1.7"0.1 1.7"0.9 3.3"1.0 1.2"0.7 1.1"0.0 1.2"0.0 1.3"0.5 2.5"1.1 3.2"0.7

22:5(n-3) 3.9"0.4 2.5"0.1 2.1"0.2 2.0"1.2 5.4"0.2 6.2"0.2 7.5"0.3 5.4"2.2 2.0"0.3 6.4"0.6 4.7"1.2 6.8"1.0 3.5"0.9 2.8"0.2 3.5"0.4 3.8"1.0 4.8"1.2 6.1"1.3

22:2 NMI 4.0"0.5 3.3"0.5 3.0"0.0 2.5"1.2 5.0"0.4 4.7"0.3 5.2"0.3 5.7"2.1 2.4"0.3 2.5"0.0 3.3"1.0 4.4"1.0 3.6"1.3 3.9"0.3 4.5"0.6 3.7"1.0 4.1"1.4 4.5"0.4

Sum PUFA 37.5"4.1 23.8"2.3 22.5"2.6 18.2"7.2 32.3"4.6 33.2"2.5 39.8"4.9 33.6"9.0 16.6"3.03 6.6"3.0 31.3"8.0 41.5"12.5 27.8"12.7 22.7"1.8 29.6"2.2 31.6"9.5 31.6"8.23 6.9"6.2

Other 3.4 2.2 2.8 2.2 3.2 2.4 2.1 2.9 3.0 2.0 2.4 2.2 2.4 2.7 2.3 2.9 2.9 3.1

Total mg gy1 49.5"6.0 51.3"6.0 53.5"4.0 101.0"25.2 18.1"8.1 22.3"6.8 19.1"4.6 16.9"3.3 95.1"15.3 20.6"1.5 27.9"12.5 19.0"6.0 48.8"26.8 38.4"4.1 26.9"10.7 47.4"29.4 30.4"17.6 16.0"9.8

Ratio AAyEPA 2.38 2.33 1.50 1.00 2.74 2.30 2.04 1.45 1.26 0.72 1.19 1.25 2.01 1.89 1.82 1.73 3.14 2.70

Presented as mean"S.D., ns2; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; AA, arachidonic acidw20:4(n-6)x; EPA, eicosapentaenoic acidw20:5(n-3)x; Other includes all components-1%a

include: 16:1(n-5)c, 16:1(n-9)c, 17:1, 18:1(n-5), 20:0, 20:2(n-6), 22:1, 22:3(n-6), 22:4(n-3), 22:6(n-3), C PUFA.24

705M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

Table 4Percentage fatty acid composition ofHaliotis fulgens larvaea

Fatty acid

14:0 8.8"0.316:0 30.3"0.218:0 2.5"0.1

Sum saturates 41.6"0.74,8,12-TMTD 0.5"0.214:1 0.6"0.016:1(n-7)c 9.6"0.116:1(n-5)c 0.5"0.018:1(n-9)c 13.5"0.318:1(n-7)c 13.6"0.520:1(n-9)c 3.1"0.020:1(n-7)c 0.7"0.0

Sum monounsaturates 41.7"1.218:2(n-6) 1.1"0.118:3(n-3) 0.8"0.120:4(n-6) 3.5"0.320:5(n-3) 2.7"0.320:4(n-3) 0.6"0.022:4(n-6) 0.7"0.122:5(n-3) 1.8"0.122:2 NMI 2.0"0.0

Sum polyunsaturates 13.0"1.3Other 3.2Ratio AAyEPA 1.32

Presented as mean"S.D., ns2; AA, arachidonic acida

w20:4(n-6)x; EPA, eicosapentaenoic acidw20:5(n-3)x; Otherincludes components-0.5%: 12:0, 15:0, 16:1(n-9)c, 17:0,i17:0, 17:1, 18:1(n-5), 18:3(n-6), 18:4(n-3), 19:1, 20:2(n-6),20:3(n-6), 22:1, 22:3(n-6), 22:4(n-3), C PUFA.24

Table 5Percentage sterol composition ofHaliotis fulgens tissues

Sterol HepatoyGonad Foot Larvae

Dehydrocholesterol Tr 0.8 –Cholesterol 98.0 96.8 100.0Cholestanol 2.0 0.6 trDesmosterol Tr tr –24-Methylenecholesterol Tr 0.8 trFucosterol Tr 1.0 trRatio phytolysterol 0.02 – –

(–), below detection; tr, trace(below integration,-0.05%);Hepato, hepatopancreas; adult tissues from springE. menziesiitreatment.

Table 6Percentage 1-O-alkyl glyceryl ether composition ofHaliotisfulgens tissues

Ether side chain HepatoyGonad Larvae

14:0 tr Tr16:1 8.1 8.616:0 36.6 42.6i17:0 – tr17:1 tr tr17:0 tr tr18:2 – tr18:1(n-9) 35.7 36.818:0 19.6 7.520:1 tr 4.420:0 tr tr

(–), below detection; tr, trace(below integration,-0.05%);Hepato, hepatopancreas; adult tissues from springE. menziesiitreatment; Foot contained trace 16:0, 18:1 and 18:0; 18:1(n-9)Double bond position determined from comparison with pre-viously identified laboratory standard.

from 0.3% (June)–0.9% (November) (Hatae etal., 1995). In our study, HG for starved abalone(;40 mgØg ) and foot(;32 mgØg ) showed no–1 –1

change in lipid content from winter to spring. Thissuggests that mortality was not caused by lack ofenergy reserves, but was possibly correlated withabsence of specific dietary nutrients, such as essen-tial fatty acids; i.e. those FA only obtained fromthe diet(see below).

There were major differences in lipid classcomposition between HG and foot tissues ofH.fulgens. Although PL (au fond membrane phos-pholipid) were in high relative levels in all samples(Table 1), TAG also reached high levels in HG.Elevated FFA in HG samples(4–12%) reflects thehigh lipase activity found in digestive glands(Phillips et al., 2001). Abalone fedE. menziesii(27–56% TAG as % of total lipid; Table 1) andcomposite algal diets(19–32%) showed anincrease in TAG over the year, while abalone fedC. canaliculatus (4–0.5%) and U. lobata(53–0.4%) experienced a decrease. This result

cannot be directly correlated with the lipid contentof algae (Nelson et al., 2002). No TAG weredetected in foot tissue, with the exception of thestarvation treatment in spring where HG and footTAG values were equal(11%). This was likelycaused by tissue degeneration, as seen in decreasedfoot PL levels, and metabolic stresses of starvation.Degenerative effects of starvation have beenobserved in the abaloneH. kamtschatkana (Care-foot et al., 1993) and H. tuberculata (Gaty andWilson, 1986), as well as the fresh-water limpetAncylus fluviatilis (Streit, 1978) and the scallopHinnites multirigosus (Phleger et al., 1978). In theAntarctic limpet Nacella concinna, starvationreduced pedal mucus production(Simpson, 1982).In our study, starved abalone were easily detachedfrom aquaria. Decreased pedal mucus and quies-cence may have decreased their metabolic demand,with less TAG utilization.

706 M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

4.2. Sterols and phytol

Cholesterol was the major sterol(ST) (97–98%;Table 5), as is typical of abalone(Dunstan et al.,1996; Shimma and Taguchi, 1964) and most othermarine fishes, molluscs and crustaceans(Tucker,1989). Over 120 ST have been isolated in marineinvertebrates(e.g. Piretti et al., 1989; Napolitanoet al., 1993; Phleger et al., 1997). Different STpatterns have been shown for the same organismat different locations, and their patterns have beenuseful in establishing food web dynamics(Phlegeret al., 1997). ST function in the molluscan endo-crine system through biosynthesis and metabolismof steroid hormones: progesterone, estrogens, andandrogens(Ikekawa, 1985). There is evidence thatcholesterol, as well as stigmasterol and severalother steroids, function in regeneration of damagedshells in gastropods(Whitehead, 1977). Inclusionof ST, most importantly cholesterol, should be aconsideration when formulating aquaculture feed.The ratio of phytol to ST(0.02; Table 5) in HGis markedly lower than in theE. menziesii diet(2.6) (Nelson et al., 2002), which is likely due tocatabolism of phytol to 4,8,12-TMTD and providesevidence of transfer of specific molecules to thelarvae(as outlined below).

4.3. 1-O-Alkyl glyceryl ethers

Lower amounts of DAGE were present in HG(not detected by TLC-FID; Table 1), compared togonad(0.7% of total lipid) (Nelson et al., 1999).DAGE (and consequently GE) occur only in smallamounts and are rarely reported. The major 1-O-alkyl glyceryl ethers(GE) in HG were 16:0, 16:1,18:0 and 18:1(n-9) (Table 6). Results for foot aresimilar to previously analyzed samples(Nelson etal., 1999), as major GE were 16:0, 18:0 and 18:1.Interestingly, the GE profiles of HG, gonad(Nel-son et al., 1999) and larvae are quite similar. Forexample, the GE 18:1(n-9) (36% of total GE) inHG was present at the same proportions in gonad(36%) (Nelson et al., 1999) and larvae(37%).This provides further evidence for transfer ofmolecules from HG to gonad to larvae.

4.4. Fatty acids

Results for H. fulgens (Tables 2 and 3) aresimilar to those for the foot muscle ofH. laevigata

andH. rubra, where the main FA were 16:0, 18:0,18:1(n-7)c, 18:1(n-9)c, 20:4(n-6), 20:5(n-3) and22:5(n-3), independent of age(Dunstan et al.,1996). Other abalone studies are also in agreementwith our data(e.g. Shimma and Taguchi, 1964;Olley and Thrower, 1977; Uki et al., 1986b;Floreto et al., 1996; Hanna and Sinclair, 1996).Other species in the Order Archaeogastopoda showsimilar profiles(Joseph, 1982). A common resultof phaeophyte and rhodophyte algal diets is themoderate abundance of 14:0. Another major FA ingonad was 16:1(n-7)c. Oleic acidw18:1(n-9)cx andcis-vaccenic acidw18:1(n-7)cx, were also higher ingonad, and at relatively equal proportions, a com-mon indication of herbivory(Nelson et al., 2000).

DHA was low in all tissues. The higher abun-dance of DPA in abalone tissue, with none detectedin algae, further confirms the ability of abalone toretroconvert DHA to DPA, as noted by Dunstan etal. (1996). DPA in abalone is likely a retroconvertproduct of DHA, the major C PUFA of most22

marine animals(Dunstan et al., 1988). Althoughlevels of DPA in abalone have been associatedwith the occurrence of C PUFA in their diet22

(Shimma and Taguchi, 1964; Uki et al., 1986b;Viana et al., 1993; Dunstan et al., 1996), weobserved no significant correlation between levelsof C PUFA in macroalgae(0–2%) (Nelson et22

al., 2002) to levels in abalone tissues(Tables 2and 3). DPA and DHA do not appear to beessential FA inH. fulgens, as they were onlydetected inU. lobata, a chlorophyte that supportedsuboptimal growth of abalone.

22:2 NMI FA were present in abalone, whichagrees with Dunstan et al.(1996), but 20:2 NMIFA were not detected.H. discus andH. japonicus(Shimma and Taguchi, 1964) contained dienoicC and C FA, and NMI have been reported in20 22

other molluscs(Calzolari et al., 1971), whereaccumulation through diet has been suggested(Paradis and Ackman, 1975; Johns et al., 1980;Jeong et al., 1991). Low levels of 22:2 NMI FAwere previously detected in abalone(gonad, 0.9%;foot 1.5%) (Nelson et al., 1999), with moderatelevels present in this study in HG(2–6%; Table3), foot (3–6%; Table 2) and larvae(2%; Table4). The latter was likely transferred cytoplasmical-ly within the egg from HG tissues. No NMI FAwere detected in algae(Nelson et al., 2002), butthey were detected in starved abalone. Thus, NMIFA are either metabolic products of the abalone or

707M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

have come from another dietary source other thanmacroalgae fed in this study. Aquaculture larvaemay benefit from the inclusion of minor amountsof 22:2 NMI FA in the diet of abalone broodstock.

EPA and DHA are generally considered essentialFA for mariculture species(Nichols et al., 1994).The (n-3) and(n-6) PUFA were determined to beessential to abalone growth, and using diets with(n-3) at 1% of total lipid(Uki et al., 1986b) andwith 5% lipid content(% of dry mass), best growthwas induced inH. discus hannai (Uki et al.,1986a,b; Mai et al., 1995). A further increase intotal lipid in the diet may not be effective, asH.midae exhibited poor food conversion efficiencyand growth on a diet of 10% lipid(% of dry mass)in comparison to diets containing 2 and 6%(Britzand Hecht, 1997). Additionally, it has been sug-gested that only linoleic acidw18:2(n-6)x andlinolenic acidw18:3(n-3)x are essential FA in aba-lone (Hanna and Sinclair, 1996). However, it wasshown that EPA, as well as 18:2(n-6) and 18:3(n-3), from the diet, contributed to a high growth ratein H. discus hannai and H. tuberculata (Mai etal., 1996). In our study,U. lobata had the greatestamounts of 18:2(n-6) and 18:3(n-3), yet supportedpoor growth, suggesting they may not be essentialFA in H. fulgens. Also, compared to rhodophyteand chlorophyte algal diets, maximum growth injuvenile H. discus hannai was found on a diet ofthe phaeophyteUndaria pinnatifida, with growthcorrelated with high percentages of(n-3) PUFAw18:3(n-3), 18:4(n-3), EPAx and AA (Floreto etal., 1996). That study indicated that AA, and notEPA, is an essential FA. Furthermore, the starva-tion control showed no formation of 20:2(n-9) or20:3(n-9), which when detected are proposed toindicate a deficiency of(n-3) PUFA (Uki et al.,1986b; Dunstan et al., 1996).

4.5. C PUFA and body mass20

The C PUFA, AA and EPA, occurred in similar20

abundance in abalone foot(1–3 mgØg ) and algae–1

(0.3–2.7 mgØg ) (Nelson et al., 2002). In HG,–1

however, amounts of C PUFA were higher(3–920

mgØg ; Fig. 3b), and the variation over the year–1

coincides with the variation in their macroalgaldiet (Fig. 3c). This temporal variation appears tohave a strong relationship with the mean growthrate of mass(Fig. 3a). Although EPA was alsopresent in similar abundances in gonad, the pro-

portion of AA was markedly lower, thus providingrelatively lower AAyEPA ratios in HG than infoot. The ratio decreased throughout the year(2.4to 1.0). This may indicate that AA has beenconverted to prostaglandins(PG), and is functionalin gametogenesis, and that EPA, which is also atype of PG precursor, does not perform the samefunction. PG are eicosanoids, derived from C20

PUFA, and are hormone-like molecules that func-tion in the fundamental physiology in representa-tives of many invertebrate phyla(Stanley-Samuelson, 1987; Gerwick, 1994). PGhave been determined to influence reproduction inseveral molluscs, including the abaloneH. rufes-cens, the musselMytilus californianus (Morse etal., 1977) and the scallopPatinopecten yessoensis(Osada et al., 1989). AA was determined to bepreferentially incorporated in the gonads offemales of the scallopP. maximus and plays animportant role in gametogenesis(Soudant et al.,1996).

In all abalone tissues, the AAyEPA ratios werehigh, with no apparent seasonal change, with theexception of HG in animals fedE. menziesii.However, the AAyEPA ratios forC. canaliculatuswere low(0.4–1.0) (Nelson et al., 2002) comparedto high ratios in corresponding abalone tissues(1.5–3.9; Tables 2 and 3). The poor growth inabalone fedC. canaliculatus may be linked toexcess EPA, which was thought to inhibit PGproduction in the turbotScophthalmus maximus(Bell et al., 1994). We propose that abalone requirea high AAyEPA ratio and that high levels of EPA,in contrast, may inhibit growth. AA and EPA werelow in U. lobata concurrent with low animalgrowth, E. menziesii had high AAyEPA and high-est growth, whileC. canaliculatus had a low ratio,low lipid content and low growth. The compositealgal diet also produced low growth. Notwithstand-ing, the combined effects of AA and EPA appearedto have the greatest influence. The low lipidcontent inC. canaliculatus may have been insuf-ficient for the abalone, or if abalone are able touse lipid efficiently then there may have been aphysiological perturbation by the high ratio ofEPA. We observed slightly lower EPA in a prelim-inary analysis of abalone fedE. menziesii(2.6–3.2%) (Nelson, 1999), a year when gameto-genesis was successful, compared to those fedE.menziesii in this study(3.1–5.5% EPA; Tables 2

708 M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

and 3). Abalone may be extremely sensitive tolevels of specific C PUFA.20

4.6. Temperature and shell growth

The mean growth rate of shell length appears tohave a strong relationship with water temperature(Fig. 2a,b). There is a positive correlation betweentemperature and the degree of saturation in FA ofcell membranes(Phleger, 1991). Temperature maydirectly affect the FA composition of abalone.Additionally, temperature may be an indirect influ-ence, by affecting FA in their macroalgal diet(Nelson et al., 2002). Certain fish species, whichare thought to consume macroalgae, have highproportions of AA and EPA(Dunstan et al., 1988).Therefore, although temperature changes directlyimpact growth via the Q effect(metabolic change10

due to temperature), it may also dictate essentialmolecular requirements, such as PUFA. Tempera-ture may affect saturation levels in FA in themacroalgal diet and in abalone, and the amountsof C PUFA may indeed affect shell length, with20

the effects of temperature dominant. Variation(i.e.S.E.) in morphometric results is not uncommon,as large intraspecific variation in growth has beenobserved in wild abalone from the same area(Hirose, 1974). The reasons for these variationsare likely several, including food availability andcompetition. In our study, abalone were fed adlibitum so that all individuals received food. Thispoints to the impact of behavioral positive thig-motropism, the clustering of individuals typical ofabalone.

4.7. Feeding preferences

We achieved maximum growth of abalone on aphaeophyte diet. This finding may be appliedtoward formulation of aquaculture diets based uponphaeophyte lipid profiles, but also has implicationsto wild abalone. Food availability determines thenatural diet of abalone, but there are also mechan-ical (Leighton, 1966, 1968; McShane et al., 1994)and chemical constituent influences of algae(Win-ter and Estes, 1992; Fleming et al., 1996) thatdetermine their acceptability and ultimately theirvalue in nutrition. Phaeophytes comprised the mainfood of Californian abalone(Leighton and Boo-lootian, 1963; Leighton, 1966), and other studiesalso show abalone typically have preference to andbest growth with phaeophytes(Leighton and Boo-

lootian, 1963; Hirose, 1974; Uki et al., 1986a; Maiet al., 1995). Although Australasian abalone havebeen found to prefer rhodophytes(Shepherd, 1973;Tahil and Junio-Menez, 1999), this may be due tohighly elevated polyphenols in many south Pacificphaeophytes(Steinberg, 1989; Stepto and Cook,1996). In fact, one study found a preference torhodophytes, but improved food conversion effi-ciency was observed on a phaeophyte diet(Mc-Shane et al., 1994). In food selection experimentsit was found the preferred phaeophyte foods ofadultH. fulgens, in order of decreasing preference,were: Egregia laevigata, Macrocystis pyrifera,Eisenia arborea, and Laminaria farlowii (Leigh-ton, 1966). Analyses of these algae revealed thatEgregia menziesii, the most preferred alga, has oneof the highest AAyEPA ratios, and was also highin other PUFA, including 18:3(n-3) (Nelson,1999). Feeding rates of abalone fedE. menziesii(2–9%Øday ) were higher than with other diet–1

treatments(composite, 2–5%Øday ; C. canalicu-–1

latus, 1–4%Øday ; U. lobata, 1–4%Øday ) (Nel-–1 –1

son, 1999), as were gains in shell length(E.menziesii, 16.4 mm; composite, 6.1 mm;C. can-aliculatus, 1.1 mm;U. lobata, 0.8 mm), and bodymass(E. menziesii, 21.3 g; composite, 7.4 g;C.canaliculatus, y0.4; U. lobata, y3.3;). Similarly,growth rates for abalone fedE. menziesii weresuperior (Fig. 3a,b). Abalone fed E. menziesiiwere the only animals to maintain a constant massylength relationship. Not only did this macroalgaesupport maximal growth, it has also providedexcellent results in commercial aquaculture andproduction ofH. fulgens (Leighton and Peterson,1998).

4.8. Larvae and evidence of cytoplasmic transfer

In larvae ofH. fulgens, the major lipid classeswere TAG (71% of total lipid) and PL (22%;Table 1), which closely reflects the compositionof gonad material of this species(Nelson et al.,1999). The larval and gonadal profiles showedlow levels of WE (0.9% and 4.3%, respectively)and DAGE(0.7%). The lower WE in larvae mayresult from the cellular demands of gametogenesis.The lipid content of larvae(24 mgØg dry mass)–1

was much lower than in combined HG(37–131mgØg ). These energy storage molecules at least–1

partially meet the requirements of abalone larvalmetamorphosis, where dynamic metabolic requi-

709M.M. Nelson et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 695–712

rements have been documented(Jaeckle and Man-ahan, 1989; Shilling et al., 1996).

FA, ST and GE profiles for larvae(Tables 4–6)were similar to gonad(Nelson, 1999; Nelson etal., 1999). The isoprenoid fatty acid 4,8,12-TMTDwas detected at low levels(0.5%; Table 4). Thisacid results from the metabolism of the phytolside-chain of dietary chlorophyll. It was suggestedto be the only detectable product in efficientmetabolic degradation of chlorophyll and inter-mediates, including phytol(trans-3,7,11,15-tetra-methylhexadec-2-en-1-ol), pristanic acid(2,6,10,14-tetramethylpentadecanoic), and phytan-ic acid (3,7,11,15-tetramethylhexadecanoic) (Ack-man et al., 1971). Although not reportedpreviously in abalone, 4,8,12-TMTD has beendetected in other molluscs(Ackman et al., 1971;Johns et al., 1980) and in the digestive gland ofthe scallopP. magellanicus (3.1%), where it wassuggested that its presence may indicate the con-tributions of a microbial population which extra-cellularly metabolizes phytol(Napolitano andAckman, 1993). This may very well hold true forabalone, which have resident bacteria that likelyassist in digestion of complex polysaccharide(Erasmus et al., 1997). Because 4,8,12-TMTD wasnot detected in the female gonad ofP. magellani-cus, the inference was that it is not transferredalong with other lipids to the developing gonad(Napolitano and Ackman, 1993). However, inH.fulgens, phytol (Table 5) and 4,8,12-TMTD(1.2–13.9%; Table 3) were detected in HG, withanimals fed U. lobata containing the highestamounts. It is likely that 4,8,12-TMTD was trans-ferred from hepatopancreas to gonad, just as trans-ference of lipids from the pyloric caeca to gonadhas been suggested to occur in the seastarsAsteriasrubens (Oudejans and van der Sluis, 1979), A.amurensis, and Solaster paxillatus (Hayashi andKishimura, 1997). 4,8,12-TMTD was also presentin abalone gonad(0.5%) (Nelson et al., 1999) andlarvae(0.5%; Table 4), providing strong evidencefor a cytoplasmic transfer from hepatopancreas togonad and via the eggs, to larvae.

4.9. Conclusions

We examined seasonal changes in lipid compo-nents of the abalone,H. fulgens, with implicationsto reproductive physiology and lipid trophodyn-amics. The abalone tissues do, in some respects,directly reflect their macroalgal diets, as seen in

the foot; there is also accumulation and metabolismof lipids, which are more complex to interpret.The hepatopancreas serves for storage of lipid,although it appears lipids are not utilized primarilyas an energy source, rather as essential moleculesfor growth and gonad maturation. Evidence wasprovided that there is selective storage of FA inabalone tissues, and that lipids do pass from thehepatopancreas to the gonad to the larvae. Weconcluded that the essential PUFA are indeedderived from the macroalgae. This result, coupledwith the observation that the temporal variation inPUFA, those lipids most likely important to cycli-cal gonadal development(Nelson et al., 1999) andgrowth, could be an important factor in wild andaquaculture larval production and subsequentrecruitment. Inclusion of C PUFA into artificial20

feeds should be considered for all aquaculturespecies, and potential renewable sources of long-chain PUFA include microalgae(Nichols et al.,1994), bacteria(Nichols et al., 1997) and thraus-tochytrids(Lewis et al., 1999). The results of ourstudy contribute to better understanding of abalonenutritional physiology, the occurrence of lipids inthe gonad, and the role of lipids in transference tolarvae, and are applicable to other species ofHaliotis and related aquaculture species of similarreproductive behavior and economic value.

Acknowledgments

We are extremely grateful to R. McConnaughey,V. Vacquier, S. Anderson and B. Mooney for theirexpertise and invaluable assistance. G. Dunstanprovided suggestions to improve the manuscript.D. Holdsworth managed the CSIRO GC-MS. Theresearch was supported in part by a CSIRO MarineProducts Research and Travel Assistance Awardand a Strauss Foundation Award. Support was alsoprovided by a Research, Scholarship and CreativityAward (RSCA) from San Diego State University,and Fisheries Research and Development Corpo-ration (FRDC) in Australia. Comments by twoanonymous referees were extremely valuable dur-ing manuscript preparation.

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