Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298

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Comparative Biochemistry and Physiology, Part B

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Lipid, fatty acid and protein content of late larval to early juvenile stages of the
western rock lobster, Panulirus cygnus
Andrew J. Limbourn a,b,⁎, Peter D. Nichols c
a
School of Animal Biology, University of Western Australia, Crawley, Perth WA 6009, Australia
b
CSIRO Marine and Atmospheric Research, Wealth from Oceans Flagship, Private Bag No. 5, Wembley, Perth WA 6913, Australia
c
CSIRO Marine and Atmospheric Research, Food Futures Flagship, G.P.O. Box 1538, Hobart, Tasmania 7001, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Lipid, fatty acid and protein content were determined individually on 7 phyllosomata, 69 clear pueruli, 286
Received 16 October 2008 pre-moult pueruli, and 86 juvenile western rock lobster (WRL) collected from four locations between the
Received in revised form 10 December 2008 settlement seasons 2000 to 2006 to evaluate compositional changes during the non-feeding puerulus stage.
Accepted 10 December 2008
Only the lipid content, particularly the phospholipids, decreased significantly with development. Protein
Available online 24 December 2008
declined sharply following moult to the juvenile. PL comprised between 86–94% of total lipid in all animals,
Keywords:
and declined most between phyllosomata and clear pueruli (238.5 to 121.4 mg g− 1 DW) (p b 0.001).
Lipid Triacylglycerols were the only lipid to increase in absolute amounts with development, but declined 53% on
Monounsaturated fatty acid average following moult to juvenile. This increase in TAG is likely due to the conversion of phospholipids to
Nutrition triacylglycerols. Monounsaturated fatty acids were the main energy form utilised during benthic
Polyunsaturated fatty acid development while polyunsaturated fatty acids showed a high degree of sparing. The n-3:n-6 fatty acid
Protein ratio of juveniles indicates that they may be approaching critically low levels of stored lipid energy reserves.
Puerulus Both protein, and lipid, declined sharply from the final puerulus phase to the juvenile confirming that a high
Rock lobster
energetic demand is required to fuel the moulting process.
© 2009 Elsevier Inc. All rights reserved.

1. Introduction reserves accumulated during the late phyllosomata stages (McWilliam

Spiny lobsters (Decapoda; Palinuridae) have a long and complex
life cycle of three phases commencing with an extensive planktonic
larval phase or phyllosoma, a short nektonic puerulus phase, and
finally a long juvenile to adult benthic phase. The larval phase consists
of nine anamorphic phyllosomata stages for the western rock lobster,
Panulirus cygnus George, which lasts for approximately 9 to 11 months
(Booth and Phillips, 1994; McWilliam and Phillips, 2007). The final
stage phyllosoma metamorphoses into a nektonic puerulus which
swims across the continental shelf to settle on reefs and seagrass beds
of the nearshore (Jernakoff, 1990). After settling, the puerulus passes
through three morpho-phases, clear, P1 and P2, distinguished by the
colouration of their carapace (e.g. Lewis et al., 1952) and hepatopan-
creas. Two to three weeks after settling the P2 puerulus moults into a
benthic first instar juvenile (also known as a post-puerulus). Juveniles
develop over 3–4 years into sexually mature adults that dominate the
macro-invertebrate communities of coastal reefs in nearshore waters
of south-western Western Australia (Lipcius and Eggleston, 2000).
The puerulus is a non-feeding stage in the palinurid life-cycle
(Kittaka, 1990; Booth and Phillips, 1994; Lemmens, 1994; Lemmens and
Knott, 1994; Lemmens, 1995). Pueruli rely exclusively on stored energy
⁎ Corresponding author. Tel.: +61 8 92045974; fax: +61 8 93336555.
E-mail address: andrew.limbourn@csiro.au (A.J. Limbourn).
and Phillips, 1997; Butler and Herrnkind, 2000) to power their move-
ment across the continental shelf to nearshore settlement sites. After
settling, the puerulus must undergo a moult before feeding resumes, a
change that may compromise those settlers with low energy stores.
Lipids, particularly, are important sources of energy used during cross-
shelf migration in P. cygnus and other rock lobster species (Jeffs et al.,
1999; Phillips et al., 2006). However, it is not clear what energy reserves
are utilised once pueruli settle and before moulting to the first instar
juvenile when feeding resumes.
The nutritional status of the puerulus (immediately following
metamorphosis from the phyllosoma) depends on the nutritional
status of the final stage phyllosoma, which presumably reflects food
availability in the plankton. Oceanographic conditions, for example
counter-clockwise upwelling eddies, generate localised high levels of
phytoplankton (Waite et al., 2007). Whatever the initial energy status
of a phyllosoma or early puerulus might be, presumably the trends in
content of the major biochemical categories will be consistent. One
major factor which generates difficulty for elucidating understanding
of the biochemical status of phyllosomata and pueruli is the paucity of
material available for study: live final-stage phyllosomata and early
pueruli are extremely difficult to capture (Phillips, 1981; Booth, 1994).
The most reliable source of specimens in number is from puerulus
collectors, as used by WA Fisheries and Marine Research Laboratories
(WAFMRL) to quantify settlement (Morgan et al., 1982; Caputi and

1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2008.12.009
 A.J. Limbourn, P.D. Nichols / Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298
Brown, 1986; Phillips 1986; Caputi et al., 1988; Phillips et al., 1991).
Material used in this study represents pueruli just prior to moulting
into the first instar juvenile. Little material is available from any one
site in each month per year and this study perforce is developed from
assessing the nutritional content from the few phyllosomata collected
from beyond the edge of the continental shelf. This establishes a semi-
baseline condition. Consequently, the pueruli were collected from four
sites over six consecutive years at the end of their swim.
The nutritional condition of P. cygnus pueruli that reach the
settling zone is yet to be determined. Hence, our main objective in the
present study was to measure changes in the protein and lipid
composition of P. cygnus pueruli at first settlement (clear) and from
first settlement pueruli (clear, P1 and P2) to the first instar juveniles,
immediately post moult from P2. We focused on changes in the
content and composition of lipids and fatty acids to elucidate changes
in preferential utilisation of different lipid classes and individual fatty
acids, from final stage phyllosomata to first instar juveniles, because
lipids have been shown to be the major energy storage product in this
and other rock lobster species (Phleger et al., 2001; Jeffs et al., 2002;
Phillips et al., 2006). We also measured protein content because
although protein does not constitute the major source of energy in
many crustaceans, including spiny lobster, they are catabolised as a
secondary resource once lipid reserves are depleted in non-feeding
stages (Lucas et al., 1979). Carbohydrate (CHO) sources of energy,
glucose and glycogen, were not measured in this study as no
significant difference in CHO content have been shown in migrating
Jasus edwardsii Hutton pueruli (Jeffs et al., 1999), or during starvation
in P. cygnus post-pueruli juveniles (Limbourn et al., 2008). The data
used in this study provide us with information about the ranges in
nutritional components measured during development and not issues
reflecting spatial and or temporal variation.
2. Methods
2.1. Puerulus development
Staging of post-settlement pueruli represents a distinction based
on pigment levels of the carapace (e.g. Lewis et al., 1952), with three
morpho-phases distinguished: clear, the first post-settlement puer-
ulus, which is transparent with no visible pigmentation and the
hepatopancreas may be visible; P1, pigmentation has commenced
around the cephalothoracic region and appendages, hepatopancreas is
opaque, clear areas remain on the exoskeleton; P2, pigmentation is
complete, exoskeleton is opaque, body remains dorso-ventrally
flattened. Moult to the first instar juvenile occurs within a few days
following complete pigmentation. We use “stage” to refer to the
period between each moult, “phase” to changes that are taking place
within each stage.
2.2. Collection of phyllosomata, pueruli and first instar juveniles
Phyllosomata of Panulirus cygnus (George) were collected opportu-
nistically in the south-eastern Indian Ocean off the west coast of
Australia between October 2004 and December 2005. A Neuston
(1 m×1 m) surface trawl plankton net was towed at approximately
1 m s− 1, or 2 nautical miles h− 1, and towed for approximately 10 min at a
depth of between 1 to 5 m below the surface by the RV Southern
Surveyor.
Pueruli and first instar juveniles in this study were collected using
artificial seaweed, Phillips type collectors. All pueruli, and first instar
juveniles, analysed for lipid, fatty acid and protein content were
collected from four locations: the Houtman Abrolhos Islands (28°41′S,
113°50′E); Seven Mile Beach (29°10′S, 114°53′E); Jurien Bay (30°19′S,
115°00′E); and Alkimos (31°36′S, 115°38′E).
Between four and six collectors were set at the surface in inshore
waters at each of the four locations and anchored in approximately 5
293
to10 m water depth. Pueruli were collected once a month by WAFMRL
research staff around the new moon phase during the annual
settlement season (July to March) between the years 2000 to 2006.
Puerulus removal from the collectors followed the standard protocol.
The three PVC sheets with attached artificial seaweed fibres were
removed from the aluminium collector frame and hit against the side
of a plastic bin 20 times to dislodge all pueruli. All pueruli were either
frozen immediately in liquid nitrogen, frozen in a standard −15 °C
freezer, or kept alive and transported to land in a well-aerated and
insulated container before being placed into liquid nitrogen. Samples
were stored long-term at −80 °C prior to analysis.
Dry weights (DW) were determined for whole individual animals
following overnight freeze drying. Wet weights (WW) were deter-
mined for whole individual animals following thawing on ice and after
blotting dry. In 21 post-settlement pueruli collected from Jurien Bay,
DW averaged 23% of WW with a range from 19 to 25%.
2.3. Protein assays
Total protein in individual, whole bodied animals was quantified
using the Bicinchoninic Acid Assay Kit (BCA) (Sigma-Aldrich, USA).
This assay is based on the animal’s wet weight (WW) to avoid the
breakdown of protein during the drying process. Assays were
performed in duplicate with absorbance read using an ELx808 Ultra
Microplate Reader (Bio-Tek, USA). 25 µL of each protein sample was
added to individual wells in a 96 well plate assembly prior to the
addition of 2 mL of the BCA working reagent. The well plate was placed
in the reader, gently vortexed for thorough mixing, then incubated at
37 °C for 30 min. Following incubation the plate was removed from the
reader and allowed to cool for 10 min before being returned to the
reader with absorbance read at 562 nm. The absorbance of each
unknown protein sample was compared against a standard curve
prepared using BSA protein standards to determine the total protein
concentration.
2.4. Lipid and fatty acid analysis
The methods for lipid and fatty acid analyses closely followed
Limbourn et al. (2008). Total lipid content was determined gravime-
trically following an overnight methanol-chloroform-Milli-Q water
extraction. An aliquot of the total lipid extract was then analysed with
an Iatroscan MK V TH10 thin-layer chromatography-flame-ionisation
detector (TLC-FID) analyser (Iatron Laboratories, Tokyo, Japan) to
determine the abundance of individual lipid classes (Nichols et al.,
1994). An aliquot of the total lipid was trans-methylated to produce fatty
acid methyl esters (FAME) using methanol-chloroform-concentrated
HCl solution (3 ml; 10:1:1 v/v/v; 100 °C; 1.5 h). Gas chromatographic
(GC) analyses of FAME were performed using an Agilent Technologies
6890N GC (Palo Alto, CA, USA) equipped with a HP-5 cross-linked methyl
silicone fused capillary column (50 m × 0.32 mm i.d., 0.17 µm film
thickness) and a flame ionisation detector (FID) (Jeffs et al., 2002). GC-
mass spectrometric (GC-MS) analyses were performed on a Finnigan
Thermoquest GCQ GC-MS fitted with an on-column injector (Austin, TX,
USA). Thermoquest Xcalibur software was used for confirmation of peak
identification. The GC was fitted with a capillary column similar to that
described above.
2.5. Statistical analysis
One-way ANOVA was used to determine if total protein (TP) in
pueruli settled in the year 2000 varied between the developmental
stages, from clear, P1, P2 and first instar juvenile. One-way ANOVA was
also used to determine if total lipid (TL) varied with developmental
stage. Where there was significant variation, a Tukey's post hoc
comparison was used to test variability (pairwise) between stages. All
ANOVA were run using SPSS v11 statistical software.
294 A.J. Limbourn, P.D. Nichols / Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298
3. Results
3.1. Protein
There was a 24% increase in total protein (TP) as a proportion of
DW (mg TP g- 1 DW) through the clear to P2 pueruli, then a sharp
decline following moult to the first juvenile instar. First instar
juveniles had on average 33% less TP than P2 pueruli (Fig. 1). Although
TP content was similar between successive clear and P1, and between
P1 and P2 pueruli, changes were highly significant (F3, 46 = 13.576,
p b 0.001), particularly between clear and P2 pueruli (p = 0.005), and
between P2 pueruli and first instar juveniles (p b 0.001).
The mean WW± 1 SE of post-settlement pueruli varied little between
the three morpho-phases (Clear n = 69, 231.8± 6.3, P1 n = 171, 233.2 ± 4.1,
P2 n = 115, 234.8 ± 3.8 mg), and then increased significantly
(F3, 437 = 60.641, p b 0.001) following moult from P2 pueruli to the first
instar juveniles (1st instar juveniles n = 86, 303.3 ± 9.8 mg). On average,
there was a 28% increase in WW between P2 pueruli and first instar
juveniles.
3.2. Lipid
Total lipid (TL) content (Fig. 2) decreased significantly by 78% on
average from final stage phyllosoma to the first instar juvenile
(F4, 444 =104.721, pb 0.001). The greatest decrease in TL content (of 49%)
occurred between final stage phyllosomata (238.5 ±11.1 mg g− 1 DW) and
recently settled clear pueruli (121.4 ± 4.3 mg g− 1 DW) (p b 0.001). TL
content decreased significantly between the three morpho-phases:
clear — 121.4, P1 — 108.3 and P2 — 96.8 mg g− 1 DW) (clear — P1 p =0.012,
clear — P2 p b 0.001 and P1 — P2 p =0.009) and between the P2 pueruli
and first instar juveniles (p b 0.001). On average, first instar juveniles had
45% less lipid (53.6 ± 2.7 mg g− 1 DW) than P2 pueruli (96.8 ± 2.9 mg g− 1
DW).
Of the lipid classes, phospholipids (PL) were the dominant lipid
class present for each stage and phase of development: phyllosoma
~ 94%; clear pueruli ~91%; P1 ~ 89%; P2 ~ 87%; first instar juvenile ~86%
of the total lipid content. As development progressed through the post-
settlement puerulus phase the content of PL (mg g− 1 DW) decreased
significantly (F4, 444 = 108.004, p b 0.001). The content of the minor lipid
classes is shown in Fig. 3. Sterols (ST), the second most abundant lipid
class, comprised between 2–8% of the total lipid content. The majority
of the difference in ST content occurred between final stage
phyllosomata and newly settled clear pueruli, with a N100% increase.
Once settled, ST remained relatively constant with no significant
difference observed in the relative levels. Triacylglycerols (TAG), the
next major lipid class present, increased markedly from the initial clear
pueruli (1.6 mg g− 1 DW, b2% TL) to the P2 pueruli (3.9 mg g− 1 DW, 5%
Fig. 1. Panulirus cygnus. Mean proportion of total body dry weight (DW) represented by
total protein (TP) during post-settlement (mean ± 1 SE). Significant differences in total
protein during development are denoted by letters, where groups sharing the same
letter are not significantly different from one another.
Fig. 2. Panulirus cygnus. Total lipid (TL) content (mean proportion of dry body weight
(DW) ( ± 1 SE)) (bars), and the mean whole body dry weight (DW) (± 1 SE) (diamonds), of
final stage (ix) phyllosomata, newly settled clear pueruli, P1 and P2 pueruli, and
following moult to the first instar juvenile. Significant differences in TL during
development are denoted by letters, where groups sharing the same letter are not
significantly different from one another.
TL). Following moult to the first instar juvenile, TAG proportions
dropped to b3% TL. Settled pueruli also had a higher proportion of TAG
and less free fatty acids (FFA) than final stage phyllosomata. Low levels
of FFA (≤2% on average) and trace amounts (≤1% on average) of wax
esters (WE) were present in all stages and phases.
The DW of recently settled clear pueruli (51.3 ± 1.0 mg) increased
by 32% on average relative to phyllosomata DW (34.7 ± 3.2 mg)
(F1, 74 = 26.237, p b 0.001). DW increased by 24% on average between
P2 pueruli (51.6 ± 1.0 mg) and first instar juveniles (67.9 ± 1.7 mg)
(F1, 199 = 64.581, p b 0.001). During the three puerulus morpho-phases,
DW remained relatively constant (Fig. 2).
3.3. Fatty acids
Forty nine fatty acids (FA) were identified in the individual analyses
of seven final stage phyllosomata, 49 clear, 146 P1, and 101 P2 pueruli
and 21 first instar juveniles of P. cygnus. The abundance of all FA
decreased proportionally with development from 98.9 ± 9.5 mg g− 1
DW in final stage phyllosomata to 14.4 ± 1.2 mg g− 1 DW on average in
first instar juveniles (Table 1). Sixteen FA at ≥1.0% of total FA were
identified in final stage phyllosomata, recently settled pueruli and first
instar juveniles. Of these 16 FA, of which six (16:1n-7, 16:0, 18:1n-9,
18:0, 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahex-
aenoic acid, DHA) accounted for between 73–80% of the total FA. The
Fig. 3. Panulirus cygnus. Percentage lipid class composition from final stage (ix)
phyllosomata, newly settled clear pueruli, P1 and P2 pueruli, and following moult to the
first instar juvenile (mean ± 1 SE). Only the minor lipid classes are shown with
phospholipid not represented due to its dominance (from 86 to 94% of the total lipid)
during all stages of development.
 A.J. Limbourn, P.D. Nichols / Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298 295

Table 1
Panulirus cygnus

Fatty Acids Content (mg g− 1 DW) Percent composition

Phyllosoma Clear P1 P2 Juvenile Phyllosoma Clear P1 P2 Juvenile
14:0 0.9 ± 0.3 0.5 ± 0.1 0.3 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.8 ± 0.2 0.9 ± 0.1 0.7 ± 0.0 0.6 ± 0.0 0.1 ± 0.0
16:1n-7 6.9 ± 1.8 4.0 ± 0.3 2.9 ± 0.2 2.2 ± 0.1 0.4 ± 0.1 9.0 ± 0.3 6.9 ± 0.2 6.3 ± 0.1 5.7 ± 0.2 2.7 ± 0.4
16:1 / 16:2 1.1 ± 0.3 0.5 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 1.3 ± 0.0 1.0 ± 0.0 0.8 ± 0.0 0.7 ± 0.0 0.2 ± 0.1
16:0 16.1 ± 4.2 9.8 ± 0.7 7.4 ± 0.3 5.8 ± 0.3 2.1 ± 0.2 20.9 ± 0.5 17.4 ± 0.3 16.9 ± 0.2 16.4 ± 0.2 14.0 ± 0.4
17:0 0.8 ± 0.2 0.6 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 1.0 ± 0.0 1.1 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.1 ± 0.0
18:2n-6 0.8 ± 0.2 0.7 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 1.1 ± 0.1 1.2 ± 0.0 1.1 ± 0.0 1.1 ± 0.0 1.2 ± 0.1
18:1n-9 12.7 ± 3.3 8.8 ± 0.6 7.0 ± 0.3 5.8 ± 0.3 2.0 ± 0.2 15.6 ± 0.4 15.9 ± 0.2 16.0 ± 0.1 15.7 ± 0.2 13.4 ± 0.4
18:1n-7 2.8 ± 0.7 1.9 ± 0.1 1.6 ± 0.1 1.4 ± 0.1 0.6 ± 0.1 3.3 ± 0.1 3.5 ± 0.0 3.7 ± 0.0 3.9 ± 0.0 4.1 ± 0.1
18:0 8.4 ± 2.2 7.1 ± 0.4 5.4 ± 0.2 4.5 ± 0.2 1.7 ± 0.1 10.3 ± 0.2 13.1 ± 0.1 12.9 ± 0.1 13.0 ± 0.2 12.2 ± 0.2
20:4n-6, ARA 1.2 ± 0.3 1.3 ± 0.1 1.1 ± 0.0 1.0 ± 0.0 0.9 ± 0.1 1.5 ± 0.0 2.5 ± 0.1 2.7 ± 0.1 3.1 ± 0.1 6.3 ± 0.5
20:5n-3, EPA 6.9 ± 1.8 5.4 ± 0.2 4.7 ± 0.2 4.1 ± 0.2 2.5 ± 0.2 8.8 ± 0.3 10.3 ± 0.3 11.3 ± 0.2 11.9 ± 0.3 17.4 ± 0.6
20:1n-9 1.3 ± 0.5 1.1 ± 0.1 0.9 ± 0.0 0.7 ± 0.0 0.3 ± 0.0 1.6 ± 0.3 2.0 ± 0.1 2.1 ± 0.0 2.0 ± 0.1 2.2 ± 0.1
20:0 0.5 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.7 ± 0.0 1.1 ± 0.0 1.1 ± 0.0 1.2 ± 0.0 1.4 ± 0.0
22:5n-6 0.5 ± 0.1 0.4 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 0.7 ± 0.0 0.8 ± 0.0 0.8 ± 0.0 0.9 ± 0.0 0.9 ± 0.0
22:6n-3, DHA 12.0 ± 3.2 6.2 ± 0.3 5.3 ± 0.2 4.3 ± 0.2 1.9 ± 0.1 15.1 ± 0.3 11.6 ± 0.2 12.1 ± 0.1 12.0 ± 0.2 13.8 ± 0.4
22:0 0.3 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 0.9 ± 0.0 1.1 ± 0.0 1.3 ± 0.0 1.9 ± 0.1
Other 7.8 ± 1.2 5.1 ± 0.3 3.9 ± 0.2 3.2 ± 0.2 0.9 ± 0.1 0.3 ± 0.1 1.3 ± 0.1 1.2 ± 0.0 1.1 ± 0.1 0.3 ± 0.1
EPA:ARA 5.9 4.3 4.3 4.0 3.1
DHA:EPA 1.7 1.1 1.1 1.0 0.8
ARA:DHA 0.1 0.2 0.2 0.3 0.5
DHA:ARA 10.1 4.9 4.8 4.2 2.6
SUM SFA 33.6 ± 3.0 19.0 ± 1.2 14.5 ± 0.6 11.7 ± 0.6 4.5 ± 0.4 34.1 ± 0.4 34.6 ± 0.3 33.8 ± 0.2 33.5 ± 0.4 30.6 ± 0.5
SUM PUFA 26.7 ± 2.4 14.0 ± 0.7 11.9 ± 0.4 10.3 ± 0.5 5.6 ± 0.4 27.2 ± 0.6 26.4 ± 0.6 28.0 ± 0.4 29.0 ± 0.6 39.6 ± 1.0
SUM MUFA 30.6 ± 3.0 16.3 ± 1.1 12.7 ± 0.6 10.3 ± 0.6 3.4 ± 0.3 30.9 ± 0.3 29.2 ± 0.4 29.0 ± 0.2 28.0 ± 0.4 22.6 ± 0.7
SUM HUFA 25.0 ± 2.3 12.9 ± 0.6 11.1 ± 0.4 9.5 ± 0.4 5.3 ± 0.4 25.4 ± 0.6 24.4 ± 0.5 26.1 ± 0.4 27.0 ± 0.6 37.5 ± 1.0
SUM n-3 23.5 ± 2.2 11.7 ± 0.5 10.0 ± 0.4 8.5 ± 0.4 4.4 ± 0.3 23.9 ± 0.5 21.9 ± 0.5 23.4 ± 0.3 23.9 ± 0.5 31.2 ± 0.7
SUM n-6 3.2 ± 0.3 2.4 ± 0.1 1.9 ± 0.1 1.8 ± 0.1 1.2 ± 0.1 3.3 ± 0.1 4.5 ± 0.1 4.6 ± 0.1 5.1 ± 0.2 8.4 ± 0.6
n-3:n-6 7.3 4.9 5.1 4.8 4.1
Total fatty acids 98.9 ± 9.5 53.9 ± 3.2 42.6 ± 1.9 35.4 ± 2.0 14.4 ± 1.2

Fatty acid content (mg g− 1 DW) and composition (% of total fatty acids) of final stage (ix) phyllosoma, newly settled clear pueruli, P1 and P2 pueruli and recently moulted first instar
juveniles (mean ± 1 SE).
Data presented as mean ± SE. Only the major FA present at ≥1% at some stage are included. ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosapentaenoic acid; PUFA,
polyunsaturated fatty acids; MUFA, mono-unsaturated fatty acids; HUFA, highly unsaturated fatty acids. Other includes FA present at b 1%: i14:0, i15:0, 15:00, i16:0, 16:1n-9c, 16:1n-
5c, 7Me17:1, a17:0, 17:1n-8, 17:1n-6, i18:0 (+18:4n-3), a18:0, 18:1n-7t, 18:1n-5c, 19:1, 20:3n-6, 20:4n-3, 20:2NMI, 20:2n-6, 20:1n-7c, 21:5n-3, 21:0, 22:4n-6, 22:5n-3 (DPA), 22:3n-6,
22:2NMI, 22:2n-6, 22:1n-9c, 22:1n-7c/22:3n-3, 24:0.

greatest difference in content of each of the six FA was between puerulus morpho-phases (Fig. 4). In general, the monounsaturated
phyllosomata and recently settled clear pueruli and between the P2 FA (MUFA) (16:1n-7, 18:1n-9 & 18:1n-7), saturated FA (SFA) (16:0 &
pueruli and first instar juveniles, with less variation between the three 18:0) and polyunsaturated FA (PUFA) (EPA & DHA) were considerably

Fig. 4. Panulirus cygnus. Flow chart of energy use during the non-feeding puerulus stage for total lipid, total fatty acid and total protein. Width of arrows indicates where there is a
change in the relative levels of the various energetic components.
296 A.J. Limbourn, P.D. Nichols / Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298
higher, quantitatively, in final stage phyllosomata than the three
pueruli morpho-phases, with all three pueruli morpho-phases con-
siderably higher in these FA than first instar juveniles.
Arachidonic acid (ARA, 20:4n-6), 18:1n-7, EPA, 20:0 and 22:0 all
increased through the developmental sequence in their relative levels.
The FA 16:1n-7, 16:1/16:2, 14:0 and 16:0 decreased in their relative
contribution, while all others remained relatively constant through
development. DHA was lower in all puerulus stages compared to
phyllosoma and first instar juveniles.
EPA increased significantly (F4, 396 = 613.885, p b 0.001) from between
8.8% in phyllosomata to 17.4% in first instar juveniles with the greatest
increase between the P2 pueruli (11.8%) and first instar juveniles.
Similarly, the relative levels of ARA showed the greatest increase
between P2 pueruli (3.1%) and first instar juveniles (6.3%), although to a
lesser degree than EPA. ARA values increased from between 1.5% in
phyllosomata to 6.3% in first instar juveniles. Thus the EPA/ARA ratios
were highest in the late-stage phyllosomata (5.9) and decreased
subsequently thereafter (puerulus stage, 4.0–4.3; first instar juveniles,
3.1). In contrast, DHA decreased significantly (p = 0.001) from phylloso-
mata (15.1%) to recently settled clear pueruli (11.6%) before increasing
significantly (p b 0.001) in first instar juveniles (13.8%) (F4, 396 = 8.998,
p b 0.001).
Subtle differences in lipid and FA content were evident between
the four locations (Limbourn et al., in press). However, differences
between locations were less significant than the differences between
developmental stages and as such are not reported on here.
4. Discussion
On settlement in the nearshore habitat, the pueruli develop colour
(P1 and P2) over the next 13 days, on average, and prepare for
moulting into the first instar juvenile. During this period the pueruli
are presumed to be quite inactive and still not feeding (Jeffs et al.,
2002). Our data of very little variation in total dry weight through the
clear, P1 and P2 pueruli are consistent with this assumption.
4.1. Lipid content – nutritional state
In the transformation from ‘clear’ to the first instar juvenile the
lipid content was the biochemical component to show the greatest
change, largely a decrease in the PL, SFA and MUFA fractions. The
increase in mean proportion of total protein per gram of DW is likely
due to the loss of lipid between successive developmental stages
rather than a net gain in protein. The largest decline in protein
following moult to the first instar juvenile is likely due to the use of
available proteins in the synthesis of the new exoskeleton (Terwilliger,
1999).
Lipid is the main storage product in final stage phyllosoma and the
non-feeding puerulus stage of J. edwardsii, the southern rock lobster
(Jeffs et al., 1999; Jeffs et al., 2001b). Its use as the major energy store
during these phases in palinurid life-cycles reflects the high calorific
value of lipid compared to protein and carbohydrate, with lipid
combustion providing nearly twice the energy yield, per unit of
weight, of carbohydrate and protein (Crisp, 1976). Higher lipid
concentrations of final-stage P. cygnus phyllosomata are reported
here (19.8–29.3% DW) than by Phillips et al. (2006): 7–22.4% DW).
However, the values reported here are comparable to those of final
stage J. edwardsii phyllosomata (18.6–34.4% (Jeffs et al., 2004)). The
differences in TL content of P. cygnus phyllosomata between the two
studies likely reflects inter-annual variation in the lipid content of the
prey of the western rock lobster phyllosomata. However, the results
run opposite to a pattern based on ENSO climate patterns. The year
2000, when Phillips et al. (2006) collected their phyllosomata, was a
La Niña year when oceanographic conditions are believed to be at
their most favourable for optimal larval growth (Griffin et al., 2001). In
2004 (an El Niño year) and 2005 (a neutral year), when our
phyllosomata were collected, conditions should be less-than favour-
able for optimal larval growth (Pearce and Phillips, 1988), yet all our
samples were higher in TL content. Clearly, much greater insight is
required than currently is available in order to make reliable/accurate
predictions on the nutritional status of phyllosomata based on the
ENSO state of a year.
TL content in recently settled clear pueruli was low in comparison
to phyllosomata values. The TL concentrations for individual clear
pueruli reported here again are higher than values reported by Phillips
et al. (2006), 6.1 mg compared with 3.1 mg TL respectively; the values
are low compared to other spiny lobster species in terms of total lipid
per individual. For example, as mentioned above, the mean TL content
for individual P. cygnus clear pueruli is lower compared to Jasus
verreauxi Milne-Edwards (8.3 mg, Jeffs et al., 2002) and J. edwardsii
(12.5 mg, Jeffs et al., 1999), reflecting the different sizes of the pueruli:
103.7 mg DW for J. edwardsii, 70 mg DW for J. verreauxi, compared
with 51.3 mg DW for P. cygnus pueruli. The smaller size of P. cygnus
pueruli potentially limits their capacity for lipid energy storage, but
presumably the size of the pueruli reflects the length of the swim
undertaken by each of the three species. Hence, elucidation of the
comparative relationships between the size of pueruli, the length of
their swim and the swimming efficiency of all three species becomes a
relevant issue to examine in developing an understanding of the
biology of this non-feeding, highly mobile phase.
The absolute mass of lipid declined at a relatively constant rate
with development from clear to P2 pueruli. The benthic P1 and P2
pueruli are likely to be cryptic and moving very little, although this
suggestion needs experimental confirmation. The sharp decline in TL
from P2 pueruli to the first instar juveniles indicates that the moult to
the first instar juvenile is energetically demanding, possibly for
respiration and the cellular preparations for moulting.
During the post-settlement phase leading to the moult into the
first instar juvenile, P. cygnus continue to use stored lipid energy
reserves, particularly PL, to sustain metabolic activity, an energy
source consistent with pueruli of other rock lobster species (Pearce,
1997; Jeffs et al., 1999, 2001a,b; Phleger et al., 2001; Jeffs et al., 2002).
Pueruli need to settle with sufficient lipid reserves for development to
progress during this non-feeding, energetically demanding life stage
(Jeffs et al., 2002).
The decrease in TL (mg g− 1 DW) across the puerulus phase was
almost solely accounted for by the decrease in PL of the five lipid
classes identified. PL is the major constituent lipid in P. cygnus
phyllosomata (Liddy et al., 2004) and pueruli of J. edwardsii (Pearce,
1997; Jeffs et al., 1999, 2001a,b; Phleger et al., 2001), J. verreauxi (Jeffs
et al., 2002) and P. cygnus (this study). In this respect, spiny lobsters
differ from other Crustacea that use TAG and WE for short-term and
long-term energy stores respectively (Hagen, 1988). The predomi-
nance of PL as the major lipid class in palinurid phyllosoma and
pueruli has been suggested to be associated with their characteristic
transparency in vivo (Jeffs et al., 2001b) in contrast to the more opaque
neutral lipids such as TAG and WE. The transparency of phyllosoma
and pueruli, combined with diurnal vertical migration as well as
activity peaks at night and during new moon phases, greatly reduces
the risk of being detected by visually-hunting planktivores (Acosta
and Butler, 1999). Phospholipids are also more polar than other lipids
and thus more easily emulsified (Koven et al., 1993; Sargent et al.,
1999), thereby simplifying the digestive pathway. Further, PL are a
better source of essential fatty acids than neutral lipids (Sargent, 1995;
Coutteau and Mourente, 1997), especially during early development
when the digestive capabilities may not yet be fully developed.
Therefore, the lower digestibility of neutral lipids combined with the
properties of PL may result in palinurid phyllosomata and pueruli
relying on PL for both their supply of energy and essential fatty acids.
The accumulation of TAG during the pre-moult pueruli stage, some-
thing normally associated with feeding, and the ensuing decline
following moult, is likely to be due to the conversion of PL to TAG by
 A.J. Limbourn, P.D. Nichols / Comparative Biochemistry and Physiology, Part B 152 (2009) 292–298
the hepatopancreas in preparation for moult to the first instar
juvenile. This conversion of lipid storage products has been identified
biochemically and histologically in J. edwardsii pueruli (Nishida et al.,
1995; Pearce, 1997; Jeffs et al., 2001b, 2002).
4.2. Fatty acids – declining of MUFA with sparing of PUFA
The major FA in P. cygnus phyllosomata and pueruli (16:1n-7, 16:0,
18:1n-9, 18:0, EPA and DHA) are also major components in the FA
profiles of J. edwardsii (Phleger et al., 2001) and J. verreauxi pueruli
(Jeffs et al., 2002). In this study we measured the FA profile of both
phyllosoma and puerulus stages. We conclude that the major FA
components used in pueruli, 16:1n-7, 16:1/16:2, 16:0, 18:1n-9, 18:1n-7,
and 22:6n-3, which were all reduced by N50% by the first instar
juvenile, are the major lipid components catabolised for metabolic
maintenance.
The maintenance of ARA levels between phyllosomata and first
instar juveniles highlights the importance of maintaining levels of
essential (not synthesised de novo) FA over other FA during
development. ARA is an important precursor for signalling molecules
including prostaglandins, prostacyclins, leukotrienes and thrombox-
anes (Sargent et al., 2002) and consequently a major structural
component of membranes. Many crustaceans are low in ARA relative
to EPA (Smith et al., 2003) which is also the case in our study.
DHA, the major PUFA present in phyllosomata (15.1%) and pueruli
(11.6–12.0%), declined significantly with development. Following
moult to the first instar juvenile EPA (17.4%) replaced DHA (13.8%) as
the dominant FA. We propose that this shift in DHA use in first stage
juveniles is possibly related to the change in source of dietary lipids i.e.
from solely pelagic to wholly benthic diet, with the high ratio of DHA
to EPA, and DHA to ARA, in phyllosomata a signature of their pelagic
diet of dinoflagellates and other plankton or zooplankton rich in DHA.
The reduction in DHA levels relative to EPA and other essential PUFA
may also indicate a lower requirement for DHA as development
progresses. Nonetheless, the importance of the DHA:EPA ratio in
animal nutrition has been recognised (Kanazawa, 1993; Watanabe,
1993; Coutteau and Mourente, 1997). Ratios of 1.5–2 occur in n-3
PUFA-rich copepods (Fraser et al., 1989; Naess et al., 1995), a likely
food source for P. cygnus phyllosomata (Danaher, 2003). Similar ratios
of DHA to EPA have also been shown to produce superior growth rates
and survival in larval fishes (Lokman, 1993; Sargent et al., 1999) and
prawns (see Castell, 1982).
The reduction in MUFA in P. cygnus phyllosomata through to first
instar juveniles indicates their use as a major source of energy, a
common strategy used by other crustaceans (Castell, 1982; Querijero
et al., 1997; Smith, 2004). MUFA constitute ideal energy substrates for
early development (Takeushi and Watanabe, 1982; Mourente et al.,
1991; Mourente and Tocher, 1992; Abi-ayad et al., 2000, 2004)
especially during the larval and post-larval stages where rock lobsters
need energy for metamorphosis and fast growth along with basal
metabolic activity. Contrary to what has been suggested by Phillips
et al. (2006), the high prevalence of the MUFA 18:1n-9 (oleic acid) in
phyllosomata suggests a diet rich in zooplankton other than
gelatinous zooplankton. Herbivorous salps generally have low (3–
7%) levels of oleic acid (Phleger et al., 2000), whereas signature lipid
profiles found in phyllosomata in this study show little correlation
with high levels (14.6–17.8%) of oleic acid recorded. Similarly, the
signature lipid profiles of J. edwardsii phyllosomata show distinct
differences to those of gelatinous zooplankton (Jeffs et al., 2004) and
of other known crustacean predators of gelatinous zooplankton
(Phleger et al., 2000). The natural diet of rock lobster phyllosomata
is presently unknown. As such, it is plausible that gelatinous
zooplankton may represent a component of the diet of phyllosoma
due to their dominance in the zooplankton where rock lobster
phyllosomata have been found (pers comm. T Koslow), albeit a minor
component.
297
Catabolism of SFA was low in comparison to MUFA despite SFA
being considered good substrates for energy production (Sargent,
1995).
4.3. Ratio n-3:n-6 — nutritional index for Panulirus cygnus
The n-3:n-6 ratio is an important indicator of the requirements for
specific fatty acid groups and has been used as a nutritional index in
marine fish (Mourente and Odriozola, 1990; Sargent, 1995) and
crustaceans (Lytle et al., 1990). If palinurid lobsters, such as P. cygnus,
follow a similar nutritional index then final stage phyllosomata, at 7.3,
through to the first instar juveniles, at 4.1, all fall within the reported
ideal ratio of between 4 and 10. The lower n-3:n-6 ratio in first instar
juveniles is largely due to the decline in DHA and increase in ARA
levels with development which may suggest that lipids available for
energy production are approaching critically low levels that can only
be resolved with the resumption of feeding. ARA is also supplied by an
increasingly benthic diet (Nichols et al., 1998).
In conclusion, the results presented here of changes in the
biochemical composition in P. cygnus in developing from phyllosoma
through puerulus to first instar juvenile suggest that poor nutrition is
a limiting factor responsible for driving the huge inter-annual variation
in puerulus recruitment to coastal waters of Western Australia. As
pueruli recruit over a long coastline that is subject to considerable
variation in oceanographic conditions, it is important to understand
how variation in oceanographic conditions between settlement
locations, season and between years impacts on settlement numbers
as well as nutritional condition. A greater understanding of larval
biology can allow better prediction of the impact of mass removal of
such a valuable fisheries species, and a dominant macroinvertebrate
species to coastal benthic communities, on sustaining rock lobster
populations.
Acknowledgements
We greatly acknowledge the generous financial support provided
by the Department of Animal Biology, UWA and CSIRO Marine and
Atmospheric Research. We are also extremely grateful to WA Fisheries
and Marine Research Laboratories for all lobster collections. We thank
B. Knott, R.C. Babcock, and D.J. Johnston for providing helpful
comments on earlier versions of this manuscript. We would also like
to thank D. Holdsworth for technical support during running of the
GC-MS. We acknowledge the careful reading and sound suggestions
by all reviewers.
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