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Effect of water temperature on the

development and energetics of early,
mid and late-stage phyllosoma larvae
of ...
Quinn Fitzgibbon

Aquaculture

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 Aquaculture 344-349 (2012) 153–160

Contents lists available at SciVerse ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effect of water temperature on the development and energetics of early, mid and
late-stage phyllosoma larvae of spiny lobster Sagmariasus verreauxi
Q.P. Fitzgibbon ⁎, S.C. Battaglene
Institute for Marine and Antarctic Studies (IMAS), Fisheries Aquaculture and Coasts, University of Tasmania, Private Bag 49, Hobart TAS 7001

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

Article history: The effect of water temperature on the survival, growth, respiration, apparent feed intake and activity of
Received 29 November 2011 Sagmariasus verreauxi phyllosoma during early, mid and late larval development through metamorphosis
Received in revised form 24 February 2012 was examined. Early-stage phyllosoma were cultured from hatch to instar 4 at temperatures 17, 20, 23, 26
Accepted 9 March 2012
and 29 °C for between 18 and 44 days. Survival and rate of development were greatest at 26 °C, while all
Available online 16 March 2012
animals died at 29 °C. The optimum temperature for larval growth in weight was 23 °C. Optimum metabolic
Keywords:
feeding efficiency measured as the maximum convection requirement index, (CRI, quotient of feed intake
Eastern rock lobster and oxygen consumption) was from 23 to 26 °C. Reduced growth at 26 compared to 23 °C was likely due
Packhorse lobster to accelerated development, resulting in a disequilibrium between moult increment and energy storage
Phyllosoma rates, whereas, lower metabolic feeding efficiency at 17–20 °C may be attributed to increased energetic
Respiration expenditure associated with activity. Mid-stage phyllosoma were cultured from instar 8 to instars 10–11
Metabolic rate over 69 days at 19, 21, 23, 25, and 27 °C. Survival was reduced at 27 °C. The optimum temperature for growth,
Thermal tolerance development and CRI was 23 °C. Late-stage phyllosoma were cultured from instar 13 over 112 days, or until
metamorphosis, at 21, 23 and 25 °C. There was no significant difference in survival among temperatures. The
rate of development was fastest at 23 °C with 36% of phyllosoma metamorphosing to puerulus, compared to
20% at 21 °C and 28% at 25 °C. Mass of final instar phyllosoma and puerulus were greatest at 21 °C, demon-
strating a downward shift in the optimum temperature for late-stage phyllosoma. The shift in temperature
optimum for late-stage larvae involves changes in feeding and energy metabolism. The study demonstrates
that the physiological energetics of S. verreauxi changes with larval ontogenetic development, which is an
important consideration for optimizing spiny lobster propagation success.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction lobsters of 6 to 12 months in the laboratory (Phillips et al., 2006).

There is great interest in the propagation of spiny lobsters owing to
their high international demand and value. Sustainable increase in
worldwide production of spiny lobsters is likely only possible through
the production of hatchery-reared seedstock for commercial grow-out
or fishery enhancement. In temperate Australia, propagation research
initially focused on Jasus edwardsii (Ritar et al., 2006) but has recently
turned to the faster growing Sagmariasus verreauxi. The species is natu-
rally distributed in coastal waters of south-eastern Australia where it is
known as the eastern rock lobster (Montgomery and Craig, 2005) and
New Zealand where it is commonly recognized as the packhorse lobster
or pawharu (Booth, 1986). It was formerly included in the Jasus genus
but is now placed in its own genus of Sagmariasus due to marked
differences in morphology and behaviour (Booth, 2006). Sagmariasus
verreauxi possess numerous favourable characteristics for warm-
temperate aquaculture, including a moderate larval duration for spiny
⁎ Corresponding author. Tel.: + 61 3 6227 7242; fax: + 61 3 6227 8035.
E-mail address: Quinn.Fitzgibbon@utas.edu.au (Q.P. Fitzgibbon).
They also have fast juvenile growth rates (Crear, 2000), high fecundity
(Pollack, 1997) and large maximum attainable size (Phillips et al.,
1980). Furthermore, relatively high survival rates have been achieved
in the culture of S. verreauxi from egg to pueruli suggesting that this
species is a “tough species particularly suitable for larval culture”
(Kittaka et al., 1997).
Defining abiotic requirements for culture of species is an impor-
tant early step in the production of aquatic organisms. This is partic-
ularly crucial for the successful development of spiny lobster
propagation because of the apparent difficulties in the long larval
culture cycle (Matsuda and Takenouchi, 2007). Temperature is prob-
ably the single most important physical factor effecting decapod
performance in both culture and the environment (Matsuda and
Yamakawa, 1997; Richardson, 2008). It is thought that temperature
first acts to affect the success of aquatic organisms through its influ-
ence on oxygen demands and the capacity to supply oxygen to the
tissues (Pörtner and Knust, 2007; Storch et al., 2009). In culture, tem-
perature has been shown to have a profound effect on physiological
functions which in turn influence survival, rate of development, and
growth (Bermudes and Ritar, 2004; Halcrow and Boyd, 1967). The

0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.03.008
154 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
influence of temperature on energy partitioning is especially critical for
phyllosoma, which must accumulate sufficient nutritional reserves for
the energetically demanding post-metamorphosis development as a
non-feeding puerulus (Jeffs et al., 2002). In this sense, optimum temper-
atures will be those that maximize the rate of energy acquisition (i.e.
feeding and assimilation) whilst minimizing energy expenditure.
A single study has previously examined the effects of temperature
on larval S. verreauxi (Moss et al., 2001). However, that study was
restricted to the early and mid phyllosoma stages. During the long
planktonic existence of phyllosoma, different stages are likely be
exposed to different temperatures associated with locality and season,
potentially altering temperature optimums. Matsuda and Yamakawa
(1997) showed that the optimum temperature for Panulirus japonicus
larvae changes with development, demonstrating the importance of
examining all stages of development. The present study examined the
effect of temperature on growth and survival of early, mid and late
stage S. verreauxi phyllosoma through puerulus. It also aimed to better
understand the physiological basis of the effects of temperature through
the examination of aspects of energetic partitioning (oxygen consump-
tion rate, feeding rates, and activity). The present study is the first to
examine environmental effects on the energy budget of spiny lobsters
throughout early, mid and late larval development and provides impor-
tant information on the thermal requirements S. verreauxi, which is crit-
ical for optimizing performance in culture.
2. Materials and methods
2.1. Broodstock and larval culture
Broodstock animals were collected from the wild as puerulus and
held in 4000 l fibreglass tanks for nine years at the Institute for
Marine and Antarctic Studies (IMAS), in Hobart, Australia, under a
regime of simulated ambient photoperiod and water temperature.
They were fed a combination of fresh blue mussel (Mytilus edulis)
and commercial prawn pellet (Higashimaru, Vital No 12, http://
www.k-higashimaru.co.jp/), and weighed approximately 2.5 kg at
spawning. Newly-hatched phyllosoma were collected from one female
on 30 January 2008 for the early and mid-stage experiments and
another female on 10 February 2008 for the late-stage experiment.
Larvae were either used immediately from hatch (early-stage exper-
iment) or mass-cultured in 200 l tanks until they reached the appro-
priate instar for the mid and late-stage experiments. Mass cultures
were maintained at 23.0 ± 0.2 °C under a 12:12 light:dark photoperi-
od. Light was supplied by cool white florescent globes positioned
above vessels which delivered a light intensity at the water surface
of between 0.08 and 0.1 μmols. From hatch they were fed excess
2–6 mm juvenile Artemia daily. Details of Artemia production and
treatment are given in Jensen et al. (2011). Late-stage larvae were
also fed 2–5 mm pieces of blue mussel gonad daily to excess. Excess
feed was flushed from the culture systems immediately before sub-
sequent feeding and tanks were cleaned weekly. Larvae were progres-
sively acclimated to experimental temperatures over approximately 8 h
before stocking into experiments.
2.2. Experimental larval culture systems
Flow-through water was filtered and treated as described by Ritar
et al. (2006). Water was heated to 23 °C before being pumped to 120 l
sumps (one for each temperature treatment) either directly (for
treatment temperatures 23 °C and above) or, for treatments below
23 °C, via a 500 l sump where it was chilled to 19 ± 0.2 °C by a heat
chill unit (C010phh7AA, Carrier, http://www.Aquasonic.com.au).
Thermostatically controlled immersion heaters (Istra Elements and
Engineering, Caringbah, NSW) in each sump heated the water to the
desired treatment temperatures (±0.2 °C) before it was gravity or
pump supplied to experimental systems.
The early-stage experiment was conducted in replicated translucent
2 l flat bottom plastic containers with the total water volume of 1.6 l
and water flow rate of 5 exchanges h− 1. The mid-stage experiment
was conducted in similar 2.6 l flat bottom plastic containers also with
a water flow rate of 5 exchanges h− 1. The late-stage experiment was
conducted in 10 l flat bottom plastic containers with 3 exchanges h− 1.
In all experiments, feeding, cleaning and abiotic factors were as for
mass culture. Treatment water temperatures were logged hourly
(iButton, Maxim, http://www.maxim-ic.com/products/ibutton/) in
spare replicate containers for each treatment. Dissolved oxygen
(HQ10, Hach, http://www.hach.com/) and total ammonia (Test kit,
Aquasonic, http://www.aquasonic.com.au/) were measured twice
weekly. Dissolved oxygen remained between 105 and 110% saturation
(slightly super-saturated due to ozonation water pre-treatment) and
total ammonia below 0.5 mg l− 1 for the duration of the experiment.
2.3. Experimental design
In the early-stage experiment, five water temperature treatments
were examined (mean ± SD, hourly observations): 17 (17.2 ± 0.6), 20
(20.1 ± 0.2), 23 (23.3 ± 0.3), 26 (26.1 ± 0.3) and 29 (28.9 ± 0.2) °C.
This temperature range was used because it spans and exceeds the
temperature range previously used in the larval culture of S. verreauxi
(20–24 °C) (Jensen et al., 2011; Kittaka et al., 1997; Moss et al., 2001).
For each treatment, six randomly allocated replicate vessels were
stocked with newly hatched phyllosoma at a rate of 40 phylloso-
ma l − 1 (64 phyllosoma vessel − 1). Treatments were terminated
once phyllosoma reached instar 4.
In the mid-stage experiment, five water temperatures were exam-
ined (mean ± SD, hourly observations): 19 (19.0 ±0.4), 21 (20.9±0.2),
23 (22.9± 0.3), 25 (24.8 ±0.3) and 27 (27.4 ±0.2)°C. For each treat-
ment, six randomly allocated replicate vessels were stocked with
69 day old phyllosoma (instar 8.3±0.5, mean ±SD, n = 40) at 12 phyl-
losoma l− 1 (30 phyllosoma vessel− 1). The experiment was terminated
after 63 days of culture.
In the late-stage experiment, three water temperatures were exam-
ined (mean ± SD, hourly observations): 21 (21.1± 0.4), 23 (22.7± 0.2)
and 25 (25.0 ± 0.3) °C. For each treatment, five randomly allocated rep-
licate vessels were stocked with 166 day old larvae (instar 12.7± 0.5,
mean± SD, n = 40) at a rate of 1.5 phyllosoma l− 1 (15 phyllosoma -
vessel− 1). The experiment was terminated after 112 days of culture.
2.4. Survival and development
Phyllosoma mortality was assessed by counting survivors during
the transfer between two1000 ml glass beakers that were illuminated
over a light source. Phyllosoma growth and development was quanti-
fied according to length, dry mass, and instar. Length was measured
on a profile projector (Nikon 6 C, Japan) from the anterior tip of the
cephalic shield between the eyestalks to the posterior tip of the abdo-
men. Dry mass was determined by rinsing phyllosoma with 0.5 M
ammonium formate, drying at 60 °C for 24 h before being weighed
to the nearest 10 μg on a precision balance (AT261 DeltaRange,
Mettler-Toledo, Switzerland). Instar was visually assessed under a
dissecting microscope according to Kittaka et al. (1997) which de-
fines 17 distinct phyllosoma instars for S. verreauxi. In the early and
mid-stage experiments, developmental parameters of six randomly
selected phyllosoma from each replicate were measured at the end
of the experiments. In the late-stage experiment, length, width and
instar of all phyllosoma were recorded at 85 d from the start of the
experiment, and all developmental parameters were measured at
the end of the experiment a further 27 d later. In the late-stage exper-
iment, any phyllosoma that progressed through metamorphosis to
pueruli were immediately removed and dry mass recorded. Some
pueruli that were stuck in the moult were excluded from dry-mass
analysis.
 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
_ 2)
2.5. Oxygen consumption rate (Mo
Phyllosoma were selected from experimental vessels for oxygen
consumption rate (Mo _ 2 ) examination. Intermoult stage of the moult
cycle was determined by counting the number of days from the
previous moult (early-stage experiment) or by microscopic examina-
tion of the second antenna according to Anger (1983; 2001) (mid and
late-stages). Oxygen consumption rate was measured in 20 ml glass
vials fitted with adjustable plungers (early- and mid-stages) and
60 ml syringes (late-stages) as respirometer chambers. Phyllosoma,
three to five for early-stages and individuals in mid and late-stages,
were stocked into unsealed chambers and left overnight to acclima-
tize, recover from handling stress, and achieve a post-absorptive
state. The following morning, chambers were sealed and left for
1–2 h, before a first water sample was drawn (~10 ml) to determine
the initial dissolved oxygen level with an oxygen optode (HQ10,
Hach, http://www.hach.com/). Phyllosoma were then left undisturbed
(chamber water volume of 10 ml for early and mid-stages and 15 ml
for late-stages) for 60–90 min before final water samples were taken
and measured. Oxygen consumption rate was recorded for six replicate
chambers and controls without phyllosoma for each treatment. The
difference from the initial and final dissolved oxygen readings minus
background respiration measured in control respirometers was used
to calculate the Mo _ 2 of phyllosoma. All Mo _ 2 measurements were
conducted with the same abiotic factors as for experimental culture
during the light phase. Length, and dry mass of phyllosoma were
recorded at the end of respiratory trials. Oxygen consumption rate
was recorded for instars 2 and 4 in the early-stage, instars 9 and 11 in
the mid-stage, and instar 14 in the late-stage experiments.
2.6. Apparent feed intake
In early and mid-stage experiments, apparent feed intake by inter-
moult phyllosoma was determined in 70 ml flow through chambers
(plastic sample jars with 500 μm screen lids). Either two (in early-
stages) or individual (in mid-stages) phyllosoma were stocked into
chambers with live 2.5–4 mm Artemia at a density of 28 μg dry
mass ml − 1 and left for 24 h. The difference in the number of Artemia
stocked, from that remaining on completion of incubation with
phyllosoma, minus Artemia losses recorded in control chambers,
was used to calculate phyllosoma apparent Artemia intake. Artemia
intake was measured for six replicate chambers and three controls
without phyllosoma for each treatment. Dry mass of phyllosoma was
recorded at the end of feeding trials. Artemia dry mass was determined
by measuring Artemia length at the beginning of the experiment and
using a relationship developed at IMAS between Artemia length:dry
mass. Feed intake was presented in terms of μg Artemia g DM− 1. Appar-
ent hourly Artemia intake by phyllosoma was recorded for instars 2 and
4 in the early-stage and instars 9 and 11 in the mid-stage experiments.
In the late-stage experiment, phyllosoma apparent Artemia and
mussel gonad intake were measured within experimental replicate
vessels. Replicates, and one control vessel without phyllosoma for
each treatment, were fed one-thousand 4–6 mm live juvenile Artemia
and 3 g of 5 mm pieces mussel gonad simultaneously, daily. Before
subsequent feeding the next day, remaining food was removed and
frozen in a cumulative sample over 5 days. Samples were later rinsed
with 0.5 M ammonium formate, before being dried and weighed as
previously described. The difference between the mass of feed in
control and replicates was used to calculate phyllosoma apparent
intake (mg phyllosoma − 1 day − 1). Apparent Artemia and mussel
gonad intake was determined over experimental days 42 to 46 with
phyllosoma at instar (mean± SE, n = 5 replicates): 14.4± 0.1, 14.3 ±
0.1, 14.4± 0.1 for 21, 23 and 25 °C treatments, respectively. All feed
intake measurements were conducted with the same abiotic factors as
for experimental culture conditions.
155
2.7. Swimming descent velocity
Swimming descent of intermoult phyllosoma was measured as an in-
dicator of activity at different temperatures as previously described by Ott
and Forward (1976). Glass measuring cylinders of 200, 500 and 1000 ml
volume were used as vertical swimming chambers for early, mid, and
late-stage experiments, respectively. Phyllosoma are both negatively
phototactic and negatively buoyant resulting in downward movement
in culture during daylight hours. All measurements were conducted adja-
cent to experimental culture vessels during daylight hours. Individual
phyllosoma were gently introduced to the top of the chamber and their
rate of descent through the bottom three quarters of the chambers
timed (swim distance of 14.0, 23.0, and 29.0 cm for early, mid, and late-
stage experiments, respectively). Descent through the top quarter of the
chambers was excluded from analysis as this allowed phyllosoma to ori-
entate after transfer. Phyllosoma were then removed and anaesthetized
in 0.1% 2-Phenoxyethanol (P1126, Aldrich, http://www.sigmaaldrich.
com/) before measuring their passive descent rate. Paralyzed phyllosoma
are negatively buoyant and typically sink vertically through the water
column in a posture head first with the body at a ~45° angle to the vertical
plane. Phyllosoma length and instar were determined before being
released back into experimental replicates. The difference between the
descent rate of the anaesthetized and swimming phyllosoma was
used to calculate swimming descent velocity in body lengths (BL) s− 1.
Swimming descent of two phyllosoma from each replicate was measured
with instar 4 (early-stage experiment), 9 and 11 (mid-stage experiment)
and 14 (late-stage experiment) phyllosoma.
2.8. Data analysis
All analyses were performed on the means of replicates for each treat-
ment, except for development data from the late-stage experiment,
where means of individual phyllosoma or puerulus for each treatment
were compared. Development data of individual phyllosoma were ana-
lyzed in the late-stage experiment because larvae were separated and
grouped into individual instars (16 and 17) and pueruli. Data were tested
for homogeneity of variance and normal distribution of residuals using
Levene's test and the Shapiro–Wilk W test, respectively. When these as-
sumptions were not met, data were transformed by log-transformation
and retested to ensure they conformed with ANOVA assumptions. Per-
centage data was arcsine transformed. One way ANOVA and Tukey's
HSD post hoc test (significance level P b 0.05) were used to test differ-
ences in dependent variables among temperature groups. As an indicator
of metabolic feeding efficiency, the convection requirement index (CRI)
was calculated by dividing feed intake (μg mg phyllosoma DM− 1 h− 1
for early and mid-stage experiments and mg phyllosoma− 1 day− 1
for late-stage experiment) and Mo _ 2 (mg g phyllosoma DM− 1 h− 1)
(Newell and Branch, 1980). Quadratic polynomial regressions were fitted
to raw data to describe the relationship between temperature, and devel-
opment parameters and CRI. Optimum temperature (Topt) for develop-
ment parameters and CRI was calculated as the maximum zero solution
to the first derivative of the quadratic regressions. Where no maximum
zero solutions to the first derivative of the quadratic regressions were
present, Topt was assumed to be below the minimum temperature exam-
ined. Statistics were performed with the SPSS 16.0 for Window software.
Unless otherwise specified, values are given as means±SE.
3. Results
3.1. Early-stage experiment
Within temperature treatments moult increment was well synchro-
nized and inversely related to water temperature with newly-hatched
phyllosoma taking 18, 22, 28 and 44 days to reach instar 4 at 26,
23, 20 and 17 °C, respectively. Survival was significantly affected by
temperature (ANOVA, F = 25.6, df= 4, 25, P = b0.001). At 29 °C,
156 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160

phyllosoma reached instar 2 within 4 days of hatch, and all died by day
8. Survival at 26 °C was significantly greater than that at 17 and 20 °C,
but not different from 23 °C (Fig. 1). There were no differences in length
(ANOVA, F = 2.7, df = 3, 20, P = 0.072) of instar 4 phyllosoma among
temperatures (Fig. 2). Mass of phyllosoma was significantly affected
by temperature (ANOVA, F = 17.1, df= 3, 20, P = b0.001) with those
cultured at 23 °C accumulating significantly greater dry mass than at
other temperatures. Optimum temperatures (Topt) for length and
mass were 22.6, and 21.7 °C, respectively.
Apparent feed intake was significantly affected by temperature for
both instar 2 (ANOVA, F = 44.9, df = 3, 20, P = b0.001) and instar 4
(ANOVA, F = 36.2, df = 3, 20, P = b0.001) phyllosoma (Fig. 3). Appar-
ent feed intake of instar 2 phyllosoma was significantly greater at 23
and 26 °C than at 17 and 20 °C. Apparent feed intake of instar 4 phyl-
losoma was greatest at 26 °C and intake at 20 and 23 °C was greater
than 17 °C. Oxygen consumption rate of instar 2 and 4 phyllosoma
was not significantly different across temperatures (ANOVA, F = 0.8,
df = 3, 20, P = 0.535 and ANOVA, F = 1.6, df = 3, 20, P = 0.225, respec-
tively). There was a significant effect of water temperature on the
swimming descent velocity of instar 4 phyllosoma (ANOVA, F = 6.7,
df = 3, 44, P = 0.019). Phyllosoma at 23 °C swam significantly faster
than those at 17 °C and 20 °C but were not significantly different
from phyllosoma at 26 °C. Topt for CRI were 24.7 and 24.8 for instar
2 and 4 phyllosoma, respectively.

3.2. Mid-stage experiment

Survival of mid-stage phyllosoma was significantly affected by

temperature (ANOVA, F = 17.1, df = 4, 25, P = b0.001) being reduced
at 27 °C compared to all other temperatures (Fig. 4). Length, dry mass
and instar development of phyllosoma were all significantly affected
by temperature (ANOVA, F = 12.7, df = 4, 25, P = b0.001, ANOVA,
F = 8.3, df = 4, 25, P = b0.001, ANOVA, F = 8.1, df = 4, 25, P =b0.001,
respectively) (Fig. 5). Length was significantly greater at 23 °C than
at 19, 21 and 27 °C, but not 25 °C. Dry mass of phyllosoma was signif-
icantly greater at 25 °C than 19, 21 and 27 °C, but not 23 °C. Phyllosoma
instar development was reduced at 19 and 27 °C. Optimum tempera-
tures for developmental parameters were similar being 23.2, 23.6 and
22.9 °C for length, dry mass, and instar, respectively.
There was no significant effect of temperature on apparent feed
intake of instar 9 and 11 phyllosoma (ANOVA, F = 0.3, df = 4, 25,
P = 0.909 and ANOVA, F = 3.3, df = 4, 25, P = 0.891, respectively)
(Fig. 6). Temperature did not affect Mo _ 2 of instar 9 phyllosoma
(ANOVA, F = 0.7, df = 4, 25, P = 0.573), however, it did affect Mo _ 2 of
instar 11 larvae (ANOVA, F = 3.3, df = 4, 25, P = 0.25). Oxygen
Fig. 2. (A) Length and (B) dry mass of instar 4 phyllosoma at the completion of the
early-stage experiment. Data from phyllosoma at 29 °C treatment were not included
due to 100% mortality. Values are mean ± SE, n = 6. Superscripts not sharing a common
letter are significantly different at the end of the experiment (ANOVA and Tukey's HSD,
P b 0.05). Details of quadratic regressions are presented adjacent to lines. Arrows indicate
optimum temperatures (Topt).
consumption rate of instar 11 phyllosoma was greater at 27 °C than
at other temperatures. Similar to the early-stage experiment, swim-
ming descent velocity of instar 11 phyllosoma peaked at 23 °C but
was not significantly different across treatments (ANOVA, F = 1.5,
df = 4, 55, P = 0.222). Optimum temperature values for CRI were sim-
ilar to those for developmental parameters, being 23.5 and 24.2 °C for
instars 9 and 11 phyllosoma, respectively.
3.3. Late-stage experiment

There was no difference in phyllosoma survival between 21, 23

Fig. 1. Survival of phyllosoma cultured from hatch to instar 4 at different water tempera-
tures. Values are mean ± SE, n = 6. Superscripts not sharing a common letter are signifi-
cantly different at the end of the experiment (ANOVA and Tukey's HSD, P b 0.05).
and 25 °C (ANOVA, F = 1.3, df = 2, 12, P = 0.320) (Fig. 7). At termina-
tion, more phyllosoma had metamorphosed to puerulus at 23 °C
(36%), compared to 21 °C (20%) and 25 °C (28%). However, this differ-
ence was not significant (ANOVA, F = 3.323, df = 2, 12, P = 0.071).
Mean instars of remaining phyllosoma were 15.8 ± 0.13 (n = 35) at
21 °C, 16.6 ± 0.15 (n = 16) at 23 °C, 16.3 ± 0.16 (n = 19) at 25 °C.
Lengths of instar 16 and 17 phyllosoma were significantly affected
by temperature (ANOVA, F = 11.3, df = 2, 65, P = b0.000 and
ANOVA, F = 31.6, df = 2, 59, P = b0.000, respectively) being inversely
related to water temperature (Fig. 8). Mean length of instar 16
phyllosoma was significantly greater at 21 and 23 °C than at 25 °C.
Length of instar 17 increased significantly with decreasing tempera-
ture. Temperature did not affect the dry mass of instar 16 phyllosoma
(ANOVA, F = 0.9, df = 2, 19, P = 0.418) but did affect the mass of in-
star 17 phyllosoma (ANOVA, F = 11.2, df = 2, 25, P = b0.000) and
pueruli (ANOVA, F = 11.3, df = 2, 46, P = b0.000). Dry mass of instar
17 phyllosoma and pueruli was greater at 21 and 23 °C than at
25 °C. Optimum temperatures were all below 21 °C except for mass
of instar 16 (22.8 °C) and 17 (21.4 °C) phyllosoma.
 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
Fig. 3. (A) Apparent feed (Artemia) intake (AFI), (B) oxygen consumption rate (M _ o2 ),
(C) swimming velocity (body lengths s− 1, BL s− 1) and (D) convection requirement
index (CRI) of instar 2 and 4 phyllosoma (instar 4 only for swimming velocity) at
different water temperatures recorded in the early-stage experiment. Data from
phyllosoma at 29 °C treatment were not included due to 100% mortality. Values are
mean ± SE, n = 6. Superscripts not sharing a common letter are significantly different
at the end of the experiment (ANOVA and Tukey's HSD, P b 0.05), lower case for instar
2 and upper-case for instar 4. Details of quadratic regressions for CRI are presented
adjacent to lines. Arrows indicate optimum temperatures (Topt).
Fig. 4. Survival of 69 day old phyllosoma (instar 8.3) cultured for a further 63 days at
different water temperatures in the mid-stage experiment. Values are mean ± SE,
n = 6. Superscripts not sharing a common letter are significantly different at the end
of the experiment (ANOVA and Tukey's HSD, P b 0.05).
157
Fig. 5. (A) Length, (B) dry mass and (C) instar development of phyllosoma at the end of
the mid-stage experiment. Values are mean ± SE, n = 6. Superscripts not sharing a
common letter are significantly different at the end of the experiment (ANOVA and
Tukey's HSD, P b 0.05). Details of quadratic regressions are presented adjacent next to
lines. Arrows indicate optimum temperatures (Topt).
Temperature did not significantly affect apparent Artemia intake of
instar 14 phyllosoma (ANOVA, F = 1.7, df = 2, 12, P = 0.222). It did af-
fect apparent mussel gonad intake (ANOVA, F = 38.3, df = 2, 12,
P = b0.000) (Fig. 9). Instar 14 phyllosoma at 21 °C ingested more
mussel gonad than at 23 or 25 °C. Oxygen consumption rate of instar
14 phyllosoma was affected by temperature (ANOVA, F = 5.8, df = 2,
15, P = 0.015) being significantly greater at 25 °C than at 21 °C. Tem-
perature did not affect swimming descent of instar 14 phyllosoma
(ANOVA, F = 2.1, df = 2, 27, P = 0.149). Optimum temperature for
CRI of instar 14 phyllosoma was below 21 °C, mainly due to increased
feed intake at 21 °C.
4. Discussion
The survival of decapod larvae, including spiny lobster phyllo-
soma, commonly declines below and above a species specific thermal
tolerance range (Bermudes and Ritar, 2008). The thermal tolerance
window of an organism is considered to be defined by lower and
upper critical temperatures whereby with further warming or cool-
ing, limited capacity to support the aerobic scope causes the animal
to resort to anaerobic metabolism, which is not sustainable (Pörtner
and Knust, 2007; Storch et al., 2009). The thermal tolerance range
for early-stage S. verreauxi phyllosoma survival in culture appears to
be between 20 and 26 °C, with warmer temperatures resulting in
rapid mortality, while at cooler temperatures, mortality is more
chronic. Similarly, Moss et al. (2001) found reduced survival of instar
1 S. verreauxi at 18 °C. However, they found no difference in survival
158 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
Fig. 6. (A) Apparent feed (Artemia) intake (AFI), (B) oxygen consumption rate (M _ o2 ),
(C) swimming velocity (body lengths s− 1, BL s− 1) and (D) convection requirement
index (CRI) of instar 9 and 11 phyllosoma (instar 11 only for swimming velocity) at
different water temperatures recorded in the mid-stage experiment. Values are
mean ± SE, n = 6. Superscripts not sharing a common letter are significantly different
at the end of the experiment (ANOVA and Tukey's HSD, P b 0.05), lower case for instar
9 and upper-case for instar 11. Details of quadratic regressions for CRI are presented
adjacent to lines. Arrows indicate optimum temperatures (Topt).
Fig. 7. Survival of 166 day old phyllosoma (instar 12.7) cultured for a further 112 days
at different water temperatures in the late-stage experiment. Values are mean ± SE,
n = 5. Superscripts not sharing a common letter are significantly different at the end
of the experiment (ANOVA and Tukey's HSD, P b 0.05).
Fig. 8. (A) Length of instar 16 and 17 phyllosoma (combined sample of phyllosoma
measured on days 85 and 112 of the late-stage experiment) and (B) dry mass of instar
16 and 17 phyllosoma measured at the end of the late-stage experiment, and puerulus
immediately after metamorphosis. Parentheses indicate sample numbers. Values are
means± SE, superscripts not sharing a common letter are significantly different at the
end of the experiment (ANOVA and Tukey's HSD, P b 0.05), lower case for instar 16,
upper-case for instar 17, and Greek alphabet for puerulus. Details of quadratic regressions
are presented adjacent to lines. Arrows indicate optimum temperatures (Topt).
between 18, and 27 °C for instar 3, 5 and 7 phyllosoma. The lack of a
significant effect of temperature on survival was likely due to the
short duration of trials. Moss et al. (2001) cultured individual phyllo-
soma for only one moult or instar. It is clear from the present study
that the effect of temperature on survival often takes several moults
to manifest.
Within limits, increasing temperature reduces inter-moult dura-
tion and accelerates the development of decapod larvae (Bermudes
and Ritar, 2008; Johns, 1981; Matsuda and Yamakawa, 1997;
Minagawa, 1990; Paul and Paul, 1999; Rothlisberg, 1979). Fastest de-
velopment of S. verreauxi phyllosoma was achieved at 26 °C for early-
stages and at 23 °C for mid and late-stages. In commercial culture, it is
desirable to maximize the rate of development, thus reducing the du-
ration of the costly larval phase. However, it is important that acceler-
ated development does not adversely affect longer-term larvae
viability. Maximum growth (size and dry mass increase) of early-
stage larvae occurred at 23 °C. Similarly, maximum convection re-
quirement index (CRI) was between 23 and 26 °C, demonstrating its
value as an assessment of growth potential. The CRI elucidates maxi-
mum metabolic feeding efficiency at which the rate of energy acqui-
sition through feeding is optimized relative to energy expenditure
of metabolism (Newell and Branch, 1980). However, the CRI only pro-
vides an instantaneous assessment of growth potential, while the ab-
solute growth of an instar is also complicated by its duration. Faster
development at warmer temperatures reduces the time available for
energy acquisition, potentially causing the larva to moult before it
would attain its maximum size (Sweeney and Vannote, 1978). Tong
et al. (2000) found that at high temperatures overall feed consump-
tion of mid-stage J. edwardsii decreases due to reduced intermoult
duration, resulting in reduced growth. Thus, the disequilibrium be-
tween development and energy acquisition rates probably explains
 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
Fig. 9. (A) Apparent feed (Artemia and mussel gonad) intake (FI), (B) oxygen consump-
_ o2 ) and (C) convection requirement index (CRI) of instar 14 phyllosoma at dif-
tion rate (M
ferent water temperatures recorded in the late-stage experiment. Values are mean± SE,
n = 5 for AFI and n = 6 for M_ o2 . Superscripts not sharing a common letter are significantly
different at the end of the experiment (ANOVA and Tukey's HSD, P b 0.05). Detail of qua-
dratic regression for CRI is presented adjacent to the line. Arrow indicates optimum tem-
perature (Topt).
the reduced growth of early-stage S. verreauxi at 26 °C even though
the CRI was high.
Reduced growth of early-stage S. verreauxi at temperatures below
23 °C is likely due to reduced CRI resulting from proportionally high
metabolism. The Mo _ 2 of early- stage S. verreauxi actually increased
below 20 °C, possibly as a stress response and a consequence of in-
creased activity. Behavioural responses of decapod larvae can alter
dramatically with changes in temperature (Ott and Forward, 1976;
Sulkin, 1984) and to a large extent the Mo _ 2 is an expression of this
changed locomotory activity (Halcrow and Boyd, 1967). However,
downward swimming velocity of early-stage S. verreauxi was reduced
in cool water, suggesting that the increase in Mo _ 2 was not a conse-
quence of increased activity. It is possible that the lack of correlation
between Mo _ 2 and recorded swimming velocity may be because
downward swimming velocity does not always represent overall ac-
tivity of phyllosoma. Negative phototaxis combined with negative
buoyancy typically results in the downward movement of S. verreauxi
phyllosoma in daylight. The slower descent rate of S. verreauxi phyllo-
soma in cooler water may be a result of negative geotaxis with larvae
expending more energy attempting to maintain position or swim up-
wards in the water column. In some decapod larvae, cold water trig-
gers vertical swimming, presumably to reach warmer water higher in
the water column (Sulkin, 1984). Furthermore, it should be noted
159
_ 2 and activity measurements in the present study were only
that Mo
made during the light phase. Larva may behave differently in the ab-
sence of light which would likely affect their overall energy consump-
tion. Further research is required to better understand energetic costs
of activity and the effects of differing environmental conditions.
Similar to early-stage larvae, the optimum temperature for develop-
ment rate, growth and CRI of mid-stage larvae was ~23 °C. However,
trends in feed intake and Mo _ 2 were less discernible than in early-
stage larvae. For mid-stage larvae, temperature appeared to have a
less profound effect on physiological functions, which may be due to
difficulties of sampling phyllosoma at a consistent point within the
moult cycle. Both Mo _ 2 and feed intake can vary profoundly within the
moult cycle of decapod larvae (Anger and Jacobi, 1985; Anger et al.,
1989; Sasaki et al., 1986). Therefore, it is important that physiological
measurements be made at a well specified point of the moult cycle, gen-
erally during the intermoult, which is particularly stable (Anger, 2001).
Moulting of early-stage larvae was closely synchronized within repli-
cate groups thus making it easy to select intermoult individuals by
counting days since the group moulted. Moulting of mid and late-
stage phyllosoma was not synchronized and moult-cycle stage was es-
timated by microscopic examination of the second antenna based on
development in brachyuran crabs Hyas araneus (Anger, 1983, 2001).
This was not reliable, with some phyllosoma even moulting within
respiratory or feed intake chambers (which were excluded from
analysis). Inaccuracies in moult cycle staging likely contributed to in-
creased variation of physiological measurements of mid and late-stage
phyllosoma. Better moult cycle staging of spiny lobster phyllosoma
will benefit future physiological research, especially as the research
focus shifts towards mid and late-stages with the advancement of
culture success.
A downward shift of at least 2 °C in the optimum temperature of
late-stage S. verreauxi phyllosoma was observed. Size and mass of
final instar phyllosoma (instar 17) increased with decreasing temper-
ature to the minimum temperature examined of 21 °C. Furthermore,
mass of puerulus was greatest at 21 °C. A similar downward shift of
optimum temperature was recorded with P. japonicas, where maxi-
mum growth occurred at 26 °C for early-stage and 24 °C for late-
stage phyllosoma (Matsuda and Yamakawa, 1997). Matsuda and
Yamakawa (1997) hypothesized that energy acquisition becomes
more critical for late-stage phyllosoma due to the exponential in-
crease in moult increments. At warm temperatures, large phyllosoma
are unable to meet the energy demands of increased maintenance
metabolism and growth requirement. Indeed, Mo _ 2 of late-stage
S. verreauxi increases with temperature. However, improved growth
of S. verreauxi could also be attributed to an observed increase in
apparent feed intake, that when combined with reduced metabolism,
resulted in a greatly enhanced CRI at the cooler temperature. This is
similar to larval red frog crab (Ranina ranina) where maximum intake
of late instar zoeas occurs at substantially cooler temperatures than
early and mid instar larvae (Minagawa, 1990). Greater feed intake
at cooler temperatures of late-stage S. verreauxi suggests a shift in
temperature preference. Wild S. verreauxi typically spawn in the
warmer northern extent during spring/summer and larvae trans-
ported south by offshore currents over 12 months (Booth, 1986;
Montgomery, 1992; Montgomery and Craig, 2005). Therefore it is
possible that late-stage larvae are typically subject to cooler temper-
atures in the wild, which may contribute to their cooler temperature
preference.
Optimizing environmental conditions to maximize energy accumu-
lation is vital in the culture of spiny lobster larvae because the accumu-
lation of adequate energy reserves is critical for the viability of the non-
feeding pueruli stage. Based on measurement of energy budgets and dry
mass accumulation, optimum energy accumulation occurs at 23 °C for
early and mid-stage S. verreauxi, with a downward shift to 21 °C or
below in late-stage phyllosoma. The shift in temperature optimum for
late-stage larvae involves changes feeding and metabolism however
160 Q.P. Fitzgibbon, S.C. Battaglene / Aquaculture 344-349 (2012) 153–160
further research is required to fully understand the physiological pro-
cesses. Previous analysis of the biochemistry of wild pueruli suggests
that polar lipid is the primary energy storage component (Jeffs et al.,
1999, 2001, 2002; Phleger et al., 2001). The storage and utilization of
energy reserves for growth and development of wild and cultured
phyllosoma are still poorly understood, particularly the effects of envi-
ronmental factors on specific energy storage components. The culture
of spiny lobsters through the many and delicate larval development
stages has proved to be difficult. The present study demonstrates that
the physiological energetics of S. verreauxi changes with larval ontoge-
netic development, which is an important consideration for optimizing
spiny lobster propagation success. Successful development of lobster
propagation technologies will further benefit by improved understand-
ing of larval energetic requirements.
Acknowledgments
The authors thank A. Ritar for his valuable input and the rock
lobster propagation technical staff at IMAS, Craig Thomas, Blair
Smith, Julia Hunter, Karl van Drunen, Alan Beech, and Bill Wilkinson.
We also thank the anonymous reviewers for their insightful and valu-
able input. Rock lobster propagation research at IMAS was supported
by the Fisheries Research and Development Corporation Project
(project number: 2006/235) and the Australian Research Council
Linkage Project funding scheme (project number: LP0775480).
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