RESEARCH Open Access
Variation in rates of early development in
Haliotis asinina generate competent larvae
of different ages
Daniel J Jackson1,2*, Sandie M Degnan1 and Bernard M Degnan1
Abstract
Introduction: Inter-specific comparisons of metazoan developmental mechanisms have provided a wealth of data
concerning the evolution of body form and the generation of morphological novelty. Conversely, studies of intra-
specific variation in developmental programs are far fewer. Variation in the rate of development may be an
advantage to the many marine invertebrates that posses a biphasic life cycle, where fitness commonly requires the
recruitment of planktonically dispersing larvae to patchily distributed benthic environments.
Results: We have characterised differences in the rate of development between individuals originating from a
synchronised fertilisation event in the tropical abalone Haliotis asinina, a broadcast spawning lecithotrophic
vetigastropod. We observed significant differences in the time taken to complete early developmental events (time
taken to complete third cleavage and to hatch from the vitelline envelope), mid-larval events (variation in larval
shell development) and late larval events (the acquisition of competence to respond to a metamorphosis inducing
cue). We also provide estimates of the variation in maternally provided energy reserves that suggest maternal
provisioning is unlikely to explain the majority of the variation in developmental rate we report here.
Conclusions: Significant differences in the rates of development exist both within and between cohorts of
synchronously fertilised H. asinina gametes. These differences can be detected shortly after fertilisation and
generate larvae of increasingly divergent development states. We discuss the significance of our results within an
ecological context, the adaptive significance of mechanisms that might maintain this variation, and potential
sources of this variation.
Keywords: Developmental variation, Developmental timing, Invertebrate, Competence, Heterochrony, Metamor-
phosis, Dispersal, Plankton, Larva
Introduction
The study and comparison of metazoan developmental
programs has provided biologists with many insights
into the ways in which evolution acts to generate mor-
phological novelty. For example, minor differences in
the genetic instructions that regulate development can
yield an adult phenotype that differs significantly from a
closely related species (for examples see [1-3]). In our
studies with the tropical abalone Haliotis asinina (a
marine gastropod with a biphasic life cycle and
planktonically dispersing larvae), we frequently observe
considerable variation in rates of development within a
cohort of synchronously fertilised gametes [4], a phe-
nomenon that can be observed in many species of indir-
ect developing invertebrates [5].
For many benthic marine invertebrates, dispersal is
achieved by a planktonic larval phase that may or may
not feed [6-10]. Factors that can affect the length of
time spent in the plankton and thus dispersal potential
[11,12] may include variation in larval size [13], growth
[14], maternal investment [11,15] and behaviour [16,17].
Variation in dispersal and larval recruitment are in turn
well known to be mechanisms that can structure adult
populations [18,19]. Related to these observations is the
* Correspondence: djackso@uni-goettingen.de
1School of Biological Sciences, University of Queensland, St. Lucia 4072,
Queensland, Australia
Full list of author information is available at the end of the article
Jackson et al. Frontiers in Zoology 2012, 9:2
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© 2012 Jackson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
fact that many marine invertebrate larvae must be
exposed to specific and ‘patchily’ distributed chemical or
physical cues in order for the processes of settlement
and metamorphosis to be initiated [20-22]. These cues
must often be encountered during a certain develop-
mental state known as ‘competence’ in order for the cue
to engender a response [23-25]. Variation in the rate of
embryonic and larval development during the dispersal
phase, and therefore in the time to acquire competence,
therefore has the potential to affect the likelihood of
encountering a suitable metamorphosis inducing cue in
a patchy environment. Interestingly, it has long been
known for some species that the rate of development is
under genetic control. In Drosophila melanogaster
developmental rate has a selectable and heritable com-
ponent [26-30]; for example after 125 generations of
selection for faster development, Chippendale et al.
established two lines of fruit flies that completed larval
development 25-30% faster than their non-selected con-
trols/ancestors [27]. This acceleration in developmental
time came at a cost, with pre-adult survivorship reduced
by more than 10% relative to controls [27]. More recent
studies have identified some of the genes (for example
Merlin and Karl) linked to this phenotypic variation
[31]. However it is clear that for Drosophila develop-
mental timing is a complex trait influenced by many
pleiotropic genes and environmental interactions. In the
first attempt to do so with a marine invertebrate, Had-
field [32] reported that after 27 generations of intense
selection, lines of faster and slower developing nudi-
branchs (Phestilla sibogae) could not be generated. Due
to the paucity of data, cross species comparisons of the
molecular mechanisms that underlie developmental tim-
ing are not yet possible, however it is clear that environ-
mental factors can directly affect developmental rate,
with temperature perhaps being the best studied and
understood for the widest variety of taxa [33-36].
We previously demonstrated that within a cohort of
synchronously fertilised H. asinina individuals some lar-
vae achieve competence at an earlier age than others
despite uniform environmental conditions [4]. Here we
demonstrate that variation in developmental rate is
manifested during early cell cleavages, with the discre-
pancy between slow and fast developing larvae increas-
ing as development proceeds.
Results
Variation in the time to onset of 3rd cleavage
Variation in the rate at which embryos progressed from
4 to 8 cells (as defined by the number of distinct nuclei)
was observed with the nuclear stain propidium iodide
(Figure 1A-F). Using intervals of 5 min between obser-
vations, we found that a period of 15 min was required
for all sampled embryos to complete the third round of
anaphase, spanning from 75 to 90 min post-fertilization
(Figure 1G). This pattern was observed across three
independent fertilizations.
Variation in the time to hatching
Observing the time taken for every individual larva
(from a cohort of synchronously fertilised eggs) to hatch
from the vitelline envelope revealed a larger degree of
chronological variation. A small proportion of indivi-
duals (2%) hatched from the vitelline envelope by 5.5 h
post-fertilization (hpf), while it took 7.0 hpf for over
75% of individuals to hatch, and 7.5 hpf for every indivi-
dual to emerge from the vitelline envelope (Figure 2).
Variation in the rate of larval shell development revealed
by Has-Ubfm1 expression
The expression of the gene Has-Ubfm1 [GenBank:
DW986191] is detected in the lateral and posterior per-
iphery of the evaginated shell field of young trochophore
larvae (Figure 3 and [37]). Normal shell development is
initiated as a thickening and then an invagination of the
dorsal ectoderm of the trochophore larva. Organic mate-
rial (likely the primordial periostracum) is secreted by
these cells. This dorsal ectoderm then evaginates to
form the shell field which then expands in all directions
concomitantly with calcification of the periostracum and
formation of the larval shell field [38]. Among a sample
of H. asinina larvae of exactly the same chronological
age (10 hpf), a range of stages of shell development can
be observed (Figure 3B-E). In some individuals, Has-
Ubfm1 expression is localized to the periphery of the
recently evaginated shell gland (Figure 3B), and thus
appears as an almost a uniform field of expression. In
other individuals of the same age, Has-Ubfm1 expres-
sion already has expanded such that a well-defined aper-
ture, completely devoid of cells expressing Has-Ubfm1,
can be identified (Figure 3E). While variation in the
intensity of staining patterns generated by in situ hybri-
disation experiments are a well known phenomena for
model organisms, here we are primarily interested in
the spatial variation in gene expression rather than
quantitative variation.
Variation in late larval development - variable rates of
metamorphosis amongst larvae of the same age
Significant variation in the percentage of larvae of the
same age initiating metamorphosis (as observed by the
initiation of post-larval shell growth) was observed after
exposure to the inductive crustose coralline algae (CCA)
Mastophora pacifica [4,39]. We commonly employ this
method of scoring metamorphosis because more
immediate responses to settlement inducing cues (such
as velar abscission) are highly unreliable [4]. Because
unambiguous post-larval shell growth takes at minimum
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Figure 1 Variation in the rate of third cleavage revealed by nuclear staining. (A - F) Propidium iodide staining of early H. asinina embryos
allowed the number of discrete nuclei to be accurately determined. All views are lateral except the inset in (B) which is from the animal pole.
(A) A four-cell embryo with two distinct nuclei in the focal plane (four nuclei in total). (B) A morphologically similar embryo to that shown in (A)
with four nuclei in the focal plane. When the same embryo is viewed from the animal pole (inset) eight nuclei are visible. Embryos shown in (A)
and (B) are of the same chronological age. (C) Following cytokinesis, eight distinct cells can be seen with four of the eight nuclei visible in this
focal plane. (D-F) Bright field views of the corresponding embryos as in (A-C). (G) Embryos from three unique fertilizations were monitored at
five-minute intervals for the time taken to progress from four discrete nuclei to eight discrete nuclei. Each point represents a minimum of 20
observations
Jackson et al. Frontiers in Zoology 2012, 9:2
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several hours post-induction to observe, we report our
results as the proportion of animals metamorphosed
post-induction (Table 1 and Figure 4). The total larval
age can be calculated by adding the time post induction
to the age at induction. We have conducted experiments
like these across multiple spawning events and multiple
spawning seasons and consistently see the same pattern
- some larvae are able to commence metamorphosis sig-
nificantly earlier than others [4]. In this experiment,
37% of larvae aged 60 hpf at the time of induction (72 h
total age) had initiated metamorphosis 12 h after induc-
tion. In contrast, 83% of larvae aged 96 hpf at the time
of induction (108 h total age) showed signs of having
initiated metamorphosis 12 h after induction (Table 1
and Figure 4).
Discussion
In H. asinina, as in other marine invertebrates, intra-
cohort variation in rates of settlement and metamorphosis
are commonly observed [5,23]. We have previously shown
that larval age is not a good predictor of whether an indivi-
dual will initiate metamorphosis when presented with an
appropriate cue [4]. Here we show that variation in rates
of embryogenesis and larval development may explain a
-10
0
10
20
30
40
50
60
70
80
90
100
370 380 390 400 410 420 430 440 450 460
%
 o
f l
ar
va
e 
ha
tc
he
d
Time post fertilisation (minutes)
0
2
19
76
94
100
60 minutes
Figure 2 Variation in the age at hatching from the vitelline
envelope. The age at which larvae derived from a single and
synchronous fertilisation event hatched from the vitelline envelope
was monitored in 15 minute intervals. Six replicates of 20 larvae
from this fertilisation were monitored. Error bars are one standard
deviation of the mean
Figure 3 Variation in rates of larval shell deposition between larvae of the same age (10 hpf) as revealed by in situ hybridisation
against Has-Ubfm [GenBank:DW986191]. (A) An SEM image of a H. asinina trochophore reveals the position of the shell field (sf). (B - D)
Representative variation in the spatial expression of Has-Ubfm
Table 1 Proportion (% ± SD) of metamorphosed H.asinina following induction by the crustose coralline algae
Mastophora pacifica as a function of age
Hours after induction
Age at induction (hours) 12 24 36 48 60
36 0.0 ± 0 0.0 ± 0 13.9 ± 22 58.4 ± 15 82.5 ± 20
42 0.0 ± 0 0.0 ± 0 62.9 ± 20 78.9 ± 15 92.1 ± 5
48 0.0 ± 0 39.5 ± 24 64.9 ± 8 89.3 ± 4 92.9 ± 5
54 0.0 ± 0 65.7 ± 21 73.8 ± 10 93.4 ± 8 95.4 ± 5
60 37.1 ± 23 70.2 ± 13 88.8 ± 5 96.4 ± 4 100.0 ± 0
66 57.6 ± 18 76.6 ± 12 93.5 ± 6 96.4 ± 3 98.0 ± 3
72 67.4 ± 21 92.5 ± 6 95.1 ± 5 98.3 ± 3 100.0 ± 0
84 76.1 ± 15 95.4 ± 5 97.0 ± 3 100.0 ± 0 100.0 ± 0
90 81.3 ± 6 92.1 ± 4 97.3 ± 3 98.2 ± 3 99.2 ± 2
96 83.0 ± 9 96.4 ± 3 98.4 ± 3 100.0 ± 0 100.0 ± 0
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proportion of the variation in the age at which competence
is acquired, and hence the typically variable rates of meta-
morphosis observed within and between larval cohorts of
the same age. Variation in the chronology of early embryo-
logical and larval developmental events (early cell divisions,
hatching, rate of larval shell deposition) suggests that from
fertilisation, idiosyncratic rates of development are present
within a single cohort of synchronously fertilised, and
genetically related (full sibling) H. asinina larvae. Differ-
ences in the rate of development produce larvae that dis-
play broader and broader ranges of developmental states
as ontogeny proceeds; individuals complete early develop-
mental events (3rd cleavage and hatching) within narrower
time spans (15 min and 60 min respectively) than the time
taken for all individuals to undergo metamorphosis (36+
h). Because we have used a variety of non-equivalent char-
acters (cleavage, hatching, gene expression and compe-
tency) at different time points we have not attempted to
quantify this increasing discrepancy through development.
To do this in a meaningful way, a character (ideally sev-
eral) that can be accurately quantified on individual larvae
at all stages of development first needs to be identified.
Additionally, with the methods we have employed we can-
not exclude the possibility that larvae which initially
develop rapidly, may slow their rate of development and
be among the last to acquire competence. Nonetheless the
final outcome is the same; the total discrepancy between
chronological age vs. developmental stage broadens as
development proceeds. These results should also be con-
sidered in light of the fact that early developmental events
are completed in less time than later events; a single cell
division (third cleavage) takes on the order of minutes to
complete, while the acquisition of competence requires
hours to develop. The endpoint to this discrepancy is
hinted at in the time taken for all larvae in a synchronously
fertilised cohort to complete metamorphosis; given enough
time, all larvae will eventually become competent to settle
and metamorphose, as suggested by the asymptote-like
characteristic of the curves in Figure 4. Conceptually
extending these results to the field (albeit in the presence
of unrealistic uniform oceanographic processes) would see
larvae that take more time to attain competence settle and
metamorphose a greater distance from the parent reef
than larvae that develop more rapidly.
Although we provide evidence for variation in rates of
development among full siblings of H. asinina, we can-
not account for the mechanism that generates it. One
potential epigenetic source could be variation in mater-
nal provisioning. Indeed, such maternal effects have
been shown to significantly influence larval and post-lar-
val performance. For example Marshall and Keough
demonstrated that larval size, a rough proxy for mater-
nal provisioning in lecithotrophic larvae, can influence
survival to adulthood and post-metamorphic growth
[11]. However these affects diminish following metamor-
phosis and depend upon the environmental context the
experiment is conducted in. Furthermore, that study
examined larvae of mixed parentage, and investigated a
species whose larval size (volume) varies by as much as
2.5 fold [11]. By comparing full siblings (see materials
and methods) we reduce the impact of such maternal
effects. Furthermore, eggs spawned by H. asinina vary
in their diameter by less than 1.2 fold (1.2, 1.1 and 1.1
fold for three independent cohorts). We also demon-
strate that much of the calculated egg volume variation
is the result of systematic error (Table 2). If we assume
that variation in maternal provisioning across a cohort
of H. asinina gametes is minimal (at least significantly
less than in those species for which maternal provision-
ing is known to affect larval and juvenile performance),
then what is the source of the variation we report here?
While our data cannot identify the source of this varia-
tion, it is tempting to speculate that a proportion of it
may be genetically encoded, as recently suggested for a
gastropod by Tills et al. [40]. Indeed variation in growth
is known to be a heritable trait for juvenile H. asinina
[41], a phenomenon that poses a significant challenge
for efficient aquaculture practises. We propose that this
post-metamorphic variation is simply an extension of
the pre-metamorphic variation in developmental rate
that we report here. Importantly, any mechanism that
produces a cohort of larvae that develops at varying
%
 a
ni
m
al
s 
m
et
am
or
ph
os
ed
Hours after induction
Age at induction (hpf)
0
10
20
30
40
50
60
70
80
90
100
6 18 30 42 54 66
36
42
48
54
60
66
72
84
90
96
Figure 4 Variation in the percentage of larvae of the same age
initiating metamorphosis following induction by the crustose
coralline alga (CCA) Mastophora pacifica. Larvae of various ages
(36 - 96 hpf) were induced to metamorphose and their response, as
indicated by postlarval shell growth, monitored at 12 h intervals
from 12 to 60 hours after induction. Error bars are omitted for clarity
of presentation. Note that the response to induction across all ages
is never uniform. i.e. some larvae are able to respond to the CCA
inductive cue faster than others
Jackson et al. Frontiers in Zoology 2012, 9:2
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rates may have significant ecological consequences as it
could provide a means of ensuring that progeny will
attain competency at variable times and distances from
the location of conception. The developmental state in
which a given larva encounters a suitable inductive cue
can have a profound impact on the process of metamor-
phosis and post-metamorphic performance. For abalone
if it is encountered too early, a habituation-like response
is generated [4,42], and may result in subsequent
encounters with a suitable cue being ignored. If it is
encountered too late, post-metamorphic growth and
survival is compromised [43]. In the case of H. asinina
it could therefore be advantageous to generate a cohort
of larvae that encounter suitable settlement environ-
ments in a range of developmental states at varying dis-
tances from the point of inception. The observation that
larvae from various phyla display variable rates of meta-
morphosis suggests that this strategy may be commonly
employed [23,44,45]. If the source of this variation is in
part genetically encoded, the identification of the mole-
cular mechanisms that generate and/or maintain this
variation would be an exciting contribution to our
understanding of the way in which marine invertebrates
are able to colonise patchily distributed habitats. Identi-
fication of the genes that encode hatching enzymes,
receptors and/or signal transducers for metamorphosis
inducing cues and other stage specific molecules would
be a first step towards this goal.
More than 35 years ago Strathmann reviewed the phe-
nomenon of variation (spread) among cohorts of sibling
marine invertebrate larvae, and concluded that “...there
is probably a bias in the literature against records of
variability in behaviour or rates of development. Most
marine biologists have been unaware of the possible
adaptive significance of spreading siblings, so the inves-
tigator is likely to note only the mode, mean, minimum,
or maximum, depending on which is felt to represent
natural conditions.” [5]. Since this time mechanisms of
larval dispersal and recruitment have received increased
attention because these processes are known to directly
affect adult population structures (for example see
[46,47] however see [48,49] for exceptions). Variation in
the timing of competence acquisition could serve to
increase the spatial selection by larvae of distinct settle-
ment sites, thereby minimising the chances of extinction
due to local catastrophes [50-52]. While the data we
present here may play a role in dispersal (and other)
processes, we cannot say to what degree they do so.
Many other factors are well known to affect the disper-
sal patterns of broadcast spawned gametes, for example
currents and tidal movements [53], maternal provision-
ing [54] and paternal affects [55]. To quantify the con-
tribution that variable rates of development might make
to the fitness of planktonically dispersing organisms
remains an exciting challenge for future marine ecolo-
gists, population geneticists and developmental
biologists.
Conclusions
Using a variety of methods we have demonstrated that
substantial differences in the rate of developmental can
be observed between individual H. asinina larvae origi-
nating from a synchronous, full sibling fertilisation
event. The discrepancy between developmental state and
chronological age appears to increase as development
proceeds, and is likely to be a factor that contributes to
the variation in rates of metamorphosis commonly
observed within and between similar aged cohorts of
abalone larvae.
Methods
Production of embryos and larvae
All gametes were produced from natural spawnings of
H. asinina following methods previously described
Table 2 Estimates of inter- and intra-cohort egg volume variation in H.asinina and systematic error
n Mean diameter
(μm)
Std. dev.
(μm)
Min/Max
(μm)
Mean vol.
(μm3)
Std. Dev.
(μm3)
Std. Error 1
(μm3)
Systematic
error (%) 2
Cohort 1 measurement 1 46 136.0 4.2 120.0/144.4 1,321,897.9 117,418.7 35.6
Cohort 1 measurement 2 46 133.5 4.5 114.0/144.4 1,249,694.1 121,253.2 34.5
Cohort 1 measurement 3 46 133.5 4.2 117.2/141.1 1,249,108.2 112,218.9 41,857.1 37.3
Cohort 2 measurement 1 25 140.9 3.6 132.0/146.2 1,466,635.5 112,840.8 31.4
Cohort 2 measurement 2 25 138.9 2.9 132.0/144.8 1,404,027.4 88,846.1 39.9
Cohort 2 measurement 3 25 138.9 4.2 129.2/148.5 1,406,434.3 126,670.2 35,472.4 28.0
Cohort 3 measurement 1 27 138.0 4.7 129.7/147.6 1,381,673.6 142,105.0 15.8
Cohort 3 measurement 2 27 136.6 3.6 131.0/143.9 1,336,833.8 105,865.6 21.2
Cohort 3 measurement 3 27 137.3 4.7 126.4/146.2 1,360,755.8 136,909.9 22,436.7 16.4
1 The standard error is the standard deviation of sample means reported in column 6 (Mean vol.)
2 The systematic error is the fraction of the standard deviation accounted for by the standard error (i.e. the variation obtained by measuring the same eggs 3
times as a proportion of the estimated variation in egg volume). This was calculated as the Std. Error (column 8) divided by the Std. Dev. (column 7) expressed
as a percentage. Note that these values can only be underestimates of the total systematic error, additional sources of error will inflate these values
Jackson et al. Frontiers in Zoology 2012, 9:2
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[4,56]. Gravid broodstock were maintained in individual
spawning aquaria in order to control for the effects of
parentage (and therefore differing nutritional histories
between individuals) and timing of each fertilisation
event. Six mature individuals (3 females and 3 males)
were used to perform three unique and independent fer-
tilization events. Sperm was added to unfertilized eggs,
gently but thoroughly mixed, allowed to stand for 1
minute, and was then thoroughly washed out to reduce
the chances of delayed fertilization events. By minimis-
ing the insemination time in this way, non-synchronous
fertilisation events (± 1 min) were eliminated. For each
experiment described, a sample of unfertilized eggs were
set aside from each spawning event to monitor for signs
of cleavage as an indicator of unsolicited fertilization.
Zygotes from each fertilization were maintained in sepa-
rate aquaria as previously described [4,41].
Measuring variation in the rate of 3rd cleavage
Embryos from each of the three fertilization events were
allowed to develop for 60 min after which approximately
100 individuals were placed in 4% paraformaldehyde in
0.2 μm filtered seawater (FSW) for approximately 10
min. Embryos were then briefly washed with FSW 3
times, phosphate buffered saline (PBS) 3 times, and then
stored in PBS with 0.1% Tween (PBTw) at 4°C until
further processing. This sampling procedure was
repeated every 5 min from 60 to 100 min post fertiliza-
tion (mpf). Nuclei were subsequently stained with 10
μg/ml propidium iodide in PBS for 0.5 - 2 h at room
temperature, and were washed with PBTw 3 times over
30 min. Embryos were mounted in 60% glycerol in PBS
and viewed under standard fluorescent optics. For each
time point a minimum of 20 embryos from each fertili-
zation were viewed laterally and from the animal pole
and scored for the number of discrete nuclei.
Variation in the time taken to hatch
Six replicates of 20 synchronously fertilized, normally
cleaving embryos derived from a single cross, were
reared in 10 ml FSW in a 6 well tissue culture dish at
approximately 28°C for 6 h. Observations were then
made every 15 minutes for hatched, swimming larvae.
At each time point, all hatched and swimming larvae
were counted and removed, with care taken to avoid
physical disturbance of unhatched larvae.
Variation in spatial gene expression during development
A genetic marker was used to monitor the variation in
development of H. asinina trochophores. Following an
EST screen for genes involved in shell formation
[37,57], we uncovered a gene (Has-Ubfm1, GenBank
accession number DW986191) that is expressed in the
developing shell field of the trochophore larva. Using an
in situ hybridisation probe against Has-Ubfm1 we found
we could make relative comparisons of larval shell
development.
For each whole mount in situ hybridization (WMISH)
experiment thousands of actively swimming, morpholo-
gically normal larvae from a synchronous fertilization
event derived from a single cross were collected and
fixed and processed as previously described [57].
Although the aim of these experiments was not to quan-
titate gene expression, the colour reaction was termi-
nated synchronously to allow direct comparisons
between individual larvae to be made. Larvae were dehy-
drated through an ethanol series, mounted and cleared
in benzyl benzoate:benzyl alcohol, and viewed under
Nomarski optics with an Olympus DP10 digital camera
connected to a BX40 microscope.
Settlement assays
Settlement assays were performed as described in [4].
Briefly, embryos derived from a single cross were
allowed to develop in 10 litres of static 5 μm filtered
seawater. Hatched and normal swimming larvae were
collected approximately 9 h later and transferred into a
300 mm diameter culture chamber with a 190 μm
screen floor and flowing 5 μm filtered seawater. Larvae
were maintained in this way until used in settlement
assays. Beginning at 36 hpf and at 6 h intervals there-
after up to 96 hpf, a sample of 120 larvae was divided
into 6 replicates (20 larvae/replicate) and were exposed
to the CCA identified as Mastophora pacifica [4,39].
Careful attention was paid to ensure that every larva
contacted the CCA when introduced into the experi-
ment. Observations of metamorphosis were made 12 h
after induction at 12 h intervals to 60 h after induction.
Larvae were visually inspected under a dissecting micro-
scope for signs of postlarval shell growth, an unambigu-
ous morphological marker of metamorphosis [4].
Egg volume measurements
Three cohorts of unfertilised eggs from 3 separate
mothers were collected and photographs of individual
eggs taken using an Olympus DP10 digital camera con-
nected to a BX40 microscope. Individual egg diameters
were measured independently 3 times using Macnifica-
tion v1.7.1 [58]. Means, standard deviations and stan-
dard errors were calculated using Excel for Mac 2008 v
12.2.4.
Acknowledgements
We are grateful to the staff of Heron Island Research Station for their
assistance with collection and maintenance of abalone broodstock. The
Bribie Island Aquaculture Research Centre provided tanks and seawater
systems with which experiments were conducted. We thank Kathryn Green
for the SEM image. This research was support by Australian Research Council
grants to DJJ, SMD, BMD, and Deutsche Forschungsgemeinschaft funding to
Jackson et al. Frontiers in Zoology 2012, 9:2
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DJJ through the University of Göttingen Excellence Initiative. The
constructive criticisms of several anonymous reviewers also greatly
contributed to the improvement of this manuscript.
Author details
1School of Biological Sciences, University of Queensland, St. Lucia 4072,
Queensland, Australia. 2Courant Research Centre Geobiology, Georg-August
University of Göttingen, Goldschmidtstr.3, 37077 Göttingen, Germany.
Authors’ contributions
DJJ conducted the experimental work and drafted the manuscript. DJJ, SMD
and BMD conceived the study and revised the manuscript. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 3 December 2011 Accepted: 17 February 2012
Published: 17 February 2012
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Cite this article as: Jackson et al.: Variation in rates of early
development in Haliotis asinina generate competent larvae of different
ages. Frontiers in Zoology 2012 9:2.
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