Mar Biol (2012) 159:455465

DOI 10.1007/s00227-011-1823-3

ORIGINAL PAPER

A new quantitative analysis of ovarian development

in echinoderms: the maturity stage index
Gina M. Doyle Jean-Francois Hamel
Annie Mercier

Received: 12 July 2011 / Accepted: 16 October 2011 / Published online: 2 November 2011
Springer-Verlag 2011

Abstract This study developed an objective quantitative

method for detecting small-scale temporal or spatial differences in gametogenesis in echinoderms. The method
was applied to conventional monthly samples of the
planktotrophic brittle star, Ophiopholis aculeata, collected
at a single site in Newfoundland (eastern Canada) at
1015 m depth. The samples were analysed to determine
gonad index, oocyte size and gonadal stage using histology. The maturity stage index (MSI) was developed to
integrate a measure of brittle star size (disc diameter),
oocyte size and oocyte density. The MSIs ranged from 0 to
800 and had significantly different means among the four
gametogenic stages (early growth, growth, mature and
spent). The MSI was more sensitive in revealing significant
differences between consecutive stages than any of its
individual constituents. The MSI was also applied to
gametogenic data from the lecithotrophic holothuroid,
Mesothuria lactea, again revealing significant differences
between successive oogenic stages. This method is
expected to be useful in field and experimental studies of
gametogenesis in echinoderms (and possibly other taxa),
where it is important to detect not just the timing of annual

Communicated by J. P. Grassle.

Electronic supplementary material The online version of this

article (doi:10.1007/s00227-011-1823-3) contains supplementary
material, which is available to authorized users.
G. M. Doyle  A. Mercier (&)
Ocean Sciences Centre (OSC), Memorial University,
St. Johns, NL A1C 5S7, Canada
e-mail: amercier@mun.ca
J.-F. Hamel
Society for the Exploration and Valuing of the Environment
(SEVE), St. Philips, NL A1M 2B7, Canada

peaks in reproduction but small differences in reproductive

status among individuals or populations (e.g. from different
habitats or feeding regimes).

Introduction
Over the years, many techniques have been developed to
elucidate different aspects of reproductive cycles in marine
invertebrates. While the primary incentive has been to
detect breeding seasons and shifts in spawning peaks
among populations, studies attempting to identify variations at finer scales, down to inter-individual synchrony,
have struggled with the objective quantification of gametogenesis. Common methods used in echinoderms and
other taxa include gonad indices (GI), scanning and
transmission electron microscopy (SEM, TEM), and histology (Giese and Pearse 1974; Mercier and Hamel 2009).
The GI and other indices typically compare the fluctuation
in a chosen index (e.g. ratio of gonad weight on body wall
weight) across the reproductive period under the assumption that changes in the index are attributable to gamete
maturation. One way to confirm this is through standard
histology or more sophisticated EM methods to visually
compare the reproductive organs as they proceed through
the reproductive cycle. Many histological studies base their
analysis on methods developed for various echinoderms by
Yoshida (1952), Fuji (1960), Patent (1969), Fenaux (1970)
and others, who divided male and female gametogenic
cycles into stages based on combinations of characters,
including size, abundance, and organization of gametes,
staining properties, thickness of reproductive tissues (e.g.
gonad wall) and presence of phagocytes. This system has
been modified to fit many species by different researchers
(see review by Mercier and Hamel 2009). Results are often

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presented in tables or graphics showing proportions of

individuals in four to six predefined stages at any given
time.
Studies on reproduction in echinoderms largely focus on
the use of gametogenic staging, commonly supplemented
by gonado-somatic indices and oocyte size frequency distributions (Table S1; Mercier and Hamel 2009) to characterize the gametogenic cycle. Drawbacks have been
identified with the GI because changes in the weight of the
gonad do not always reflect reproductive processes, and
also because of inherent issues with weight measurements
in several marine invertebrates, including echinoderms
(Mercier and Hamel 2009). While qualitative methods and
the GI work well for detecting the periodicity of the
gametogenic cycle, or differences in spawning peaks
between populations, quantifying minute shifts through
statistical comparisons remains difficult. The index developed in this study is potentially useful for the assessment of
inter-individual synchrony on small temporal or geographical scales and/or in experimental trials.
Relatively few studies on marine invertebrates have
tried to circumvent difficulties associated with the qualitative staging system and the GI by developing other
quantitative methods. The most notable examples come
from studies of commercial bivalves. One method commonly used is gamete volume fraction (GVF), defined as
the proportion of germinal tissue containing follicles with
developing and/or mature gametes, or proportions of connective tissue and lumen space. This is determined by
performing histology and analysing the tissues using stereology (Bayne et al. 1978; Lowe et al. 1982; Newell et al.
1982; MacDonald and Thompson 1986). The GVF is most
often estimated visually, with methods such as a pointcount technique (Newell et al. 1982; MacDonald and
Thompson 1986), though newer studies have used imaging
software (Toro et al. 2002). A derivation of the GVF is
seen in Buchanan (2001), who measured three indices in a
mussel, including (1) proportion of histological sections
comprised of follicles, (2) proportion of section comprised
of gametes and (3) proportion of follicles comprised of
gametes. Recently, Gomez-Robles et al. (2005) used digital
imagery to measure lipid and protein indices to indicate
oocyte quality in an oyster. Osada et al. (2007) and Enrquez-Daz et al. (2009) used mitotically active gonial cells
and stereologically measured gonad biomass, respectively.
These methods are quite useful, but the approach of Osada
et al. (2007) entailed extensive manipulation of live animals (injection and later detection of an incorporated
chemical compound) and that of Enrquez-Daz et al.
(2009) was still based on analysis of whole gonad size/
weight using histology and image analysis. In a study of
holothuroids, Singh et al. (2001) used several quantitative
methods to assess reproductive condition, including a

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derivation of the GVF. However, most of his methods are

specific to the study of holothuroids (focusing on tubule
area) and are not applicable to other taxa. Statistical techniques were developed for asteroids by Schoenmakers et al.
(1984) and Grant and Tyler (1986) to delineate the reproductive cycle, but they are not adapted for fine comparative
studies.
The majority of quantitative studies still rely primarily
on oocyte size frequencies to define gametogenic development, although these can be problematic when trying to
distinguish between mature and spent individuals, which
often exhibit the same size range of oocytes due to partial
spawning. In 1952, Yoshida developed a maturity index
(MI) that was adapted by Patent (1969), Tyler (1977) and
Bowmer (1982), among others. The MI is based on the
number of individuals in each of the predefined qualitative
gametogenic stages (usually numbered 16, from recovery
to spent). It calculates the average maturity, based on
which stages are displayed in a given month. This type of
maturity index works well but has the disadvantage of
being based on qualitative gametogenic stages. It is adequate for studies using monthly samples to assess the
overall reproductive cycle, but presents difficulties for field
or experimental studies elucidating differences at finer
scales.
Our goal was to develop a straightforward quantitative
determination of gametogenic development that would
lend itself to robust statistical analysis and be useful for
fine-scale studies of reproductive synchrony at various
levels. Increasing research on the effects of spatial distribution, environmental factors and pheromones on gametogenesis would benefit from such a method. The present
study was carried out during an investigation into the
reproductive cycle of the brittle star Ophiopholis aculeata.
The species was chosen due to its high density and easily
accessible habitat. It is common in the benthic macrofaunal
assemblages around Newfoundland (eastern Canada),
where it occupies rocky substrates of the upper sublittoral
zone (Falk-Petersen 1982; Gosner 1978). Ophiopholis
aculeata lives at relatively high densities ([80 individuals
m-2 at 712 m depth in the Gulf of St. Lawrence;
Himmelman et al. 2008), preferentially under rocks and
shells and inside coralline algae (Fell 1966; Drolet et al.
2004; GM Doyle pers. obs.). Oocytes of O. aculeata have a
maximum diameter of 120 lm and planktotrophic (feeding) larvae (Falk-Petersen 1982; Hendler 1991). Large
variations in its spawning periodicity have been reported,
with spawning presumed to occur sometime between April
and September (Boolootian 1966; Blake 1978; Falk-Petersen 1982).
We determined gonadal development of O. aculeata in
monthly samples using conventional methods (GI, histological stages and oocyte sizes). Then, the different

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variables (disc diameter, oocyte density, various measures

of oocyte size and coefficients of variation) were used in a
number of different maturity stage index (MSI) formulae.
Initial assessment of female gonadal sections of O. aculeata indicated an inverse relationship between oocyte density (decreasing as gametogenesis progresses) and oocyte
diameter (increasing as gametogenesis progresses) in this
species, and that each phase of growth (as defined by
qualitatively determined stages of maturity) was difficult to
characterize based on either of these variables alone. It was
hypothesized that a functional formula would yield significantly different ranges of values for each of the qualitatively determined stages of maturity, thus becoming a
quantitative measure of gametogenic development. This
new quantitative index was subsequently compared with
traditional indices and singular parameters of the formula
(oocyte density, oocyte diameter), to determine whether it
was more sensitive. Finally, the use of the MSI was tested
in the holothuroid Mesothuria lactea to preliminarily
assess whether/how the MSI formula could be adapted to
species with a different reproductive mode. Mesothuria
lactea has large lipid-rich oocytes (to 700 lm) typical of
lecithotrophic larval development that develop synchronously among gonad tubules and asynchronously among
females (Baillon et al. 2011).

Materials and methods

Sampling
Samples of O. aculeata were collected off St. Philips on
Conception Bay, in south-east Newfoundland (4735.5 N;
5253.5 W) via mid-monthly dives from June 2008 to June
2009, with an extra sample at the end of August 2008 in the
midst of the anticipated spawning period. On each occasion, a minimum of 2030 individuals were collected from
an area of small rocks on a low-grade slope at 1015 m
depth, measuring approximately 100 m 9 100 m. Only
sexually mature females (disc diameter C8.5 mm) were
used for the present analysis.
Biometrical analysis
Photographs were taken of each specimen under a Nikon
SMZ1500 stereomicroscope attached to a Nikon
DXM1200F digital camera. Photographs were used to
take two perpendicular measurements of disc diameter
using the imaging software Simple PCI (v. 6.0). Each
individual was then preserved in 4% formaldehyde for a
minimum of 7 days. Gonads were removed using forceps
to extract both gonadal lobes in each bursa (including the
genital rachis).

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Traditional methods
For comparative purposes, all samples were analysed using
two traditional methods of studying gametogenesis: gonad
index and stage determination from histological sections. A
gonad index (GI) was calculated for a monthly minimum of
10 sexually mature females. All gonadal lobes were carefully removed, blotted to remove excess liquid and
weighed. The GI of each individual was calculated as the
per cent wet weight of all gonads to wet weight of the
central disc (with arms removed). Two gonadal lobes (from
the same bursa) were used for histological analysis of each
individual. A preliminary assessment had shown that all
gonads in an individual were in the same developmental
state. Samples were dehydrated in a Leica TP1020 Semienclosed Benchtop Tissue Processor that moved them
through a graded series of baths (70% ethanol, Flex 80 and
95, and 100% ethanol), two baths of Neo-Clear Clarifier,
followed by paraffin infiltration and embedding via two
vacuum-baths of Paraplast. Three 1214 lm sections
were made from each paraffin block (2 gonads 9 3 histological sections each = 6 sections ind-1), which were
mounted and stained using the periodic acid-Schiff (PAS)
method (Junqueira et al. 1986).
Four main oogenic stages were defined: early growth,
growth, mature and spawning/spent, as described below,
based on the work of Patent (1969), Fenaux (1970) and
Falkner and Byrne (2003). Some studies use five or six
stages, typically including an indeterminate stage (often a
post-spawning or recovery stage) or two separate stages for
spawning (i.e. partially spent and post-spawning). However, a four-stage system was determined to work best for
the current study. The indeterminate stage was seen in so
few samples that they were not considered useful for
quantitative analysis and were discarded. In addition, the
population of O. aculeata used in the current study appears
to exhibit partial spawning, so that fully spent individuals
were rare. All samples in any state of spawning were thus
considered in the spent category. The stages used were as
follows:
Stage 1Early Growth (Fig. 1a): The ovaries are very
small, though the gonad walls and germinal epithelium are
at their thickest. Oocytes are generally teardrop shaped,
measuring 1030 lm at the longest axis (maximum Feret
diameter), and are intimately linked with the germinal
epithelium. They are basophilic, staining dark blue/purple
with PAS.
Stage 2Growth (Fig. 1b): Early on, oocytes are still
closely linked to the germinal epithelium, but they are
larger (3050 lm) and more abundant. Most are teardrop
shaped, but some are elongated and spindle shaped. In the
later part of the growth stage, the ovaries become larger
and the gonad wall thinner and less prominent. Some

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Mar Biol (2012) 159:455465

Fig. 1 Oogenic stages in

female Ophiopholis aculeata.
a early growth [with very small
oocytes (O) growing from
germinal epithelium (GE)];
b growth [with first appearance
of early vitellogenic (EV)
oocytes]; c mature [with largest
vitellogenic (V) oocytes];
d spent [with residual
(R) oocytes]. Scale bar
represents 400 lm and applies
to panels ad

oocytes are still closely associated with the germinal epithelium, but most are spread throughout the ovarian lumen.
Oocytes are 3070 lm. Most large oocytes are spindle
shaped or polygonal. Most have begun vitellogenesis, have
a clearly visible germinal vesicle and single nucleolus, and
are much less basophilic, staining light purple with PAS.
Stage 3Mature (Fig. 1c): The ovaries are swollen, and
the gonad wall and germinal epithelium are very thin. The
lumen of the ovary is packed with large vitellogenic
oocytes, with almost no connective tissue visible. Virtually,
all oocytes are fully grown, with a uniformly polygonal or
ovular shape and maximum Feret diameters of 70120 lm.
Very few small oocytes (1050 lm) may occur along the
germinal epithelium. PAS staining imparts a light pink
shade to mature oocytes. This stage is very brief and was
only seen in a few individuals.
Stage 4Spent (Fig. 1d): In the early part of this stage,
the ovary is still swollen, but smaller than when mature.
The ovary contains residual vitellogenic oocytes
(80120 lm) in the process of being lysed by phagocytes,
which are less densely packed than in the mature stage,
with significant gaps between them. As this stage progresses, the ovary is no longer swollen and returns to a
relatively small size, and the gonad wall remains thin.
Maturity stage index
A quantitative measure of oogenesis was developed using
digital photographs of the histological sections obtained

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with the previously described imaging system. The surface

area of each digital micrograph depended on the size and
maturity of the gonad: 0.834 mm2 (at 109), 0.208 mm2
(209) or 0.005 mm2 (409). All complete oocytes present
in the field were counted and measured at the longest axis
(maximum Feret diameter). This process was repeated for
each of the six histological sections from each individual.
As a quantitative measurement of gametogenic development, several formulae were compared which incorporate size and different variables in the gametogenic cycle
which are quantified easily (as per the classical methods).
There is an inverse relationship between oocyte density and
size, and it was clear that these factors should be used
together. Initial assessment of population structure and size
at sexual maturity suggested a wide size range for sexually
mature individuals of this species, based on disc diameter,
so this factor was included in several of the formulae. The
coefficient of variation (CV; ratio of standard deviation of
oocyte diameter to the mean) was also a potential variable,
assessing variability in oocyte size at different gametogenic
stages (high variance would lead to high CV). Also considered were oocyte surface area and square value of
oocyte diameter to test for the potential effect of oocyte
size variation (to determine which representation of oocyte
size is most effective for statistical analysis). These variables were arranged in multiple formulations:
(1)
(2)

Oocyte density 9 oocyte diameter 9 CV 9 0.001

Oocyte density 9 (oocyte surface area 9 0.01) 9
CV 9 0.001

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(3)
(4)
(5)

(Oocyte density 9 size of individual-1) 9 oocyte

diameter 9 CV 9 0.01
(Oocyte density 9 size of individual-1) 9 oocyte
surface area 9 0.01
(Oocyte density 9 size of individual-1) 9 oocyte
diameter2 9 0.01

Oocyte density is the number of oocytes present per

mm2 of ovarian tissue, oocyte diameter is the mean maximum Feret diameter of all oocytes present, size of individual is the disc diameter, oocyte surface area is
p 9 oocyte diameter2, and CV (coefficient of variation)
is SD of oocyte diameter 9 mean-1 9 100. Each of these
maturity stage indices (MSI) was calculated in triplicate for
each individual (from different gonad subsamples)
throughout the sampling period to compare them with
traditional methods of studying gametogenesis. No significant differences in MSI were found among replicates,
indicating that gametogenic development was uniform
within individuals.
Applicability using another species
Ophiopholis aculeata possesses small transparent oocytes
(to 120 lm in diameter) with limited lipid reserves typical
of planktotrophic larval development, which will likely
affect which of the formulae works best to describe
gametogenic development. For comparison with a species
with a different reproductive strategy, data were obtained
on the holothuroid Mesothuria (=Zygothuria) lactea, which
has larger opaque lipid-rich oocytes (to 700 lm) typical of
lecithotrophic larval development. This data set was compiled in a project on reproductive synchrony in deep-water
echinoderms (Baillon et al. 2011) in which M. lactea was
the only species with a different reproductive strategy with
enough samples. Samples were collected from depths of
7981,337 m off the south-west Grand Banks in October
2005. Standard histological and examination techniques
were used on all specimens (as in the present study), and
four maturity stages were defined, analogous to those for
O. aculeata. Oocyte density and the diameters of the first
100 oocytes were measured as previously described. Data
were used to calculate an MSI with the formulae above,
although size of individual in this case was body length in
cm between mouth and anus. Each set of MSI was compared to assess which formula provided the best results.
Statistical analyses
A one-way ANOVA, or its non-parametric counterpart
(KruskalWallis ANOVA on Ranks), was used to determine whether the respective indices would discriminate
among the gametogenic stages (early growth, growth,

459

mature and spent). Conventional methods (GI, oocyte

diameter and oocyte density) were tested in the same
manner. Post hoc analyses were used to compare specific
groups: StudentNewmanKeuls (SNK) for ANOVAs and
Dunns method for ANOVAs on ranks. Pearson and
Spearman correlations were used to investigate the relationships between the different indices. Statistical analyses
were performed with the software package Sigmaplot
(v. 11.0; Systat, Inc.).

Results
The five MSI formulae generated a continuum of values
that effectively translated the progression of oogenesis
from post-spawning to the fully mature stages, but with
variable amplitude. Statistical analysis was used to determine how clearly MSI values obtained with the different
formulae delineated the corresponding qualitative stages.
The most discriminating formula was subsequently used to
compare the MSI with other variables (GI, oocyte diameter
and oocyte density) for its ability to quantitatively define
gametogenic development.
Comparison of MSI obtained with different formulae
All the MSI formulae generated ranges of values with
significantly different means among qualitative gametogenic stages in O. aculeata, but not all of them clearly
distinguished between adjacent stages (Fig. 2). The MSI
calculated with Formula 1 established significant differences between stages (KruskalWallis test, H3 = 94.7,
p \ 0.001), but MSI values between growth and mature
and between mature and early growth stages were not
distinct (Dunns test, p [ 0.05). Results for the MSI
obtained with Formula 2 were similar, with differences
evident among stages (H3 = 124.1, p \ 0.001) and post
hoc tests showing that MSI failed to differentiate growth
and mature as well as spent and early growth stages
(Dunns test, p [ 0.05). While clear distinctions among
stages were obtained using Formula 3 (H3 = 84.3,
p \ 0.001), the post hoc analysis only found differences
between the MSI of the spent stage and all others (Dunns
test, p \ 0.05). Formula 4 was the only one that provided
significant differences among stages (H3 = 157.8,
p \ 0.001) with post hoc tests showing distinctions
between all stages in pairwise comparisons (Dunns test,
p \ 0.05). Formula 5 also provided distinct MSI values
among stages (H3 = 107.3, p \ 0.001), although it did not
differentiate between the spent and early growth stages
(Dunns test, p [ 0.05). Furthermore, results of MSI
comparisons using Formula 4 were significant when all
data points were used, whereas Formula 5 only yielded

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significant pairwise differences when outliers were

removed.
Formula 4 was therefore chosen for comparison of the
MSI with conventional methods of characterizing

Fig. 2 Maturity stage indices (MSI) obtained with different formulae

and corresponding oogenic stages of Ophiopholis aculeata, where EG
is early growth, G is growth, M is mature, and S is spent. Data
presented as mean SE, N = 13101
Fig. 3 Mean MSI f female
Ophiopholis aculeata ( SE;
N = 816) with corresponding
proportion of individuals in
each oogenic stage (early
growth, growth, mature and
spent) throughout sampling
period

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gametogenic maturity. Overall, the quantitative MSI values

projected a well-defined annual cycle that also mirrored the
qualitative staging patterns in monthly samples (Fig. 3).
The following stage equivalencies were found: early
growth = MSI of 100200, growth = MSI of 200400,
mature = MSI of 400800 and spent = MSI of 0100.
The MSI is small for early growth due to the dominance of
very small oocytes. It is larger in the growth stage due to
the proliferation of larger oocytes, though density remains
relatively low. The mature stage has large MSI values due
to the high density of uniformly large oocytes. Most individuals in this stage had MSIs of 400500, though a few
had much larger values, having higher densities and larger
oocytes. The maximum MSI found was *800, representing the highest measurements of oocyte size and density.
The spent stage had a low MSI due to the relatively low
density of oocytes, with residual mature oocytes in the
process of being lysed.
Comparison between MSI and other variables
For O. aculeata, the four gametogenic stages established
visually from ovary sections were well distinguished by the

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461

Fig. 4 Oocyte density (oocytes mm-2), oocyte diameter (lm), gonad

index (% wet weight) and maturity stage index (MSI) across oogenic
stages for Ophiopholis aculeata, where EG is early growth, G is
growth, M is mature, and S is spent. In box plots, horizontal line
shows median, lower and upper limits of boxes represent 25th and

75th percentiles, whiskers represent 10th and 90th percentiles and

outliers appear as open circles. Scatter plot shows mean and SE
(N = 1193). Means with different letters (a, b, c, d) are significantly
different (Dunns or StudentNewmanKeuls, p \ 0.05)

MSI but were not as clearly defined by oocyte density,

oocyte diameter or GI alone (Fig. 4). While there was a
significant difference among stages using oocyte density
(KruskalWallis test, H3 = 92.5, p \ 0.001), the early
growth and growth stages, and the growth and mature
stages did not differ significantly (Dunns test, p [ 0.05).
The same test with oocyte diameter found a significant
difference among stages (H3 = 97.3, p \ 0.001), except
between the growth and spent stages (Dunns test,
p [ 0.05). Analysis of the GI also found significant differences among stages (one-way ANOVA, F3,80 = 29.4,
p \ 0.001), except between growth and mature stages
(SNK, p = 0.132) and between spent and early growth
stages (SNK, p = 0.236). There were generally significant
positive correlations between MSI and GI (Spearman rank
correlation, rs = 0.505, N = 141, p \ 0.001), MSI and
oocyte density (rs = 0.402, N = 223, p \ 0.001), and MSI
and oocyte diameter (rs = 0.677, N = 205, p \ 0.001).
While the GI showed a positive correlation with disc

diameter of individuals (Pearson correlation, r = 0.386,

N = 82, p \ 0.001), the MSI did not (Spearman rank
correlation, rs = 0.119, N = 147, p = 0.155), confirming
that the latter was independent of body size (Fig. 5).
Determination of MSI in another species
All MSI values calculated with the holothuroid M. lactea
exhibited a downward trend as the gametogenic cycle
progressed from early growth to spent (as opposed to
increasing from spent to mature in O. aculeata), due to the
decreased impact of density (fewer larger oocytes) and the
added influence of variance on mean oocyte size. Nevertheless, clear distinctions among gametogenic stages were
obtained when using MSI values calculated with the various formulae. Again, formulae were not equally successful
in delineating adjacent stages. Values of MSI established
with Formulae 1 and 2 worked best in this particular case,
despite not taking individual size into consideration

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Fig. 5 Relationships between disc diameter (mm) and maturity stage

index (MSI; upper panel) and gonad index (GI, % wet weight; lower
panel) in Ophiopholis aculeata, where lines represent linear regressions. For MSI, rs = 0.119, N = 147, p = 0.155; for GI, r = 0.386,
N = 82, p = 0.0004

(Fig. 6). Overall significant differences among stages were

shown for both Formula 1 (one-way ANOVA,
F2,15 = 37.9, p \ 0.001) and Formula 2 (F2,15 = 10.0,
p = 0.002). Post hoc tests confirmed that all stages were
significantly different in pairwise comparisons (SNK,
p \ 0.05). For Formula 3, significant differences among
stages occurred (F2,15 = 10.2, p = 0.002), and post hoc
tests revealed differences between all stages (SNK,
p \ 0.05) except mature and spent (p = 0.230). Formula 4
resulted in a significant difference among stages
(F2,15 = 4.602, p = 0.028), but a post hoc test found no
pairwise differences between any stages (SNK, p [ 0.05).
Formula 5 showed no significant difference among stages
in this species (F2,15 = 3.344, p = 0.065).

Discussion
The maturity stage index (MSI) effectively translated the
progression of oogenesis on a continuous scale, between 0

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Fig. 6 Comparison of maturity stage indices (MSI) defined by

Formulae 1, 2 and 3 across oogenic stages of Mesothuria lactea,
where EG is early growth, G is growth, M is mature, and S is spent.
Horizontal line shows median, lower and upper limits of boxes
represent 25th and 75th percentiles, whiskers represent 10th and 90th
percentiles and outliers appear as open circles (N = 213). Different
letters (a, b, c, d) indicate significantly different MSI (Student
NewmanKeuls, p \ 0.05)

and *800 in the planktotroph O. aculeata and between 0

and *70 in the lecithotroph M. lactea. Values assigned by
the MSI also generated significantly different means for
each of the qualitative oogenic stages. The MSI was more
sensitive than gonad index (GI), oocyte density or oocyte
diameter alone; unlike the MSI, none of these could distinguish between all stages in pairwise comparisons. The
MSI was easily calculated, using the same data as traditional techniques (disc diameter, oocyte diameter and
oocyte density), thus not requiring any increase in workload or development of new methods. In contrast to the
visual assessment of the gonad to which it was compared,
the MSI is a quantitative method based on objective counts
and measurements that are more likely to be consistent
among investigators. Hence, it provides a reliable alternative (quantitative) methodology for studying reproductive
cycles, particularly for studying finer variations in gametogenic maturity, including small-scale spatial differences
and inter-individual synchrony.
Most of the traditional methods of studying gametogenesis are reasonably reliable, especially when used
together, although there are some drawbacks. Gonad
weight does not always reflect gamete maturity, and

Mar Biol (2012) 159:455465

gonado-somatic indices can be affected by intrinsic variability in the chosen denominators. For instance, the variable water content in organs and tissues, or the transfer of
energetic reserves between them, make it hard to standardize the GI. Gonad indices can also be influenced by the
size of the organism, especially since the relationship
between gonad size and body size can change throughout
ontogeny (Devlaming et al. 1982; Hughes et al. 2006;
Mercier and Hamel 2009; Ebert et al. 2011). Histological
studies using gametogenic stages are essentially qualitative
assessments, which make it difficult to quantitatively study
reproductive cycles and to make fine comparisons between
samples. The maturity index (MI) defined by Yoshida
(1952) is a quasi-quantitative analysis, assigning a number
(19 or 16) to each qualitative gametogenic stage in order
to calculate an average. It is useful to assess the overall
gonad maturity state of a group and make broad comparisons. However, it is subjective (based on the users ability
to visually assess which developmental stage each individual is in) rather than a true quantitative measure and
skews the data away from the first and last stages, being
based on the average value. Tyler (1977) used Yoshidas
MI in ophiuroids in conjunction with oocyte counts and the
per cent of individuals in each stage in a given month to
increase the reliability of the analyses. The MSI effectively
consolidates this approach by bringing together, in one
simple formula, parameters that are not as sensitive when
used alone as when they are combined.
Studies that truly quantify gametogenic development in
some way are rare. The gamete volume fraction (GVF)
used in bivalve research is a reliable method for comparing
genders or locations (Bayne et al. 1978; Lowe et al. 1982;
Newell et al. 1982; MacDonald and Thompson 1986);
however, it requires extensive additional analysis with
imaging software, and issues have been identified with its
use (Urrutia et al. 1999). The incorporation of 5-bromo-20 deoxyuridine (BrdU) to detect mitotically active gonial
cells (Osada et al. 2007) is useful but can only be done in
laboratory trials. Enrquez-Daz et al. (2009) measured
gonadal biomass, soft tissue production and spawning
intensity in oysters, a technique that still relies significantly
on the weight and size of whole gonads (similar issues as
with the GI). Singh et al. (2001) used measurements similar
to GVF in holothuroids, as well as gonad dry weight,
tubule area and tubule wall area, and levels of haemal fluid.
Some of these measures are partly qualitative in nature, and
many of them are very specific to holothuroid anatomy.
Statistical approaches were also developed for asteroids:
Grant and Tyler (1986) used residuals and G-statistics to
examine variability in monthly ovarian samples of Astropecten irregularis, and Schoenmakers et al. (1984) measured a complex of variables in the ovaries of Asterias
rubens to make correlations between them. Both methods

463

focused on oocyte size (see below) and were used to trace

the seasonal cycle of gonad maturation. Schoenmakers
et al. (1984) distinguished four gametogenic stages (and
eight substages) based on quantitative and semi-quantitative measurements but were criticized by Grant (1986),
because the main variable measured, oocyte diameter, is
continuous rather than disjoint, so the division into stages is
incorrect because it represents a dissection of a continuum. A key advantage of the MSI is that it quantifies
gonad development on a continuous scale.
One of the oldest and most commonly used quantitative
methods in studies of the reproductive cycle is analysis of
oocyte size frequency distributions, used extensively in
echinoderms (Mercier and Hamel 2009). This measure is
generally a good indicator of gametogenic activity and
spawning, but not always (e.g. Grant and Tyler 1986). The
present work indicated that oocyte size is less sensitive
than the MSI in partial-spawning species such as
O. aculeata. As most species of marine invertebrates do not
completely shed all gametes upon spawning, the lingering
presence of large mature oocytes in the spent stage is a
generalized concern with the use of oocyte size as a sole
parameter.
In the present study, all MSI formulae provided a
quantitative reflection of oogenic maturation, but Formula 4 (using oocyte density, size of individual and oocyte
surface area) was the most discriminating proxy for
oogenic development in O. aculeata and thus was chosen
as the definition of MSI. The MSI represents a simple
multi-parameter approach, in that it takes separate measurements and combines them into a single formula. While
oocyte density and diameter provide some measure of
gametogenic development individually, neither can consistently distinguish each of the gametogenic stages on its
own (i.e. all late gametogenic stages are hard to separate on
the basis of oocyte density, whereas the growth and spent
stages often exhibit similar mean oocyte diameters). The
MSI combines these two variables into a more powerful
quantitative index. Another important advantage of this
formula is that it takes the size of individual females into
consideration. As in many species, this was desirable in
O. aculeata because sexually mature individuals had disc
diameters of 8.520.0 mm (though most were 1014 mm).
The GI was strongly correlated with disc diameter and was
therefore size biased. Using the same data set, appropriate
use of the GI would have required decreasing the number
of samples in order to narrow the size range as indicated by
Blake (1978), Hughes et al. (2006) and Mercier and Hamel
(2009). The MSI incorporated disc diameter; therefore, it
made fuller use of the samples. This can be particularly
useful when sample sizes are low. More importantly, the
wide range of disc diameters in our study represents natural
variation in O. aculeata, so that limiting the size range

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464

could have biased the study towards a select group, instead

of being representative of the entire population.
One parameter that was integrated into several MSI formulae but was found not to be a significant determinant of
gametogenic development for O. aculeata was the coefficient of variation (CV) of the mean oocyte diameter. The
oocytes measured in this study were small (10120 lm), as is
typical of species exhibiting planktotrophic development.
Oocyte size frequencies had a relatively narrow range
(46.0 20.8 lm). Consequently, the CV of mean oocyte
diameter had a relatively small range (mean CV was
32.5 10.7%) that did not make a major contribution to the
MSI relative to other parameters. A review of ophiuroids
(Hendler 1991) indicated that maximum oocyte diameter in
planktotrophic species was 70170 lm with a maximum of
2.4 9 1052.6 9 106 oocytes female-1, whereas lecithotrophic species had a maximum oocyte diameter of
130250 lm and 4 9 1033 9 104 oocytes female-1.
Because lecithotrophic species have fewer, larger oocytes,
greater variations in oocyte size likely occur, making the CV
more relevant for these species (see below).
We hypothesize that the MSI formulated in this study
will provide a sensitive proxy of reproductive activity in
other species with similar types of gonads and oocyte size
frequencies. Furthermore, the MSI can easily be adapted to
species with different reproductive strategies, as demonstrated here with M. lactea. The formulae that worked best
for M. lactea contained a measure of variance, supporting
the hypothesis of CV being relevant for species with larger
oocytes. The fact that Formulae 1, 2 and 3 worked for this
species despite not taking body size variation into account
is likely because the size range of sampled individuals was
relatively narrow (913 cm in length).
This is by no means a definitive study: the MSI formulae
developed for O. aculeata and M. lactea may not apply
directly to other marine invertebrates, although the basic
variables will be the same. Analyses of a variety of species
with small and large oocytes will establish the value of this
novel method. However, the principles can provide a useful
framework for developing similar quantitative methodologies for echinoderms and species in other phyla. The MSI
is not meant to replace established methods that work well
for large-scale studies when combined. The true advantage
of the MSI lies in field and experimental studies that
require statistical analysis of samples collected from different locations within a month or of samples collected
simultaneously within a location (e.g. Doyle 2011). Ideally,
the MSI can overcome some of the limitations of other
methods for quantifying small-scale differences in gametogenic development. For instance, we could have quantified developmental asynchronies in previous studies (e.g.
Hamel and Mercier 1996; Baillon et al. 2011). The
potential benefits of such a system include expediency of

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Mar Biol (2012) 159:455465

processing samples with standard methods, consistent and

easily replicable methodologies, and quantitative determination of gametogenic cycles with minimum skew or bias.
The number of independent gonad samples examined and
the number of oocytes measured in each of them can be
increased to scale up the resolution of the MSI (e.g. to
establish intra-individual variance). Future studies could
also develop variants of the MSI for male specimens. The
present work focused on females because it was more
useful and more easily achievable. Many investigations of
reproductive cycles rely on females through the use of
oocyte size frequency distributions (Mercier and Hamel
2009), which have no direct equivalent in males. Nevertheless, it might be possible to find similar quantitative
features in spermatogenesis (e.g. thickness of the germinal
epithelium and of layers of spermatogonia, spermatocytes
and spermatozoa; abundance of nutritive phagocytes).
Acknowledgments Special thanks to P. Gagnon for logistic
support, guidance and valued help in the field, and to S. Caines,
W. Coffey, R. Guest, R. Hooper, K. Matheson, R. ODonnell,
P. Sargent, and C. Vickers for assistance with field work and sample
collection. Many thanks as well to OSC Laboratory Services, the MI
Centre for Aquaculture and Development, J. Foote, N. Laite, C. Short,
J. So and Z. Sun for technical and moral support and to S. Baillon for
her work on M. lactea. This study was financially supported by grants
from the Natural Sciences and Engineering Research Council of
Canada (NSERC) and the Canada Foundation for Innovation (CFI) to
A. Mercier. It was conducted in partial fulfilment of the MSc degree
of G. Doyle, who wishes to thank examiners for helping to improve
earlier versions of this manuscript. We also thank two anonymous
reviewers for constructive comments.

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