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Marine Biology 104, 275-289 (1990)

@ Spr[nger-Verlag 1990

Annual reproductive cycles of the commercial sea urchin

Paracentrotus lividus from an exposed intertidal
and a sheltered subtidal habitat on the west coast of Ireland
M . Byrne *
Department of Zoology, University College Galway, Galway, Ireland

Reproduction of the commercial sea urchin Paracentrotus
lividus (Lamarck) from contrasting habitats on the west
coast of Ireland was examined from May 1986 through
August 1988. Urchins were collected intertidally from an
exposed rocky shore and subtidally from a protected bay.
Monthly measurements of the gonad index and histological
examination of the gonads demonstrated that P. lividus has
an annual reproductive cycle. Although the two populations
exhibited similar reproductive patterns over three breeding
seasons, there were inter-annual and inter-population differences in the amplitude of gonadal growth and in the time
when spawning started. The subtidal urchins had significantly larger gonads and exhibited a longer period of reproductive maturity compared with the intertidal urchins. This
difference was also evident in the histology of the ovaries at
the beginning of breeding, when most of the subtidal females
contained mature ovaries, whereas most of the intertidal
females contained premature ovaries. An inter-annual difference in the onset of spawning was observed, with the start
of gamete release differing by as much as four weeks between years. It appears that inter-annual differences in sea
temperature influence the temporal variation in spawning
by P. lividus and that increasing temperature may serve as
an exogenous cue for gamete release. The inter-annual variability in the onset of spawning suggests that photoperiod
does not cue gamete release. Gonadal growth, on the other
hand, occurs during the coldest months of the year and during the period of shortest days, suggesting that temperature
and photoperiod may both influence gonadal growth during
the winter. Oogenesis and spermatogenesis of P. lividus were
examined histologically. During the post-spawning recovery
and growth stages, the gonads gained weight through
growth of the nutritive phagocytes and the accumulation of
periodic acid Schiff (PAS)+ droplets by these cells. The
* Present address: School of Biological Sciences, Zoology AO8,
University of Sydney, Sydney, New South Wales 2006, Australia

PAS + material appears to play a nutritive role in gametogenesis. For the females, the frequencies of six ovarian maturity stages was assessed at approximately monthly intervals.
Small oocytes were present throughout the year and clusters
of early oocytes were most apparent during the spent and
recovery stages. With the onset of vitellogenesis and subsequent accumulation of ova, the nutritive phagocytes and
their PAS + droplets became depleted. The ovarian condition at the onset of breeding was variable, due to differences
in the number of vitellogenic oocytes, differences in the
number of ova in storage, and differences in the amount of
PAS + material. In general, the nutritive phagocyte tissue is
reduced by the onset of spawning and is exhausted by the
termination of breeding. A similar series of events occurs
during spermatogenesis. The relevance of this study for the
use of P. lividus as a brood-stock organism for mariculture
is discussed.

The sea urchin Paracentrotus lividus (Lamarck) has a boreomediterranean distribution (Mortensen 1943, Southward
and Crisp 1954) inhabiting intertidal rock pools and shallow
subtidal habitats. In Europe, P. lividus has been an important source of gametes for embryological research since the
turn of the century (Czihak 1971) and it is also of commercial importance, with a high market demand for its roe,
particularly in France. Destructive harvesting methods employed in France caused the collapse of the P. lividus fishery
in the 1970's with a complete disappearance of urchins from
areas of former abundance (Allain 1975, Southward and
Southward 1975, R~gis et al. 1986). An Irish fishery for
P. lividus supplied the French market until recent times, but
now, after 20 yr intensive fishing, this fishery has largely
ceased due to the depletion of commercial stocks (own personal observation). The depletion of wild stocks of P. lividus
and the lucrative market for this urchin have generated

interest in the potential of P. lividus for mariculture (R6gis
1980, Birais and Le Gall 1986).
There are several studies on the pattern of gonadal
growth of Paracentrotus lividus. An annual cycle of gonadal
growth is documented for Mediterranean populations of
P. lividus, with two spawning periods each year (Fenaux
1968, R6gis 1979). A single and prolonged spawning period
is reported for P. lividus in Brittany (Dominique 1973). In
Ireland, both single and double spawning periods are reported for the species (Crapp and Willis 1975, Willis 1976).
Despite the economic importance of P. lividus, there is a
paucity of information on the cellular events of gonadal
development in this echinoid.
The aims of this investigation were to determine the
annual cycle of gonadal growth of Paracentrotus lividus
from contrasting habitats on the west coast of Ireland and
to document gametogenesis through histological examination of the gonads. Particular attention was paid to the
development of nutritive, non-gametogenic tissue of the gonads which is the major component of the gonads during the
fishing season. Three breeding seasons were studied to establish the regularity and amplitude of gonadal growth between years and between populations. The influence of environmental seasonality on reproduction of P. lividus was also
examined. This study is intended to constitute a guide to the
use of P. lividus as a brood-stock organism for mariculture.

Materials and methods

Reproduction of Paracentrotus lividus (Lamarck) was examined from May 1986 through August 1988 at two sites on the
west coast of Ireland. At Ballynahown (93 I'W; 5313.4'N),
urchins were collected from two adjacent mid-shore tide
pools. At the second site near Glinsk Pier (950.7'W;
5322.4'N), urchins were collected from depths of 3 to 5 m
with the aid of SCUBA. The two study areas are separated
by 30 km and represent contrasting habitats. Ballynahown
is an exposed rocky shore and the tide goes out beyond the
urchin pools most days of the year. The pools were devoid
of macroalgae and had a pink colour due to a crust of
coralline algae lining the substratum. At this site, the urchins
fed on drift sea weed and covered themselves with ensnared
pieces of Fucus and Laminaria spp. In contrast, Glinsk lies
in a sheltered part of Bertraghbouy Bay and the urchins
were only exposed during the lowest spring tides, two to
three weeks a year. At this site the urchins were located in
a kelp bed under and around Laminaria-covered boulders.
Thirty Paracentrotus lividus were collected from each site
at 4 to 6 wk intervals from May 1986 through December
1987 and from February 1988 through August 1988. Their
reproductive cycle was monitored by the gonad index method and by histological examination of the ovaries. Sex determinations were made on freshly dissected specimens by examining the gonads for extruded gametes. From each
urchin, four gonads were dried and the fifth was preserved
in Bouin's fluid. The viscera and test were also dried. After
drying to constant weight, the gonads and the body were

M. Byrne: Reproduction of Paraeentrotus lividus

weighed. The total dry-weight of the gonads was estimated
by correcting for the one-fifth portion removed and the
gonad index (GI) was calculated as:
dry weight gonads x 100%
total dry weight
Inter-annual and inter-population differences in the gonad
indices were analysed by Student's t-test.
The test diameter of each urchin was measured to the
nearest millimeter. The two populations of Paracentrotus
lividus differed in their size, with the maximum test diameter
of the subtidal urchins (ca. 80 mm) being larger than that of
the rock pool urchins (ca. 50 ram). Urchins collected from
Ballynahown had test diameters ranging from 30 to 50 mm,
while those from Glinsk had diameters ranging from 50 to
65 ram. The size range of the urchins collected from each site
was made as small as possible to avoid the influence that
urchin size may exert on the gonad index (Gonor 1972), and
reflects the sizes of urchins that were readily available.
The temperature of the water in the tide pools was measured at each sampling date, and the sea temperature at
Glinsk was measured at high tide at 2 to 4 wk intervals.
Times of sun-rise and sun-set, obtained from the Dunsink
Observatory, Dublin, were used to estimate day length.
The preserved gonads were dehydrated, embedded in
paraffin and sectioned at 7 #m. The sections were stained
with haematoxylin and eosin (H/E) and with the alcian blueperiodic acid Schiff reagent (AB/PAS) method. Initial examination of sections through entire ovaries (n = 20) revealed
that the gametogenic condition was homogeneous throughout the gonad. Thereafter, only the middle third portion of
the ovaries was sectioned. At approximately monthly intervals, the oogenic condition of ten females from each site was
assessed. The ovaries were categorized according to morphologically defined criteria on a scale of I to 6, modified
from the staging methods used by Fuji (1960a), Chatlynne
(1969) and Pearse (1969). This method was also used to
describe spermatogenesis.
Information on the size frequencies of the oocytes was
obtained by measuring with an ocular micrometer the diameter of the first 50 oocytes encountered in each ovary.
These included primary oocytes sectioned through the nucleolus and ova sectioned through the nucleus. For nonspherical oocytes, the eggs were measured along their
longest and shortest axes and the sum of these measures was
halved to calculate the diameter. Relict oocytes exhibiting
obvious signs of degeneration or resorption were not measured. Data from ten females (500 oocytes/sample) were
pooled and used to determine the oocyte size-frequency distributions.


Gonad index (GI)

The GI of Paracentrotus lividus undergoes an annual cycle
and the pattern of gonadal growth was similar over the three

M. Byrne: Reproduction of Paracentrotus lividus


Table 1. Paracentrotus lividus. Sea Water temperatures and day

length before and after onset of spawning at Ballynahown (intertidal) and Glinsk (subtidal). "Date", is when maximim gonad index
(GI) was recorded and when pre-spawning temperatures and day
lengths were recorded; the post-spawning temperatures and day
lengths were recorded when a decrease in GI due to spawning was
first noted, approximately I m after date listed in first column
and date

Temperature (C):

Day length (min):

after initial
spawning spawning

after initial
spawning spawning


10 i







19 June 1986
1 May 1987
16 May 1988
20 June 1986
9 June 1987
1 June 1988





M.J. J. A.S,O.N.D.J. F.M.A.M.J. J. A.S.O.N.D.J. F.M.A.M.J. J. A.S.O.











Table 2. Paracentrotus lividus. Maximum gonad indices of female

and male urchins from Ballynahown and Glinsk. Standard errors in
Location and date




22 May 1986
1 May 1987
16 May 1988

7.3 (0.65)
8.8 (0.36)
7.7 (0.42)

7.2 (0.48)
9.3 (0.53)
7.7 (0.50)

7.2 (0.30)
8.3 (0.29)
7.6 (0.31)

20 June 1986
10 May 1987
1 June 1988

10.9 (0.52)
9.0 (0.55)
10.7 (0.75)

11.1 (0.97)
8.6 (0.47)
9.3 (0.51)

11.0 (0.46)
8.5 (0.33)
10.0 (0.45)


A:M: J. J. A. S: O . N . D . J . F . M . A . M . J . J . A . S . O .





M.' J.' J.' A: S.'O: N.' D.' J.'F.'M.' A:M: J.' J. ' A.' S.' O: N.' D.' J.' F.'M.' A~M.' J.' J.'A.' S.' O.

breeding seasons examined (Fig. ] a). In both populations,

the GI had a pre-spawning peak in early summer ( M a y June) and declined to a minimum after the final spawn-out
in late summer (August-September). Thereafter, the index
increased during the recovery and growth stages with the
gonads gaining weight over the winter and spring ( O c t o b e r April). Although the overall pattern was consistent, there
were inter-annual and inter-population differences in the
For the intertidal population at Ballynahown the pattern
of gonadal growth was similar over the three breeding seasons, but the time when spawning started, marked by a drop
in the GI, differed between years (Fig. 1 a; Table 1). In 1987
and 1988, spawning of the rock-pool urchins began between
1 May and 1 June and between 16 M a y and 20 June, respectively. In 1986, however, spawning began between 19 June
and 23 July (Table 1). Although the sampling intervals do
not provide precise information on the inter-annual differences in spawning, it is clear that spawning in 1986 began
approximately a month later than spawning in the subsequent two summers. This was verified by histological examination of the gonads which also showed that the GI decrease in March 1987 did not reflect a spawning event.

Fig. 1. Paracentrotus lividus. (a) Annual reproductive cycles of sea

urchins from Ballynahown and Glinsk, west coast of Ireland, May
1986 to August 1988; data points are means, bars show SD,
n = 30. (b) Surface sea-temperature measured at Glinsk, data points
indicate temperatures of rock pools at Ballynahown when these
differed from sea temperature at Glinsk. (c) Day length

Spawning in the intertidal was episodic over the summer

(Fig. 1 a). The GI of the rock pool urchins did not differ
significantly between years, with minima ranging from 2.8 to
3.2 and maxima ranging rom 7.2 to 8.3. Comparisons of the
G I of female and male urchins from the intertidal indicated
that the GI maxima of the two sexes did not differ (Table 2).
For the subtidal population at Glinsk (Fig. I a; Table i),
the onset of spawning in 1986 and 1988 was marked by a
distinct GI maximum followed by a sharp drop in the index
value, whereas in the breeding season of 1987 the GI did not
exhibit a distinct peak. The GI stayed at the same level from
April through July, 1987 and the major spawning event occurred in July. In 1986, spawning started between 20 June and
30 July and in 1988 spawning started between I and 26 June
(Table 1). These spawning times were verified by the histo-

logical condition of the gonads (Fig. 2f, g, 30. For 1987,
histological examination of the ovaries revealed that gamete
release started in June. Spawning in the subtidal was
also episodic over the summer. The minimum GI of the
subtidal urchins ranged from 3.3 to 4.6 and was similar
between years. The maximum GI ranged from 8.6 to 11, and
the peak value obtained for 1987 (8.6) was significantly
lower compared with the peak indices of 1986 and 1988 (11,
p=0.0001, 10, p=0.08, respectively). Comparisons of the
GI of female and male urchins from the subtidal indicated
that the GI maxima of the two sexes did not differ (Table 2).
The dates when spawning was first detected show that
the two populations started gamete release around the same
time in 1986 and 1988, but in 1987 the intertidal urchins
spawned a month earlier than the subtidal conspecifics
(Table 1). In general, gamete release terminated in late August or early September, although some of the subtidal
urchins continued to spawn through September, with the
minimum GI recorded in October, 1986 (Fig. I a).
The maximum GI of the subtidal urchins was significantly higher than that of their subtidal conspecifics in 1986
and 1988 (p = 0.0001, p = 0.002, respectively). This difference
was most apparent in the 1987/1988 period of gonadal
growth (October-March). In 1987 there was no difference in
the maximum GI of the two populations, but the GI of the
subtidal urchins remained significantly higher for the duration of the breeding period (p = 0.002).

M. Byrne: Reproduction of Paracentrotus lividus

be present. Nutritive phagocytes pack the ovary with
eosinophilic, and intensely PAS + droplets. Relict oocytes
may be present.

Stage IIL" premature stage

Vitellogenesis continues and oocytes at all stages of development (10 to 90 #m diam) are present in the ovary (Fig. 2d).
The large primary oocytes are ovoid and project towards the
centre of the ascinus. At this stage, they stain pink with H/E
and purple with AB/PAS, detach from the ascinal wall, and
move to a central position. As vitellogenesis proceeds, the
nutritive phagocytes are displaced from their central position by large ooeytes and their PAS + material is reduced.
Once the primary oocytes reach maximum size they promptly undergo maturation and ova accumulate in the lumen of
the ovary.

Stage IV." mature stage

Prespawning, Stage IV ovaries are filled with closely-packed
ova that stain purple with AB/PAS and are strongly
eosinophilic (Fig. 2 e). These ovaries contain a large cohort
of ova (90/~m diam) and there are a few small oocytes (10
to 60 pm diam) along the ascinal wall. The nutritive phagocytes are either absent or form a thin, pale meshwork
around the small oocytes. The PAS + material is reduced or
absent. Early primary oocytes may be present.

Histology of the ovaries

The pattern of ovarian growth was divided into six stages
(Fig. 2), and these are used to describe the histology of the
oogenesis and to document the annual oogenic cycle of
Paracentrotus lividus.

Stage I: recovery stage

Ovaries in Stage I (Fig. 2 a, b) contain primary oocytes (5 to
30 #m diam) and clusters of early oocytes along the acinal
wall. The oocytes are strongly basophilic, staining dark purple with H/E, and blue with AB/PAS. Nutritive phagocytes,
the non-germinal accessory cells, form a meshwork across
the ascinus, giving the ovary a vacuolated appearance.
These cells contain eosinophilic-PAS + droplets, some of
which are derived from phagocytosis of relict oocytes
(Fig. 2a). The ovary may contain unspawned ova in the
process of lysis and being engulfed by the phagocytes.

Stage II: growing stage

With the onset of vitellogenesis the primary oocytes increase
in size (10 to 50pro diam) and become decreasingly
basophilic, taking on a light purple hue (Fig. 2 c). They remain attached to the ascinal wall and are surrounded by
nutritive phagocytes. Clusters of early primary oocytes may

Stage V: partly spawned stage

In Stage V ovaries, the ova are loosely packed with spaces
vacated by spawned ova (Fig. 2 f, g). Ova may also be pres-

Fig. 2. Paracentrotus lividus. Histology of ovaries. (a) Stage I:

cross-section through ascinus of recovering ovary showing periodic
acid Schiff-positiveglobules (arrowheads) derived from lysis of relict oocytes; extensions of nutritive phagocytes (NP) project into
lumen; small previtellogenic oocytes (PO) occur along ovary wall.
(b) Stage I: ascini are filled with eosinophitic nutritive phagocytes;
previtellogenic oocytes line ascinal wall. (c) Stage II: growing ovary
with early vitellogenic oocytes (EV) and nutritive phagocytes; N:
nucleus. (d) Stage III: premature ovary with oocytes at all stages of
development; nutritive phagocytes surround vitellogenic oocytes
(VO) which detach from ascinal wall, and ova (O) accumulate in the
lumen. (e) Stage IV: mature ovary packed with ova, nutritive
phagocytes are reduced to a thin layer along ascinal wall. (f)
Stage V: partly spawned ovary with loosely packed ova and a
paucity of nutritive material; except for the spaces vacated by
spawned ova, ovary is similar to Stage IV. (g) Stage V: partly
spawned ovary in Stage III condition, with oocytes at different
stages of development and nutritive phagocytes; most vitellogenic
oocytes will eventually mature and move to lumen. (h) Stage VI:
spent ovary containing unspawned relict ova (R) that will be resorbed; nutritive phagocytes form an eosinophilic meshwork across
some ascini. (i) Stage IV: spent ovary largely devoid of ova and
nutritive phagocytes; all vitellogenic oocytes and relict ova will be
resorbed. (j) Ovary intermediate between Stages VI and I, with relict
ova undergoing lysis (L); lysed material is taken up by nutritive
phagocytes. Scale bars: (a) 120 #m, (b), (c) 100/~m, (d)-(j) 180 #m

ent in the oviduct. Partly spawned ovaries are variable in
appearance. Some ovaries (Fig. 2 g) contain oocytes at all
stages of development, as described for Stage III, whereas
other ovaries (Fig. 2 f) contain a large store of ova, as decribed for Stage IV. Thus, at the beginning of the breeding
season, it appears that females with Stage III and Stage IV
ovaries both commence spawning.
Partly spawned ovaries with the Stage III condition
(Fig. 2 g), have a small number of ova at the onset of spawning and primary oocytes replace ova as they are shed; In
these ovaries, vitellogenesis continues during the early part
of the breeding season, with vitellogenic oocytes surrounded
by nutritive phagocytes. There is a progressive decrease in
the PAS + material in the phagocytes as oocyte growth continues. The oocytes mature successively and spawned ova
are replaced as long as there are large primary oocytes remaining in the ovary. By the end of spawning, most of the
fully-grown oocytes have undergone maturation and the
nutritive phagocytes are reduced or absent.
By contrast, ovaries that progress from Stage IV to Stage
V have a large store of ova at the onset of spawning and
there are few or no primary oocytes to replace them as they
are shed (Fig. 2f). In these ovaries, vitellogenesis is mostly
finished and the nutritive phagocytes are reduced or absent.

M. Byrne: Reproduction of Paracentrotus lividus

Stage II: growing stage

The basophilic layer increases in depth as columns of spermatocytes project centrally (Fig. 3 c). The ascinus is filled
with nutritive phagocytes containing intensely PAS + and
eosinophilic material.

Stage III: premature stage

Premature testes contain columns of spermatocytes along
the ascinal wall and spermatozoa accumulate in the lumen
(Fig. 3 d). Nutritive phagocytes are still present, although
displaced from the centre by the spermatozoa.

Stage IV." mature stage

Mature testes are packed with spermatozoa and the nutritive phagocytes are limited to the periphery (Fig. 3 e). At this
stage the testes are largely devoid of the eosinophilic-PAS +

Stage V: partly spawned stage

Stage VL" spent stage
Spent ovaries have thin ascinal walls and appear empty
except for relict oocytes (Fig. 2 h-j). The number and type
of oocytes present in the ovary at the end of the breeding
season is variable. Some ovaries contain unspawned ova,
whereas others are largely devoid of large oocytes. Any
vitellogenic oocytes and ova present in the ovary at this
stage are eventually resorbed and phagocytosis of relict
oocyte material may be evident. Some ovaries contained a
pale meshwork of nutritive phagocytes around the periphery
that may have started to sequester reserves for the next
oogenic cycle, including material phagocytosed from relict
oocytes. Clusters of primary oocytes occur along the ovary

Histology of the testes

The pattern of testis growth in Paracentrotus lividus was also
divided into six stages (Fig. 3), and these are used to describe
the histological events of spermatogenesis.

Stage I: recovery stage

In Stage I testes the ascinal wall is lined with a thinbasophilic layer of spermatogonia and primary spermatocytes (Fig. 3 a, b). Nutritive phagocytes containing eosinophilic and PAS + droplets form a meshwork across the ascinus. Relict spermatozoa may be present.

Partly spawned testes are similar to those of Stage IV, except

that there are spaces in the ascinal lumen and spermatozoa
may be less concentrated (Fig. 3 f). The ascinal wall appears
thin and spermatozoa may be present in the gonoduct.

Stage VI." spent stage

Spent testes have thin ascinal walls and a pale meshwork of
nutritive phagocytes around the periphery (Fig. 3 g). They
appear to be devoid of contents, although relict spermatozoa may be present.

The gametogenic cycle

The relative frequencies of the ovarian maturity stages in
female Paracentrotus lividus from Ballynahown and Glinsk
over the three breeding seasons are illustrated in Fig. 4. The
histograms show an annual oogenic cycle that was similar in
both populations. Although small primary oocytes were
present throughout the year, clusters of early oocytes were
most apparent in the spent and recovery stages (VI and I,
respectively), from August to December. During the recovery and growth stages (I and II, respectively), from October
to March, the nutritive phagocytes develop and fill the
ascini. From the GI data it is evident that during these stages
the gonads attained 80 to 98% of their maximum weight
(Table 3). This increase in weight was due to the accumulation and storage of PAS + droplets by the nutritive phagocytes. The build-up of PAS + material was followed by vitellogenesis which started in February, in Stage II ovaries. By

M. Byrne: Reproduction of Paracentrotus lividus


Fig. 3. Paracentrotus lividus. Histology of testes. (a) Stage I: crosssection through ascinus of recovering testis containing relict spermatozoa (R) and nutritive phagocytes (NP) which form an
eosinophilic meshwork. (b) Stage I: primary spermatocytes along
ascinal wall (arrowheads). (c) Stage II: columns of spermatocytes
project centrally (arrowheads) in growing testes, nutritive phagocytes fill ascini. (d) Stage III: premature testis with spermatozoa (S)

in centre and nutritive phagocytes around periphery. (e) Stage IV:

mature testis filled with spermatozoa and largely devoid of nutritive
tissue. (f) Stage V: partly spawned testis with spaces vacated by
spawned spermatozoa. (g) Stage VI: spent testis largely devoid of
contents; L: lumen. Scale bars: (a), (b) 150#m, (c) 200#m, (d)
170 #m, (e) 180 #m, (f) 210 #m, (g) 140 #m

March, ova had started to accumulate in the premature

(Stage III) ovaries of subtidal females, indicating that vitellogenesis in P. lividus takes a minimum of one month. The
accumulation of PAS + material by the nutritive phagocytes
continued through Stage III (April-June). In each year, ova

were prevalent in the subtidal females from March onwards,

whereas they did not appear in the intertidal females until
April. The subtidal females came into breeding condition
approximately a month prior to the rock-pool urchins.
Comparison of the histograms shows that mature Stage IV


M. Byrne: Reproduction of Paracentrotus lividus


(I) Recovering

] Premature


(V) Partially Spawned



(IV) Mature


(Vl) Spent




' M.' J, ' J. ' A,' S.' O.' N.' D.' J.' F.' M.' A.' M.' J. ' J. ' A.' S.' O.' N.' D.' J. ' F.' M.' A." M.' J.








M.' J.' J. 'A.' S O. N. D.' . ' F . ' M ' A ' M


J 'J ' A ' S I ' O . ' N . ' D . ' J . ' F ,



. A.

Fig. 4. Paracentrotus lividus. Annual oogenic cycle. Histograms

show relative frequencies of ovarian stages in histological sections
of female urchins from intertidal (Ballynahown) and subtidal
(Glinsk) populations

Table 3. Paracentrotus lividus. Gonad growth during period of day

length < 12 h (October--March). Gonad indices (GI), and temperatures recorded in October and March are shown, together with
maximum GIs and minimum temperatures
and year


Temperature (C)

Oct. Mar.




ferences at other times of the year were due to differences in

the number of ova present, the number of ova available for
spawning and the amount of nutritive tissue present.
Histological examination of the females from Glinsk at
the onset of spawning revealed that, over the three breeding
seasons examined, 80 to 100% of them attained the Stage IV
condition. In contrast, only 30 to 50% of the females from
Ballynahown had fully mature ovaries at the beginning of
the breeding season. The remaining females in both populations were characterised by a successive maturation of latevitellogenic oocytes positioned around the periphery of the
During Stages V I - I I , resorption of relict oocytes is a
continuous and variable process. Although most females
had few late-vitellogenic oocytes and ova remaining in the
ovary by the end of spawning, specimens with ovaries filled
with unspawned oocytes were present through December.
Relict oocyte material was evident through March, 7 mo
after the termination of spawning.
The spermatogenic cycle is similar to the ovarian cycle
except that the accumulation of PAS + material by the nutritive phagocytes during the winter months is followed by
the appearance of spermatozoa. In the males, however, spermatogenesis does not appear to continue during the breeding season. By the onset of spawning all the males examined
(n = 10) had Stage IV testes with a large store of spermatozoa and little nutritive tissue.
Hermaphrodites were present in both populations, and
the incidence of hermaphrodism was 1% at both Ballynahown (n= 150) and Glinsk (n= 170). The hermaphrodites
possessed ovotestes (Fig. 5 a) that were predominantly female with a few testicular ascini. Occasionally, ascini with
female and male portions were encountered.

Gross appearance of dissected urchins















females were present for 3 to 4 mo at Glinsk, but for only 2

to 3 mo at Ballynahown (Fig. 4). Partly spawned (Stage V)
females were present in the intertidal, starting in June 1986
and 1987 and in May 1988. For the subtidal population,
partly spawned females were present in June of each year.
Spent (Stage VI) females usually appeared in August or
September, although in 1988 some intertidal females were
spent in July.
Oogenesis was not synchronous in either population,
with inter-individual differences in the ovarian condition in
most samples (Fig. 4). The only samples in which all females
from both populations exhibited the same stage of maturity
were those collected in February 1987. During this period all
the females were in the recovery stage. Inter-individual dif-

Mature gonads ooze gametes on dissection. Ripe ovaries are

orange, while the testes are cream or yellow. For 6 to 8 mo
each year the sex of the subtidal urchins could be determined
by visual inspection, whereas the intertidal urchins could
only be sexed for 3 to 5 mo. Spent and recovering specimens
could not be sexed visually due to the dark brown colour of
their gonads. The gross appearance of the gonads of the
Glinsk population suggested that the subtidal urchins came
into breeding condition at least one month prior to the rock
pool urchins. Moreover, the gonads of the subtidal urchins
exhibited the spent condition approximately one month after their intertidal conspecifics.

Spermatozoa in the oviduct

Histological examination of the ovaries during the summer
months revealed the presence of sperm in the oviduct of
several females (Fig. 5 b d). This prompted a closer examination of the ovaries of other females for the presence of
spermatozoa. The spermatozoa were identified by their

M. Byrne: Reproduction of Paracentrotus lividus


Fig. 5. Paracentrotus lividus. (a) Ovotestes of a hermaphrodite; O:

ova, SC: spermatocytes. (b) Mass of spermatozoa (S) surrounding
ova in oviduct; J: jelly coat. (c) Spermatozoa in middle of an ovary.

(d) Detail of spermatozoa (arrowheads) and ova. (e) Parasite (P)

near ascinal wall of testes. Scale bars: (a) 112 #m, (b) 135 #m, (c)
90 #m, (d) 45 #m, (e) 40 #m

basophilic-conical head region (Fig. 5 d), identical in length

(ca. 3.0/~m) and shape to the head region of the spermatozoa in the testes. Females with spermatozoa in the oviduct
were observed throughout the year and in each monthly
sample of 10 females, a mean of 6.4 specimens ( S E = 1.1)
from Ballynahown and a mean of 8.2 specimens (SE = 0.6)
from Glinsk were found to have spermatozoa in the oviduct.
Although this phenomenon was first detected in females
where the spermatozoa were obvious (Fig. 5 b, c), there were
usually only a few spermatozoa embedded in cell debris or
in mucous-like material in the oviduct (Fig. 5 d). Occasionally, spermatozoa were attached to oocytes in the oviduct, but
their heads were not orientated at a 90 angle to the oocyte
surface as would be expected in fertilization, and fertilization membranes were not observed.

and that they were the most numerous size-class present

(Fig. 6). The method of measurement, however, underestimated the number of ova due to their comparatively small
nucleus (Fig. 2e). A seasonal change in the oocyte size-frequencies was evident, with large oocytes (80 to 90/~m diam)
present from May to August. The large oocytes recorded in
September and October were unspawned oocytes that did
not have the appearance of relict oocytes. The oocyte sizefrequencies (Fig. 6) show that large oocytes and ova were
present in females from the subtidal one month prior to their
presence in females from the intertidal.

Oocyte size-frequencies
Examination of the oocyte size-frequency distributions reveals that small oocytes were present throughout the year

Temperature and day length

Although the pattern of the increase and decrease in sea
temperature was similar each year, there were inter-annual
differences in the minimum and maximum temperatures
recorded (Table 3; Fig. 1 b). In 1986 and 1987, the maximum
sea temperatures, 15.8 and 16.2C, respectively, were


M. Byrne: Reproduction of Paracentrotus lividus





















~ 60

~ so








~< 60



~ 29

~ 20























Mar May



Fig. 6. Paracentrotus lividus. Oocyte size-frequencydistributions of females from Ballynahown and Glinsk

recorded in August, whereas in 1988 the maximum sea temperature, 17.5 C, was recorded in June. Temperature minima of 5.5 and 6.0 C were recorded in February and December 1987, respectively. It should be noted, however, that
compared with the subtidal urchins, the rock pool urchins at
Ballynahown experienced a fluctuating temperature regime
due to their intertidal location. On several occasions the
rock pools were noted to have a higher temperature than the
sea in the summer and a lower temperature in the winter
(Fig. 1 b).
The GI shows that maximum gonadal growth occurred
between October and March (Table 3). This period coincides with the shorter days of the year (day length _< 12 h;
Fig. 1 c; Table 3). It also coincides with the period of decreasing sea temperatures, and gonadal growth exhibits an
inverse relationship with temperature (Fig. 2 a, b; 7). By the
period of minimum sea temperatures in February 1987, the
gonad indices for Ballynahown and Glinsk were 7.3 and 7.5,
respectively, approximately double the GI minima and close
to the GI maxima for 1987 - 8.3 and 8.6, respectively.
In each year, spawning commenced before the sea-temperature maxima and around the shortest days of the year.
The sea temperatures recorded at GI maxima and after the
onset of spawning are listed in Table 1. Over the three breeding seasons studied, spawning was bracketed on one side by
temperatures of 11.5 to 14.5 C measured before spawning
started, and on the other by temperatures of 13.4 to 16.0 C
measured one month later when spawning was first recorded. If gamete release is influenced by temperature, then tern-



8<> I




. . . . . . . .
y=-,416X+9.131; R2=.377


. . . .










16 .

14 t











Temperature C

Fig. 7. Paracentrotus lividus. Gonad indices at Ballynahown

(n=240) and Glinsk (n=210) vs temperature during OctoberMarch (1986/1987 and 1987/1988)

M. Byrne: Reproduction of Paracentrotus lividus

peratures between 13 to 15C may be required for the
commencement of spawning.
Gonadal parasite
Vermiform parasites (100 to 120/~m diam) were observed in
the gonads of several Paracentrotus lividus (Fig. 5 e). These
parasites, usually near the edge of the ascini, were easily
detected because their cuticle is brightly eosinophilic. The
incidence of parasitism differed in the two populations, with
infestation rates of 4% in the intertidal urchins (n = 150) and
38% in the subtidal urchins (n= 170). Although parasites
were encountered with a relatively high frequency in the
subtidal urchins, they had no discernable effect on the host's


Reproduction of Paracentrotus lividus on the west coast of

Ireland exhibits an annual cycle of gonadal growth and
maturation and the overall pattern of the cycle was similar
in the intertidal and subtidal populations during the three
breeding seasons examined. Although there was temporal
variation in the reproductive events, in general, spawning
began in May or June and ended in August or September.
The termination of breeding is followed by gonadal growth
from September to May and ova start to accumulate in the
ovaries in March.
Comparison of the gonad indices revealed inter-population differences in reproductive activity. The significantly
higher gonad production and the prolonged period of reproductive maturity of the subtidal urchins compared with their
intertidal conspecifics suggest that the subtidal urchins may
have been in a better nutritional state. Field observations
provide circumstantial evidence for a difference in the
availability of food to the two populations which may contribute to a difference in their nutritional states. The rockpool urchins, depending on ephemeral drift algae, contended with low food availability. By contrast, the subtidal
urchins, positioned in a Laminaria spp. bed, appeared to
have an abundance of food. Differences in gonadal growth
of urchins from populations separated by relatively short
distances are also reported for several echinoids and are
considered to be influenced by differences in food availability (Fuji 1960b, Ebert 1968, Gonor 1973a, Vadas 1977,
Pearse 1981, Keats et al. 1984). There are numerous studies
showing that the quantity and quality of food influences
gonadal growth in echinoids (Vadas 1977, Larson et al.
1980, Lawrence and Lane 1982, Keats et al. 1983, Andrew
1986). As for the Ballynahown population, intertidal
urchins are often smaller that their subtidal conspecifics and
this may be due to a reduced food supply in intertidal habitats (Ebert 1968, Lawrence and Lane 1982, Gonor 1973a)
and is regarded as an adaptation to exposed habitats where
hydrodynamic conditions may set a mechanical limit to
maximum size (Gonor 1973 a, Denny et al. 1985). Gonadal
growth may also be influenced by population density, as

demonstrated for Evechinus chloroticus where there is an
inverse relationship between density and gonad size in the
presence of equivalent food (Andrew 1986). For Paracentrotus lividus, high densities resulted in suboptimal growth in
culture, despite the enhanced availability of preferred food
(O'Sullivan 1988). The population densities of P. lividus
were not measured, because the urchin stocks at Glinsk were
harvested by divers during the investigation. For the intertidal and subtidal populations of P. lividus, it appears that
differences in food availability, wave action and density
probably influence the inter-site differences in gonadal
growth and reproduction. The influence of these factors
needs to be examined through a replicate study of several
populations that experience differences in exposure and
food availability.
Aspects of oogenesis differed in the two populations of
Paracentrotus lividus. Most of the subtidal females started to
accumulate ova in March and attained the Stage IV condition with a large store of ova a month prior to spawning.
The intertidal females lagged behind their subtidal conspecifics and, during the early portion of the breeding period, were characterized by continuous vitellogenesis and
oocyte maturation. This difference in the histological state
of the ovaries suggests that the subtidal urchins may have
accumulated a comparatively higher store of reserves during
the recovery and growth stages which promoted vitellogenesis of a large number of oocytes. Once initiated, vitellogenesis of P. lividus may be completed within a month, as reported for Strongylocentrotus purpuratus (Gonor 1973 a).
The within-sample difference in the state of gonadal maturity of Paracentrotus lividus is largely due to an intrapopulation variability in the development of the nutritive,
nongametogenic tissue. This variability was evident at the
onset of the ovarian cycle when some females had ovaries
that appeared empty, while others had ovaries containing a
well-developed layer of nutritive tissue and relict oocytes.
Relict oocytes appear to be recycled by the nutritive phagocytes and serve as a source of nutrients for the next vitellogenic cycle (Walker 1982). The variability in the amount of
reserves present in the ovaries in Stages VI and I and in the
amount of PAS + material accumulated during the growth
stage, undoubtedly influences the gonadal condition at the
onset of breeding. A within-population variability in the
gonadal condition is also reported for other populations of
P. lividus (Allain 1975, Crapp and Willis 1975) and for several echinoid species (Fuji 1960b, Pearse 1969, Chiu 1988).
As noted for other echinoids (Giese and Pearse 1974),
the gonad index method in conjunction with histology provided a good estimate of the reproductive cycle of Paraeentrotus lividus. The ovarian maturity method utilizes convenient, morphological criteria to assess gonadal development
and revealed the within-sample variability in gonadal condition. Ovarian development of P. lividus is an integrated sequence of events and many females had ovaries in a transitional state, particularly before and during breeding when
vitellogenesis, oocyte maturation and spawning are events
along a continuum. Post-spawning growth in P. lividus gonads occurs rapidly, as it does in Strongylocentrotusfrancis-

canus (Bernard 1977), but not as reported for other echinoids where a quiescent period of minimal gonadal growth
follows spawning (Fuji 1960b, Gonor 1973a).
The accumulation and storage of eosinophilic-PAS +
droplets by the nutritive phagocytes before and during
gametogenesis, the depletion of these droplets with oocyte
growth, and the phagocytosis of unspawned oocytes, are
characteristic features of echinoid reproduction (Walker
1982, Pearse and Cameron in press). Several immunocytochemical and ultrastructural studies show evidence that the
PAS + material plays a nutritive role in gametogenesis
(Walker 1982). Since the oocytes of several echinoids are
PAS + , it is inferred that the PAS + material stored by the
nutritive phagocytes is taken up by vitellogenic oocytes
(Holland 1967, Chatlynne 1969, Tyler and Gage 1984).
Moreover, recent biochemical investigations demonstrate
that vitellogenin is present in the nutritive phagocytes, where
in the females it provides a source of yolk precursors for
oogenesis (Ozaki et al. 1986, Shyu et al. 1986) and in the
males it appears to support spermatogenesis (R. Raft personal communication). Although it is likely that the PAS +
material in the nutritive tissue is also taken up by Paracentrotus lividus oocytes, the vitellogenic oocytes and ova did
not exhibit a PAS + tinctoral response.
The oocyte size-frequency data demonstrated that small,
previtellogenic oocytes were present throughout the year in
Paracentrotus lividus ovaries and that they were the most
abundant oocytes, even in mature ovaries, as reported for
Stylocidaris affinis (Holland 1967). This contrasts with the
oocyte size-frequency data of Strongylocentrotus purpuratus,
where mature ovaries contain a dominant class of large
oocytes (Gonor 1973b).
There are several reports of hermaphroditic individuals
in populations of normally dioecious echinoids (Boolootian
and Moore 1956, Lawrence 1987, Pearse and Cameron in
press) and, as noted for Paracentrotus lividus (Drzewina and
Bohn 1924), the incidence of hermaphroditism was low.
Most reports of hermaphroditic urchins note the presence of
ovaries and testes in the same animal (Boolootian and
Moore 1956). By contrast, the hermaphroditic P. lividus described here possessed ovotestes and, as for hermaphroditic
Strongylocentrotus purpuratus, the gonads were predominantly female (Gonor 1973 c). As noted by Gonor (1973 c),
ovotestes can be detected only through histological examination of the mosaic portion of the gonad, and may be a
more common expression of hermaphroditism in echinoids
than previously thought.
The seasonality of gametogenesis in several echinoid species raises questions about the role that seasonal environmental events play in controlling reproduction (Pearse 1981,
Pearse and Cameron in press). For Paracentrotus lividus, the
maximal period of gonadal growth coincides with decreasing sea temperatures and a day length of less than 12 h. This
suggests that temperature and photoperiod may both influence gonadal development. A relationship between decreasing temperature and gonadal growth has been noted for
Mediterranean populations of P. lividus and for Strongylocentrotus droebachiensis (R6gis 1979, Himmelman 1975). By

M, Byrne: Reproduction of Paracentrotus lividus

contrast, gonadal growth of S. purpuratus is not correlated
with sea temperature (Gonor 1973 a, Pearse 1981). For this
species, Pearse and his colleagues (Pearse et al. 1986, BaySchmith and Pearse 1987) have demonstrated that gonadal
growth is under photoperiodic control, with short days serving as an exogenous cue. A comparison of the temperature
data shows that on the west coast of Ireland, P. lividus experiences marked seasonal changes in sea temperature, whereas on the west coast of North America, S. purpuratus inhabits environments with poorly defined seasonal temperature
changes (Gonor 1973a, Pearse 1981, Pearse etal. 1986).
Given this difference, it might be expected that reproductive
events of P. lividus would be influenced more by temperature than those of S. purpuratus. It is also interesting that the
timing of their reproduction differs, with gonadal growth of
S. pwTuratus in the summer followed by vitellogenesis in the
autumn and spawning in the winter (Gonor 1973 a, Pearse
1981), six months after these events in P. lividus. Although
the consistency of the pattern of reproduction suggests that
gonadal growth and gametogenesis in P. lividus are entrained by exogenous cues, experiments such as those undertaken with S. purpuratus (Pearse et al. 1986) are required to
establish the influence that temperature and photoperiod
many exert on reproduction of P. lividus. The role of endogenous factors on gonadal growth must also be considered (Pearse 1981, Giese and Kanatani 1987).
As suggested by Lane and Lawrence (1979), the environmental factors that trigger spawning may differ from those
that influence gonadal growth. For Paracentrotus lividus,
the temporal variability in the onset of spawning between
years suggests that photoperiod may not serve as the
proximate cue for gamete release. Each year, spawning
started before the maximum sea temperature, the time of
which was variable. In 1988, the maximum temperature
occurred in June, approximately two months earlier than
in the previous two years. Correspondingly, spawning in
1988 commenced at least a month earlier than in 1986.
Due to the prolonged storage of gametes by the subtidal
urchins in 1987, the influence that temperature may have
exerted on spawning in the subtidal that year is not clear.
It appears that rising sea temperature may serve as a
proximate cue for the induction of spawning, a suggestion
made for other populations of P. lividus (Fenaux 1968, Dominique 1973). In Irish waters, temperatures of 13 to 15 C
may be required for the commencement of spawning. For
Mediterranean and Breton populations of P. lividus, temperatures of 16 C and 14 to 15 C, respectively, are suggested to be required for spawning (Fenaux 1968, Dominique
A prolonged spawning period over the summer months
is reported for Irish and Breton populations of Paracentrotus lividus (Dominique 1973, Willis 1976), whereas in the
Mediterranean two spawning periods occur, one in early
summer and a second in the autumn (Fenaux 1968, R6gis
1979). Fenaux suggests that in the Mediterranean the coldest temperatures of the winter (8 C) and the warmest temperatures in the summer (24 C) both inhibit spawning. The
biannual spawning appears to be influenced by the increase

M. Byrne: Reproduction of Paracentrotus lividus

in temperature to a critical point in June, and then a decrease past this critical temperature again in August (Fenaux 1968). If spawning of P. lividus is cued by increasing
sea temperatures, then the early spawning of both populations in 1988 may be attributed to the especially high and
early temperature maximum (17.5 C) in June of that year.
Latitudinal differences in the time of spawning have been
noted for several marine invertebrates with boreo-mediterranean distributions (Runnstr6m 1927, Giese and Pearse
1974, Costelloe 1988) and, as suggested for P. lividus (Fenaux 1968), the zoogeographic differences in spawning may
reflect the existence of different "physiological races" of this
urchin at different latitudes which spawn at different temperatures. The results of the present study contrast with the
results of Crapp and Willis (1975), who report winter and
summer spawning in an Irish population of P. lividus.
Although increasing temperature appears to be a proximate cue for spawning, other factors such as illumination,
phermones or gametes in the water, and endogenous factors
may also play a role (Fox 1924, Himmelman 1975, Minchen
1987, McEuen 1988, Pearse et al. 1988). Minchen (personal
communication) observed spawning of Paracentrotus lividus
on several occasions in June on days of long sunshine. The
intensity of illumination is suggested to influence spawning
in several echinoderms (Costelloe 1988, McEuen 1988,
Pearse et al. 1988). An increase in phytoplankton levels induces spawning in Strongyloeentrotus droebachiensis (Himmelmann 1975, 1978). Spawning of P. lividus on the west
coast of Ireland, however, occurs well after the spring phytoplankton bloom at Glinsk and nearby areas (Roden et al.
1987). Unlike that documented for several echinoids (Fox
1923, Pearse 1975), reproductive activity of P. lividus does
not exhibit lunar periodicity (Fox 1923, Ke6he~ 1966, Allain
The presence of spermatozoa in the oviduct during the
breeding season may be due to males spawning during collection. Spermatozoa were also present in the oviduct during
the winter, six months after the termination of spawning,
suggesting that these observations may not be entirely
anomalous. Although it is unlikely that these spermatozoa
fertilize ova, their presence in the oviduct is evidence of
chemotaxis in the spermatozoa of Paracentrotus lividus. To
gain entrance to the oviduct, the sperm must be carried
towards the females and then swim into the oviduct. The
sperm of several asteroid, ophiuroid and holothuroid species
are attracted to molecules extracted from homologous and
heterologous ovaries (Miller 1985) and sperm chemotaxis
has recently been reported for an echinoid (Rink 1988). It is
possible that spermatozoa present in the oviduct of other
echinoids have not been detected due to their low numbers
and small size. For P. lividus, sperm chemotaxis may increase the chances of fertilization in situ and may alleviate
somewhat the problem of fertilization in conditions of low
gamete density (Pennington 1985).
As noted for other urchin fisheries (Bernard 1977, Chiu
1986), harvesting of Paracentrotus lividus occurs during winter (Allain 1975, R6gis 1979, and own personal observation).
In P. lividus this winter fishery coincides with the recovery

and growing stages of gonadal growth, when nutritive material is being elaborated and stored. During this period, the
gonads are filled with nutritive phagocytes and there are few
or no ripe gametes in the gonads. The urchin harvest declines in April and terminates in May, coinciding with the
period when the nutritive tissue is being converted into
gametes. Gonads at an advanced stage of sexual maturity
are not acceptable due to their soft texture.
The depletion of commercial stocks of Paracentrotus
lividus in European waters and the continued demand for
urchin roe has stimulated interest in the potential of this
urchin for mariculture (R6gis 1980, Birais and Le Gall
1986). It is clear from this investigation that the large subtidal urchins would be a superior source of gametes for
brood-stock purposes, compared with their intertidal conspecifics. The subtidal females possessed a larger store of
ova, and mature gametes were available in the subtidal population for at least five months of the year. By contrast, the
rock pool urchins were seldom fully mature at their GI
maximum and their ovaries contained an abundance of immature late vitellogenic oocytes. It is unfortunate that the
populations of P. lividus which appear to be the best source
of brood stock for mariculture have been depleted. The
temporal variability in the breeding period of P. lividus and
the possibility that sea temperature serves as an exogenous
cue for the induction of spawning have relevance for the use
of this urchin for brood stock. For the mariculturist, both
sea temperature and gonadal condition should be monitored
closely to assess the availability of mature gametes, particularly at the approach of the breeding season.
Acknowledgements. This work was supported by a postdoctoral
fellowship from the Department of Education, Dublin. I thank
Professor P. O. C6idigh and Dr. B. F. Keegan for support and use
of facilities. Thanks to Dr. D. McGrath for introducing me so
promptly to Ballynahown. The suggestions of Dr. V. Morris and
Dr. N. Andrew improved the manuscript. Thanks to Mr. A. Lawless, Mr. J. Galvin and Ms. E. Moylan for technical assistance and
to Mr. D. Burke, skipper of the R. V. "Ona 3". Mr. M. Ricketts and
Mrs. J. Jeffrey also provided technicaI assistance. The Dunsink
Observatory, Dublin, provided day-length information. This paper
is Contribution No. 316 from the University College Galway School
of Marine Sciences.
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Communicated by G. F. Humphrey, Sydney