Annual Reproductive Cycles of the Commercial Sea Urchin

Marine Biology 104, 275-289 (1990) Marine BiOlOgy @ 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

Abstract

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 subse- quent 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.

Introduction

The sea urchin Paracentrotus lividus (Lamarck) has a boreo- mediterranean distribution (Mortensen 1943, Southward and Crisp 1954) inhabiting intertidal rock pools and shallow subtidal habitats. In Europe, P. lividus has been an impor- tant 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 em- ployed 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

276 M. Byrne: Reproduction of Paraeentrotus lividus

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 (9°3 I'W; 53°13.4'N), urchins were collected from two adjacent mid-shore tide pools. At the second site near Glinsk Pier (9°50.7'W; 53°22.4'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 deter- minations were made on freshly dissected specimens by ex- amining 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

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 blue- periodic 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 mor- phologically 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 nu- cleolus and ova sectioned through the nucleus. For non- spherical 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.

Results

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 277

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

Location Temperature (°C): Day length (min): and date

before after initial before after initial spawning spawning spawning spawning

Ballynahown 19 June 1986 13.0 15.0 1027 942

1 May 1987 11.5 13.4 950 996 16 May 1988 12.4 16.0 955 1015

Glinsk 20 June 1986 13.0 15.0 1027 942

9 June 1987 13.4 15.5 996 1016 1 June 1988 14.5 16.0 1001 1015

Table 2. Paracentrotus lividus. Maximum gonad indices of female and male urchins from Ballynahown and Glinsk. Standard errors in parentheses

Location and date Females Males Combined

g

121 <

a

,11 g

b

11

10 i

9

8

7

6

5

4

3

! . . . . . BALLYNAHOWN GLINSK

,di

2 0

18

16

14

12

10

8

6

4

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. 1986 1987 1988

e • • •

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 .

Ballynahown 22 May 1986 7.3 (0.65) 7.2 (0.48) 7.2 (0.30)

1 May 1987 8.8 (0.36) 9.3 (0.53) 8.3 (0.29) 16 May 1988 7.7 (0.42) 7.7 (0.50) 7.6 (0.31)

Glinsk 20 June 1986 10.9 (0.52) 11.1 (0.97) 11.0 (0.46) 10 May 1987 9.0 (0.55) 8.6 (0.47) 8.5 (0.33) 1 June 1988 10.7 (0.75) 9.3 (0.51) 10.0 (0.45)

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 (October - April). Although the overall pattern was consistent, there were inter-annual and inter-population differences in the GI.

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 May 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 subse- quent 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.

IOO0

900 2g

800 I"

7 0 0

6 0 0

500

400

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.

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 GI 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-

278 M. Byrne: Reproduction of Paracentrotus lividus

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 Au- gust 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 dura- tion of the breeding period (p = 0.002).

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 re- main attached to the ascinal wall and are surrounded by nutritive phagocytes. Clusters of early primary oocytes may

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.

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-positive globules (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 de- cribed 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.

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 + material.

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 wall.

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

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 thin- basophilic 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.

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


Fig. 3. Paracentrotus lividus. Histology of testes. (a) Stage I: cross- section 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 (Apri l-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


100

LU 50

100

LU =o 50

[ ] (I) Recovering

[ ] (11) Growing

BALLYNAHOWN

I q:::

' M.' J, ' J. ' A,' S.' O.' N.' D.' J . ' F.' M.' 1986 1987

• ] (111) Premature [ ] (V) Partially Spawned

• (IV) Mature [ ] (Vl) Spent

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

1988

GLINSK

1111 M.' J.' J. 'A. ' S

1986

!111!i ° O. N. D.' . ' F . ' M ' A ' M J 'J ' A ' S I ' O . ' N . ' D . ' J . ' F , A.' J . A.

1987 1988

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

Location GI Temperature (°C) and year

Oct. Mar. max. Oct. Mar. min.

Ballynahown 1986-1987 4.0 7.1 8.3 12.0 6.8 5.5 1987-1988 3.2 6.1 7.6 13.8 7.8 6.0

Glinsk 1986-1987 4.4 7.6 8.6 12.0 6.6 5.5 1987-1988 5.4 9.8 10.0 10.7 8.3 6.0

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-

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 late- vitellogenic oocytes positioned around the periphery of the ascini.

During Stages VI - 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 Ballyna- hown (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

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


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 (SE= 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). Occasional- ly, 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.

Oocyte size-frequencies

Examination of the oocyte size-frequency distributions re- veals that small oocytes were present throughout the year

and that they were the most numerous size-class present (Fig. 6). The method of measurement, however, underesti- mated 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 size- frequencies (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.

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 16.2°C, respectively, were


100

80

BALLYNAHOWN 2O%

40

2o r3 May Jun 1986

too

~ 60

o 40

~ 29 8

Jan Mar May Jun 1987

1°°i s°i t 401

Mar May Jun Aug 1988

July Aug Oct

July Sept

Dec

120 ,

100-

80"

6O

4O

20

E•.lO0 ~ so F- ~ 60 <

4 0

~ 20 0 0

100

80

60

40

20

GLINSK 2O% -"---I

May Jun 1986

Feb Apr 1987

Mar May Jun Aug 1988

7

JUly/ Aug Sept

\

/ May Jun July Sept

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

Oct Nov

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 De- cember 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 de- creasing 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-

BALLYNAHOWN

11 . . . . . . . . . . . . . . . . . 10 ~ oO O y=-,416X+9.131; R2=.377

° 8 8<> I , . tl

~ 4

3

2

1 . . . . . . . . . . . . . . . . . . . 6 7 8 9 10 11 12 13 14 15

T e m p e r a t u r e ° C

GLINSK 16 . . . . . . . . . i . i , i . i .

14 t 8 y = - . 5 0 7 x + 1 1 . 1 4 8 ; R 2 = . 1 9 1

'2t 8 I s "~clO g Q

J , u !

6 7 8 9 t0 11 12 13 14

Temperature °C

Fig. 7. Paracentrotus lividus. Gonad indices at Ballynahown (n=240) and Glinsk (n=210) vs temperature during October- March (1986/1987 and 1987/1988)


peratures between 13 ° to 15°C 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.

Discussion

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 con- tribute to a difference in their nutritional states. The rock- pool urchins, depending on ephemeral drift algae, contend- ed 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 Paracentro- tus 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 intra- population 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 Paraeen- trotus lividus. The ovarian maturity method utilizes conve- nient, morphological criteria to assess gonadal development and revealed the within-sample variability in gonadal condition. Ovarian development of P. lividus is an integrated se- quence of events and many females had ovaries in a transi- tional 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-

286 M, Byrne: Reproduction of Paracentrotus lividus

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 immunocyto- chemical 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 Paracen- trotus 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 decreas- ing 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 decreas- ing temperature and gonadal growth has been noted for Mediterranean populations of P. lividus and for Strongylo- centrotus droebachiensis (R6gis 1979, Himmelman 1975). By

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, Bay- Schmith and Pearse 1987) have demonstrated that gonadal growth is under photoperiodic control, with short days serv- ing as an exogenous cue. A comparison of the temperature data shows that on the west coast of Ireland, P. lividus expe- riences marked seasonal changes in sea temperature, whereas on the west coast of North America, S. purpuratus inhab- its 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 en- trained by exogenous cues, experiments such as those under- taken 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, Do- minique 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 1973).

A prolonged spawning period over the summer months is reported for Irish and Breton populations of Paracentro- tus 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


in temperature to a critical point in June, and then a decrease past this critical temperature again in August (Fe- naux 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 (Fe- naux 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 in- duces spawning in Strongyloeentrotus droebachiensis (Him- melmann 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 1975).

The presence of spermatozoa in the oviduct during the breeding season may be due to males spawning during col- lection. 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 de- clines 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 im- mature 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. Law- less, 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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Date of final manuscript acceptance: November 3, 1989. Communicated by G. F. Humphrey, Sydney

Annual Reproductive Cycles of the Commercial Sea Urchin

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