P.S.Z.N.

1: Marine Ecology, 7 (2): 115-122 (1986) Accepted: August 27,1985

Q 1986 Paul Parey Scientific Publishers, Berlin and Hamburg
ISSN 0173-9565

The Reproductive Cycle of

Ophioderma brevispinum
(Echinodermata: Ophiuroidea)
HENDLER'*
GORDON & P. A. TYLER?

' Smithsonian Oceanographic Sorting Center, National Museum of Natural History,

Smithsonian Institution, Washington, D. C. 20560, U.S.A.
Department of Oceanography, University College of Swansea, Swansea SA2 8PP,
U.K.

With 3 figures

Key words: Ophioderma brevispinum, ophiuroid, echinoderm, reproduction, reproduc-

tive cycle, gametogenesis, spawning, hermaphrodite.

Abstract. The reproductive cycle of the brittlestar Ophioderma brevispin~trnis described using
histological and organ index data for a population in Massachusetts, U.S.A. The cycle consists of a
one month mid-summer spawning phase followed by gametogenesis and gradual gonadal growth
during the winter, and greatly accelerated gonadal growth from May to June. At the end of the
spawning season, oogonia proliferate near the base of the ovary, and a continuous layer of
spermatogonia lines the testis. As oocytes grow to a maximum diameter of 350 pm, yolk granules
accumulate and the cytoplasm becomes less basophilic. Prior to spawning, the testis becomes
branched and sulcate, and a whorl of spermatozoa produced by columns of spermatids accumulates
in the lumen. Comparisons between the reproductive cycles of different populations of 0. brevi-
spinum and its congeners support the hypothesis that temperature may be a critical exogenous
factor, but definitely not the only factor, in the initiation and duration of the growth and spawning
phases of the ophiuroid reproductive cycle.

Problem
Since BOOLOOTIAN'S (1966) review of echinoderm reproductive physiology,
knowlei ?e of ophiuroid reproduction has increased as new, more reliable
techniques have been developed to examine the reproduction of marine inverte-
brates. Published accounts of ophiuroid reproductive cycles have accumulated
slowly from studies based either on histological analysis or on indices of gonadal
weight, length and volume (e.g. BOWMER,1982; MLADENOV, 1983). Here,
gonadal indices and histology are used to describe the reproductive cycles of
male and female Ophioderrna brevispinum (SAY). The annual reproductive
pattern is discussed in relation to seasonal changes in behavior and environ-
ment, and compared with cycles of other Ophioderma species, to identify the
proximate factors governing reproductive periodicity.
Present address: Natural History Museum of Los Angeles County, 900 Exposition Boulevard,
Los Angeles, CA 90007, U.S.A.

U.S. Copyright Clearance Center Code Statement: 0173-9565/86/0702-0115$02.50/0

116 HENDLER
& TYLER

Material and Methods

Ophiodernia brevirpinum was collected from beneath shell-rubble and clumps of algae in Waquoit
Bay, East Falmouth, Massachusetts, U.S.A. The habitat sampled was a coarse sand and shell-rubble
substratum, 2-4 m deep, with abundant seagrass (Zostera marina), macro-algae, and sessile faunae.
At the study site the water was swiftly flowing; salinity measurements ranged from 27.5 to 31.1%0
(HENDLER, 1982).
Each month, from July 1973 to August 1974, the gonad index values were determined for 5 males
and 5 females. Gonad index was calculated for each specimen as the drained wet weight of its
gonads, divided by the drained wet weight of the ophiuroid (weight of the disc and arms less the
weight of the gonads), multiplied by 100. Descriptive statistics were calculated for index values of
males and females, and for both sexes combined, in each sample.
Each month, histological samples were prepared of the gonads of 5 males and 5 females. Fresh
squash preparations were made to examine the structure of the gonads and gametes. Interradial
portions of the disc were preserved in BOWIN'S fixative (in seawater solution), dehydrated in alcohol
and xylene, embedded in paraffin, sectioned at 7 pm,and stained with 0.5 % aqueous toluidine blue.
Using light microscopy, the diameters of at least 100 oocytes, sectioned through the nucleus, were
measured for each female specimen to construct oocyte size-frequency diagrams and to calculate
descriptive statistics.

Results
1. The gametogenic cycle of Ophioderma brevispinum
Oogenesis. The proliferation of oogonia commences while relict ova and
breakdown products from the previous oogenic cycle are still in the ovary
(Fig. 1a). Oogonia appear to originate at the base of the ovary near the genital
rachis (Fig. 1a). They are < 20 pm in diameter and have a large nucleolus and a
weakly basophilic cytoplasm.
In unstained squash preparations previtellogenic oocytes are colorless or pale
white, whereas the relict, mature oocytes appear densely pigmented and filled
with yolk globules. Aggregations of yellow-brown arnoebocytes (possibly folli-
cle cells) adhere to some developing oocytes, being associated more frequently
with small, previtellogenic oocytes than with mature oocytes (Fig. 1a, b). The
ovaries contain an extracellular greenish-brown substance, probably cell-break-
down residue. The smallest oocytes, which develop from oogonia, lie along (not
embedded within) the germinal epithelium. They have distinctly basophilic
cytoplasm, a large centrally-placed germinal vesicle containing diffuse chroma-
tin strands, and a nucleolus with a single vacuole. Notably, the simultaneous
occurrence of two generations of oocytes in the same gonad suggests (but alone
does not prove) that 0. brevispinum is iteroparic.
As an oocyte grows, the ratio of cytoplasm to nucleus increases, reaching
about 1 : 1 in the 50 pm oocyte (Fig. 1a, b). At this size, some previtellogenic
oocytes begin to fill the ovarian lumen (Fig. 1b). In unstained squash prepara-
tions, the oocytes at this stage have a transparent germinal vesicle and nu-
cleolus; minute yolk granules in the cytoplasm give the cells a pale gray-green
appearance.
The oocytes of 150 pm diameter have an eccentrically-placed germinal vesicle
and a single, eccentrically-placed nucleolus. In these cells, cytoplasmic
basophilia is reduced and the cytoplasm stains very pale-green with aqueous
Ophiuroid reproductive cycle 117

Fig. 1. Ophioderma brevispinum. A. Early stage of oogenesis in two adjoining ovaries showing
oogonia proliferating at the base and previtellogenic oocytes within the lumen of the gonad; B.
Developing ovary showing previtellogenic oocytes and mature vitellogenic oocytes; C. Mature
oocyte about to undergo reduction division; D.Testis in intermediate stage of spermatogenesis.
Abbreviations: a, oocyte attachment; B, bursa1 sac; f, follicle cell; g, germinal vesicle; p, previtel-
logenic oocyte; Sg, spermatogonia; V, vitellogenic oocyte; w, ovarian wall; z, spermatozoa. Scale
bar equals 100pm.
118 & TYLER
HENDLER

toluidine blue, indicating that vitellogenesis is taking place (Fig. 1b). This is
confirmed by the markedly denser green or brown pigmentation, associated
with yolk, which tints live oocytes of this size. When ovaries at this stage are
dissected in seawater, some of the freed oocytes are buoyant, suggesting that
lipids of low specific gravity have accumulated in the cells.
The mature oocytes grow to a maximum diameter of about 350pm. Their
cytoplasm has a meshlike appearance in stained sections due to the presence of
relatively pale yolk granules in a densely staining cytoplasmic matrix. The yolk
granules proliferate first at the periphery of the oocyte and then appear toward
the center of the cell (Fig. 1c). Opaque granules, possibly cortical granules,
occupy a stratum beneath the oolemma. The germinal vesicle remains intact
even in mature oocytes, but prior to spawning (e. g., in the June, 1974 sample)
the nucleus migrates to one end of the cell, perhaps as a preliminary to
reduction division (Fig. 1c). We found that female 0. brevispinum usually have
either greenish or brownish oocytes, and sometimes both brown and green
oocytes occur in the same specimen, whereas GRAVE(1900) states that the
oocytes in one individual are all of the same color.
Spermatogenesis. In the developing testis, spermatogonia form a continuous
layer, up to two cell-layers deep, lining the periphery of the testicular lumen.
The nucleus comprises most of the volume of these cells, which are 5ym in
diameter. The spermatogonia give rise to spermatocytes, which in turn undergo
reduction division producing columns of spermatids that extend toward the
center of the lumen (Fig. 1d). In fresh tissue, anastomosing branched strands
composed of reddish-brown cells appear within the testes.
As the testes mature, the outer, cortical zone of the testis is folded (visible in
live tissue at low magnification). The inner zone, the lumen of the testis,
expands as it fills with spermatozoa. Before spawning, the spermatozoa in the
lumen are massed in a whorl of cells that is separated by a narrow gap from the
peripheral spermatogenic tissues. After spawning, relict spermatozoa and
deposits of brown material remain in the testes.

2. The reproductive cycle of Ophioderma brevispinum

The production of new oogonia is initiated in July, at the end of the spawning
period, and continues for several months (Figs.2, 3). Oogonia always are
present, but they are numerous only in the latter half of the year. The
disappearance of relict oocytes between July and August is indicated by
decreasing gonad index values (Fig. 3) and is apparent in histological prepara-
tions. In August only early vitellogenic oocytes occur in the ovary, and by
September rapid increases in mean gamete size and in the gonad index reflect an
acceleration of gametogenesis and gamete growth (Figs. 2 and 3). Following the
rapid production of previtellogenic oocytes, oocyte growth slows; by December
the mean oocyte diameter reaches 60 pm (Fig. 2).
During December, pronounced vitellogenic activity is initiated. Mean oocyte
diameter slowly increases until May, finally exceeding 100 pm (Fig. 2). The
gonad index values also gradually rise through the period of early vitellogenesis
(Fig. 3), but the changes from month to month are not statistically significant (as
shown by the overlapping 95 % confidence interval bars in Fig. 3).
Ophiuroid reproductive cycle 119

Fig. 3. Ophioderma brevi-

spinurn. Monthly data for
field temperature (asterisks)
and gonad index (GI). Val-
ues are shown for mean GI of
males and females combined
(dots) and 95% confidence
interval bars, as well as the
mean GI for females ( 0 )and
males (0).

1973 1974
120 HENDLER
& TYLER

During May, as greater numbers of oocytes accumulate yolk, the mean

oocyte diameter increases dramatically to 170 pm. At this time there actually are
two distinct cohorts of oocytes: a group of previtellogenic cells and another
group of larger vitellogenic cells (Fig. 2). Every month except June, a cohort of
the small oocytes (those 5219pm) comprises at least 90% of the oocyte
population. Between May and June some oocytes undergo rapid vitellogenesis
and the percentage of large vitellogenic oocytes in the cohort with a diameter
> 219 pm increases from 5.3 % to 43.7 %. However, large oocytes compose only
4.8 % of the population in July, reflecting a spawning event between mid-June
and mid-July. Freshly collected individuals spawned in the laboratory during
that period, supporting the notion that there is a correspondence between the
diminished numbers of large oocytes seen in histological preparations and the
occurrence of spawning events in the field.
Our interpretation of the pre-spawning histological data is supported by the
trends in monthly gonad index values. For example, the mean gonad index
value doubles from May to June in concert with the rapid increase in oocyte
diameter. In July, the gonad index plummets to half the May value; it continues
to fall in August until the gonads are nearly emptied (these differences in mean
monthly values are statistically significant, as shown by the separation between
confidence interval bars in Fig. 3). The evacuation of the ovaries is evidence that
most of the small oocytes, as well as the larger oocytes that were present in May,
mature and are shed by the end of J ~ l y .
The seasonal cycle of spermatogenesis follows the same pattern as oogenesis,
and the monthly mean gonad index values of male and female brittlestars are
not statistically different. Monthly data (for 13 months) were separately ana-
lysed, and the hypothesis that the gonad index values of males and females are
equal was not rejected (t-tests; P < 0.05). Spermatogenesis is initiated in July,
and by January histological preparations show increased sperm production. By
February the cortical zone of some testes appears sulcate under low magnifica-
tion; the additional three dimensional relief increases the surfaceholume ratio
of the gonadal tubules. Spermiogenesis is most pronounced in May and June.
Spawning occurs in late-June to early-July, followed by a short “resting” or
“early growth” period before the reinitiation of rapid spermatogenesis. Some
spermatozoa are present in the testicular lumen year-round.
An intra-gonadal hermaphrodite was discovered - a specimen collected on 26
February 1974. Histological examination revealed that it contained oocytes,
scattered amongst spermatogenic tissue, which had not entered the vitellogenic
growth phase. This specimen was one of over 300 dissected during the study and
the only hermaphrodite found among the 100 individuals examined histologi-
cally.
Discussion
In certain respects the reproductive cycle of 0. brevispinum in Waquoit Bay,
reported herein, resembles that of a Mediterranean population of Ophioderma
longicaudurn (RETZIUS) in Villefranche Bay (FENAUX, 1970, 1972). However,
the durations of the spawning and growth phases of the two species are distinctly
different. Ophioderma brevispinum has a brief spawning period (mid-June to
mid-July), while 0.longicaudum releases gametes for 2 months (July and
Ophiuroid reproductive cycle 121
August). Ophioderma brevispinum has a longer period of slow gonadal growth
(September to May) than 0.longicaudum (September to March) (as estimated
from oocyte size-frequency polygons for the latter species in FENAUX,1972:
p. 260, Fig. 2). However, the rapid gonadal growth phase of 0. brevispinum
(mid-May to midJune) is shorter than the rapid growth phase of 0. lon-
gicaudum (April through June). The duration of the growth phase does not
appear to be related to oocyte size as all Ophioderma species examined,
including 0. brevispinum and 0. longicaudum, have similar maximum oocyte
diameters (HENDLER, 1979).
Although the relationship between temperature and reproduction may be
indirect or even coincidental, the similarity of temperatures during analogous
phases of the reproductive cycles of 0. brevispinum and 0. longicaudum sug-
gests that specific temperatures are critical for the processes of gonadal growth
and spawning (vide FENAUX,1970, 1972; this paper). Differences between
reproductive patterns of the two species may be caused by dissimilarities in the
temperatures of their habitats. For instance, the relative brevity of the rapid
growth and spawning phases of 0. brevispinum is perhaps attributable to its
comparatively cooler habitat. Water temperatures are 2 13"C all year for
0.fongicaudum but 2 13 "C for only 6 months for 0. brevispinum (FENAUX,
1970; this paper).
Observations on the reproduction of different populations of 0. brevispinum
further attest to the possible significance of temperature in the timing of the
reproductive cycle. The contrast between the brief gonadal growth and spawn-
ing periods of a northern population of 0.brevispinum (GRAVE,1900; this
paper) and the longer gonadal growth and spawning periods of a more southern
population (STANCYK, 1974) points to an extension of the growth and spawning
phases in warm water environments. In addition, four tropical Ophioderma
species have longer growth and spawning phases than their temperate-zone
congeners (HENDLER, 1979), as might be predicted if duration of reproductive
phases is related to temperature.
Despite the agreement of these observations with the hypothesis that temper-
ature is a controlling factor in the spawning and growth phases of the reproduc-
tive cycle, the rates of gametogenesis and gonadal growth are definitely not
simple, direct functions of temperature. Other variables, such as day-length,
may be important. For example, in the case of 0. brevispinum the slow post-
spawning gonadal growth stage in the fall and the rapid growth in the spring
occur at the same temperature (Fig. 3), suggesting that temperature by itself
does not regulate the rate of gonadal growth. This may be true for other
ophiuroids in temperate waters. For example, YAMASHITA & IWATA(1983) have
experimentally confirmed that in Amphipholis kochii an initial period of slow
gonadal growth and spermatocyte production following spawning is tempera-
ture-independent, while a rapid growth phase and the generation of sper-
matozoa in the spring is temperature-dependent.
Summary
1. The oocytes of Ophioderrna brevispinum grow to a maximum diameter of
350 pn within eleven months. Mature oocytes contain numerous small yolk
granules that give the cell a brown or green color. Eggs are buoyant,
122 & TYLER
HENDLER

suggesting that they contain lipids of low specific gravity. As the testes
mature, the outer, cortical zone of the testis becomes folded. The lumen of
the testis fills with a mass of spermatozoa separated by a narrow gap from the
peripheral spermatogenic tissues.
2. The spawning of males and females between mid-June and mid-July is
suggested by changes in gonad index values and gonadal histology. The
brittlestars spawn in the laboratory during the same period.
3. The durations of the spawning and growth phases of the reproductive cycle of
0. brevispinurn in the Western Atlantic and 0. longicaudurn in the Eastern
Atlantic are distinctly different. The differences in the timing of reproduction
might be attributable to dissimilarities in the temperature of the habitats of
the two species. However, the occurrence of slow and rapid gonadal growth
phases of 0. brevispinurn in different seasons, but at similar temperatures,
indicates that temperature by itself does not regulate gamete growth.

Acknowledgements
We are grateful to Mr. L. SCHWARTZ for assistance in the field, to Dr. R. SCHELTEMA for providing
laboratory facilities, and to Mr. 0. KELLYwho graciously provided access to the study site. Drs.
T. BOWIIER,M. BYRNE, K. ECKELBARGER and D. PAWSON provided helpful reviews of draft manu-
scripts. A portion of this research was carried out under the auspices of a Woods Hole Oceano-
graphic Institution Scholarship to G. H.

References
BOOLOOTIAN, R. A., 1966: Reproductive Physiology. In: R. A. BOOLOOTIAN (Ed.), Physiology of
Echinoderrnata. John Wiley & Sons, N. Y . : 561-613.
BOWMER, T., 1982: Reproduction in Arnphiura filiformis (Echinoderrnala: Ophiuroidea): Seasonal-
ity in gonad development. Mar. Biol., 69: 281-290.
FENAUX, L., 1970: Maturation of the gonads and seasonal cycle of the planktonic larvae of the
ophiuroid Amphiura chiajei FORBES. Biol. Bull., 138: 262-271.
- -, 1972: Evolution saisonnitre des gonades chez I'Ophiure Ophioderrna longicauda (RETZIUS),
Ophiuroidea. Int. Rev. ges. Hydrobiol., 57: 257-262.
GRAVE,C . , 1900: Ophiura brevispina. Mem. Biol. Lab. John Hopkins Univ., 4: 79-100.
HENDLER, G., 1979: Reproductive periodicity of ophiuroids (Echinoderrnata: Ophiuroidea) on the
Atlantic and Pacific Coasts of Panam& In: S . STANCYK (Ed.), Reproductive Ecology of Marine
Invertebrates. Belle W. Baruch Library in Marine Science No. 9.; University of South Carolina
Press, Columbia, South Carolina: 145-156.
- -, 1982: The feeding biology of Ophioderrna brevispinurn (Ophiuroidea: Echinoderrnata). In:
J. M. LAWRENCE (Ed.), Echinoderms: Proceedings of the International Echinoderms Confer-
ence, Tampa Bay. A. A. Balkema, Rotterdam: 21-27.
MLADENOV, P. V., 1983: Breeding patterns of three species of Caribbean brittle stars (Echinoder-
rnara: Ophiuroidea). Bull. Mar. Sci., 33: 363-372.
STANCYK, S . E., 1974: Life history patterns of three estuarine brittlestars (Ophiuroidea) at Cedar
Key, Florida. Ph. D. Dissertation, University of Florida, Gainesville; 78 pp.
YAMASHITA, M. & F. IWATA, 1983: A quantitative analysis of the annual testicular cycle of the brittle-
star Arnphipholis kochii by means of autoradiographic investigation. Biol. Bull., 164: 327-340.