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JEZ 829
THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:189–200 (1997)
Specificity of Sperm Chemotaxis Among Great
Barrier Reef Shallow-Water Holothurians
and Ophiuroids
RICHARD L. MILLER*
Department of Biology, Temple University, Philadelphia,
Pennsylvania 19122
ABSTRACT
Sperm chemotaxis and sperm motility activation by egg or ovarian extracts were
demonstrated in 24 holothurians (six genera from three families) and 22 ophiuroids (eight genera
from five families) from the Australian Great Barrier Reef. Specificity was observed mainly at the
family level in holothurians, but a single case of specificity at the species level was found in the
genus Bohadschia. No recognizable specificity was found between any of the currently recognized
subgeneric groups within the genus Holothuria. A similar pattern of specificity was previously
observed in a group of dendrochiridotid holothurians in temperate waters.
In contrast to the holothurians, specificity in ophiuroids existed mainly at the genus or species
level. The best case of specificity at the species level was among six species in the genus Macrophiothrix which were reciprocally tested and a further three species in the same genus which were
partially tested. Examples of partial specificity at the species level were also found in the general
Ophiarthrum and Ophiocoma. The sperm chemotaxis assay reliably sorted to species a random
collection of unidentified Macrophiothrix ophiuroids. These results suggest that sperm chemotaxis
may play a role in gamete recognition prior to fertilization in one group of echinoderms. J. Exp.
Zool. 279:189–200, 1997. © 1997 Wiley-Liss, Inc.
Sperm chemotaxis has been described in one
or more genera within five invertebrate phyla
(reviewed by Miller, ’85a,b). Most of the species
of hydromedusae and hydroids, molluscs, asteroids, holothurians, and ascidians used in these
studies were collected in the eastern North Pacific (Miller, ’79, ’82a,b, ’85a,b). The patterns of
specificity differed in significant ways in each phylum. In the Cnidaria, the hydrozoa showed specificity of sperm chemotaxis mainly at the level of
the genus (Miller, ’66, ’79). In the Mollusca, specificity was completely absent in the polyplacophora
(chitons) (Miller, ’77). However, two species of
Dreissenid (zebra) mussels possessed complete
specificity of sperm chemotaxis at the species level
(Miller et al., ’94).
Sperm chemotaxis specificity in three groups of
northeast Pacific echinoderms showed that all
genera within the asteroid (starfish) families
Asteriidae and Solasteridae cross-reacted strongly
with each other, but no cross-reactivity was observed between them and members of any of the
other families tested (Miller, ’85b). The primarily
dendrochirotid holothurians (sea cucumbers) from
the northeast Pacific also showed considerable
sperm attractant cross-reactivity irrespective of
© 1997 WILEY-LISS, INC.
genus (Miller, ’85b). Two northeast Pacific ophiuroids (brittle stars) showed specificity at the genus level (Miller, ’85b). Northeast Pacific coast
tunicates showed cross-reactivity at the species
level but were specific at the family level (Miller,
’82b). These patterns fall into two groups: 1) reciprocal species or generic specificity (hydroids,
a few ophiuroids, and fresh water bivalves) and
2) specificity generally at the level of the family but sometimes between classes, with much
cross-reactivity between closely related genera or
species (corals, chitons, holothurians, asteroids,
and tunicates).
In 1992, sperm chemotaxis was found to be induced by egg extracts of many species of mass
spawning scleractinians (hard corals) of the Great
Barrier Reef (Coll and Miller, ’92; Miller, unpublished data). Although species specificity of sperm
chemotaxis seemed to be present in a few cases
(Coll and Miller, ’92; Coll et al., ’94), it was otherwise largely absent (Miller, unpublished data).
*Correspondence to: Richard L. Miller, Department of Biology,
Temple University, Philadelphia, PA 19122. Email: STARFISH@
ASTRO.OCIS.TEMPLE.EDU
Received 10 January 1997; Revision accepted 9 May 1997.
190
R.L. MILLER
Sperm chemotaxis was also demonstrated in many
shallow water reef holothurians and ophiuroids
of the Great Barrier Reef. Specificity occurred primarily at the family level in holothurians, a situation resembling the results obtained in eastern
north Pacific waters (Miller, ’85a). In contrast,
sperm chemotaxis specificity in ophiuroids occurred primarily at the genus and species level.
The specificity at the species level was almost complete between several sympatric species in the genus Macrophiothrix. The results reveal that sperm
chemotaxis has evolved in a highly specific manner in this group, possibly for gamete recognition
prior to fertilization.
MATERIALS AND METHODS
Collection of animals
Holothurians were collected during reef walks
or scuba dives at Orpheus Island (Central Section, GBR: 18° 3´S, 146° 30´E) from October to
December 1984–1989 and at Heron Island (Capricornia Section, GBR: 23° 27´S, 155° 53´E) in December 1988–1992. Deepest dives were to 15 m.
Some specimens were also collected at Davies Reef
(13° 15´S, 144° 27´E) off Townsville, Queensland.
Ophiuroids were collected by snorkeling or diving
at depths of 1–5 m at Lizard Island (Northern
Section, GBR 14° 40´S, 145° 28´E) in November
from 1992–1994 and at Orpheus Island in October 1985 and 1993. The animals were found by
turning over coral rubble blocks or breaking up
dead coral skeletons. They were held in running
seawater in the laboratory until use.
Taxonomy
Species of holothurians were easily identified
using field guides (Cannon and Silver, ’86; Guille
et al., ’86; Rowe, ’69; Rowe and Doty, ’77) and spicule analyses where visual identification was uncertain. Some color variation was observed in a
few populations. The taxonomic scheme which recognizes numerous subgenera within the large genus Holothuria (Diechmann, ’58; Rowe, ’69) has
been used in the present paper. Ophiuroids were
identified using Hoggett (’91), Guille et al. (’86),
Clark and Rowe (’71), Devaney (’70), and Clark
(’38). Although most of the ophiuroid genera and
species were well-known reef inhabitants, all identifications were confirmed by Dr. A. Hoggett.
Forty-five species of the ophiuroid genus Macrophiothrix are thought to exist within the Indo-west
Pacific. Many species show some color polymorphism and are difficult to identify using morphological criteria. Seventeen Australian species have
been carefully described recently using general
morphological criteria coupled with skeletal elements viewed with the scanning electron microscope (SEM) (Hoggett, ’91). Two were new species,
five were previously unknown in Australian waters, and seven appeared to be endemic to Australia. Allozyme studies on 21 species within the
family Ophiotrichidae have supported the validity of all but two pairs of morphologically indistinguishable species (Hoggett, ’90).
Morphological and allozyme character analysis
of the Macrophiothrix lineage within the Ophiotrichidae indicated that the genus evolved recently
relative to other genera in the family (Hoggett,
’90). The diagnostic characters of the genus are
homogeneous despite the large number of species
and subgenera described so far. The subgenera
Ophiothrix (Placophiothrix) and Ophiothrix (Keystonea) appeared to have diverged quite early in
the Macrophiothrix line, but their character states
strongly suggest that they should be merged into
the genus Macrophiothrix (Hoggett, ’90). Following this reasoning, the single Keystonea species
used in this study, Ophiothrix (Keystonea nereidina) is listed in the figures, tables, and text as
Macrophiothrix nereidina. The subgenus Ophiothrix (Placophiothrix) was unavailable. Even if the
Keystonea subgenus is removed from the Macrophiothrix species group used in the present study,
the sperm chemotaxis species-specificity pattern
described in the present paper would not be materially altered.
Obtaining gonads and gametes
No method is currently known for induction of
spawning in ophiuroids or holothurians that is as
simple and reliable as those used for sea urchins
or asteroids (Smiley and Cloney, ’85). Because the
sexes of both holothurians and ophiuroids were
externally indistinguishable, entire ovaries or testes sacs or tubules were collected through small
incisions in the body wall. This procedure produced an unambiguous sex determination, decreased the likelihood of evisceration in the
holothurians, and permitted frequent subsampling
of fresh sperm from males of both classes. The
incisions usually healed within a week, and most
animals appeared healthy when returned to the
reef at the end of the research period. In a few
cases, indicated in Figures 1 and 2, both sexes of
a particular species could not be found, and some
holothurians found in sexual condition one year
were spent during the same time period in subsequent years.
ECHINODERM SPERM CHEMOTAXIS
Eggs released from dissected ovaries were often fragile and sticky after release into seawater,
so the entire tissue mass, plus adherent seawater, was dropped into alcohol. This procedure carried over various amounts of adherent coelomic,
digestive, and ovarian somatic tissue. Although
the latter could not be removed, nearly all of the
coelomic lining and digestive material was removed from the samples before alcoholic extraction. Alcoholic extraction of somatic tissues alone
revealed the same lack of sperm-attracting activity reported previously in other echinoderm species (Miller, ’85b), as long as the ovaries were not
disturbed during the dissection. Females of the
smaller species of ophiuroids were often completely ovariectomized to obtain sufficient eggs and
oocytes and were usually killed in the process. The
diameters of at least 30 of the largest oocytes which
fell off the ovaries after slight agitation were measured under a compound microscope to provide an
estimate of the ripeness of the ovary and oocyte size
variation between species.
Testes sacs were incubated at room temperature in 0.5–1.0 ml of seawater immediately after
removal from the animal. If the sperm were not
released naturally, individual sacs were torn open
to permit the sperm to ooze out. Microliter samples of semen were diluted into a flat drop of water on a microscope slide to check sperm motility.
Sperm was generally used fresh because sperm
motility was usually lost within 30 min after dilution. Sperm longevity was considerably increased if the semen from the testes remained
undiluted until just before addition to the observation slide. Useful numbers of holothurian sperm
regained normal motility after being stored undiluted at 4°C for 12 h, but this treatment killed
ophiuroid sperm.
Preparation of extracts with sperm
attracting activity
Ovarian extracts were prepared only if the oocytes within the ovaries were large and pigmented
and had prominent germinal vesicles, but ovaries
of rare or very small species were collected and
extracted if any oocytes were visible in the ovaries. Ovaries from one or more individual females
were removed with forceps and placed directly into
five times their volume of 95% alcohol to extract
the sperm attractant. Carry-over seawater volume
ranged as high as 20% of the final extract volume, especially where ovarian material was very
limited or highly fragmented. Effects on sperm
behavior due to carry-over seawater were mini-
191
mal because of the extract dilution procedure (see
below) and the fact that excess salinity seawater
in the extracts was easily observed, since it
blocked sperm motility.
Sperm attraction bioassay
The bioassay for sperm chemotaxis was based
on visual observation of sperm behavior close to
a micropipette injecting a seawater-solubilized,
dried ovarian extract and has been described in
detail elsewhere (Miller, ’79, ’85a; Miller and King,
’83). Alcohol extracts many types of compounds
from cells. Since sperm never experience many of
these compounds or such high concentrations of
organic materials in nature, it was important to
avoid exposure of cells, glassware, and injection
micropipettes to damaged eggs or high concentrations of extract. The assay procedure always began with a test of seawater alone (blank control).
An aliquot of the extract being tested was then
diluted until its biological activity matched the
blank control. Testing for species specificity always began with extract dilutions that were just
higher than the known threshold onset of biological activity.
Sperm activation responses were also assayed
using sperm that were released from the testes
in a nonmotile condition. Injection of a biologically active extract into such a preparation induced a wave of sperm motility activation which
spread outward from the affected area with decreasing intensity (Miller, ’66). Seawater injected
in the same manner had no effect on the sperm.
In a few cases, homotypic sperm and egg extracts
tested negative for both sperm activation and
sperm chemotaxis. Such sperm were probably immature, since motility was not observed 10, 20,
or 60 min later.
The holothurian data set represents the combined results of collections and bioassays made
during October–November in the years 1984–1985
and 1987–1994. Because the number of holothurian species collected at the different locations varied, it was impossible to repeat every bioassay
combination each year, though all were repeated
several times during the course of the study. The
ophiuroid assay combinations were completely repeated using freshly prepared extracts in the years
1992–1994. The titers obtained during repeats
were generally equivalent to those of previous
years because it is almost impossible to change a
titer number in a limited capacity vial by addition of further ovaries. The amount of added extractable material has to be at least double that
192
R.L. MILLER
already present. Evaporation of the alcohol volume to allow addition of more ovaries was not attempted once the vials were more than half full
because it was not certain if the sperm-attracting
activity was stable.
Quantification of the response
A titer number was defined as the number of
dilutions which produced a just-visible attraction
or activation response. Titers were determined for
the following combinations of sperm and ovarian
extracts: 1) sperm vs. the oocyte extract of the
homotypic species (homotypic reaction), 2) sperm
vs. the oocyte extracts of each available heterotypic species (heterotypic reaction), and 3) oocyte
extracts of each species vs. sperm from each of
the other available species (heterotypic reaction). The titer number for sperm activation is
usually higher than for attraction by three to
four dilution steps (Miller, ’66; unpublished observations). The titer number can be converted
to units of activity by taking it as a power of two
(a titer of 10 = 210 = 1,024 units; a titer of 15 =
215 = 32, 768 units).
A distinction was made between low-level heterotypic cross-reactions and the normal homotypic
chemotactic response (Miller and King, ’83). The
maximum number of serial dilutions required to
activate but not attract sperm shed in an immobile condition was found to be four (equivalent to
16 units of activity) (Miller, ’66). If an obvious
sign of attraction was defined as a titer of one,
then the previous nonattraction titer was one-sixteenth, or 6.25%, of the titer required for attraction. For example, a homotypic test produced an
extract titer of 17 (131,072 units), and the same
extract gave a heterotypic titer of 12, or 4,096
units (3.1% of 131,072). Rounding off to the nearest titer number (6.25% of 131,072 = 8,192) gives
a cross-reactivity titer 13. On this basis, a heterotypic titer less than 6.25% of the homotypic assay was assumed to be due mainly to sperm
activation responses rather than attraction. It was
not possible to accurately distinguish the homotypic
sperm-attraction behavior from that of the heterotypic response because the rapid sperm flagellar movements leading to directional changes
could not be followed by eye.
RESULTS
Holothurians
Results of comparative testing of sperm-attracting or -activating activity of egg extracts from 24
Great Barrier Reef holothurians in two major
families of the order Aspidochiridota: the Stichopodidae (genera Stichopus and Thelenota) and the
Holothuriidae (genera Holothuria, Bohadschia,
and Actinopyga)—are shown in Figure 1. The
sperm chemotaxis assay generally distinguished
these animals at or above the genus level.
Stichopodidae
Reciprocal testing of species of Stichopus was
not possible because animals with ripe sperm or
ovaries were not available during most years.
Strong cross-reactivity occurred between the two
species (S. chloronotus and S. variegatus) which
were most thoroughly tested. Two Stichopus
variegatus color morphs were noted, one with a
light green integument with scattered small black
spots, the other with a darker green integument
with variegated, curved, thin black stripes. A clear
distinction between them was not made in the
available taxonomic guides, nor could any be found
using sperm chemotaxis. No significant cross-reactivity was found between the genera Thelonota
and Stichopus, but strong cross-reactivity occurred
between the two species of Thelenota tested. Complete species specificity was observed between an
egg extract of the eastern north Pacific Parastichopus californicus and the sperm of the tropical Pacific species Stichopus variegatus and S.
chloronotus. There was no significant cross-reactivity between any members of the families
Stichopodidae and Holothuriidae.
Holothuriidae
Reciprocal testing of active extracts and sperm
from one or more members of each of the subgenera within the large genus Holothuria—H. (Acanthotrapeza), H. (Platyperona), H. (Thymiosycia),
H. (Halodeima), H. (Mertensothuria), H. (Lessonothuria), and H. (Microthele)—revealed many
heterotypic cross-reactions and no obvious pattern
of specificity between these species (Fig. 1).
Twenty-seven cross-reactions out of 130 tests between species in the genus Holothuria and species in the genera Bohadschia and Actinopyga
were also found. In every case but three, the heterotypic titer was only 0.2% of the homotypic. B.
graffei sperm produced a substantial cross-reaction with H. nobilis extract (216), but lack of sperm
prevented homotypic assay of the H. nobilis extract.
Actinopyga palauensis provided sperm for testing against a wide variety of Holothuria extracts.
This species’ sperm showed some cross-reactivity
ECHINODERM SPERM CHEMOTAXIS
193
Fig. 1. Species specificity of sperm chemotaxis in shallow-water holothurians of the Great Barrier Reef of Australia. The columns and rows highlighted with bold separate
different species or groups of species that belong to different
families or are outliers used for comparative purposes.
Parastichopus californicus is one such outlier but also separates the species in the family Stichopodiidae from those in
the family Holothuriidae. Numbers are the titers (see text
for definition) of sperm-attracting activity. Spm, sperm; -, no
reaction; *, male not available; +, female not available; blank
areas indicate the test was not done; shaded areas and bold
titers indicate the homospecific test; titers in italics are >
6.25% of the corresponding homospecific test.
against the extracts of H. coluber, H. nobilis, and
Bohadschia graffei, but all the heterotypic titers
were less than 0.2% of the homotypic activity.
The only case of complete specificity at the species level in the holothuroidea was found between
two species in the genus Bohadschia. However, a
few minimal to extensive cross-reactions were
noted between one or the other of these two species and other genera: minimal (0.2% of the homotypic titer) between the ovarian extract of B.
argus and the sperm of H. verrucosa, mild (2.5%
of the homotypic titer) between the sperm of B.
graffei and the extract of H. nobilis, minimal between B. graffei sperm and the extract from
Actinopyga miliaris, and strong (100% of the B.
argus homotypic titer) between the sperm of B.
argus and the extract of H. nobilis. Only a single
member of the Chiridotidae was tested, and it
showed no cross-reactivity with any member of
the other families tested.
animals at the family level. Ophiarachnella
(Ophiodermatidae) was clearly distinguished from
Ophiolepis (Ophiuridae), which was distinguished
from Ophiarthrum (Ophiocomidae), which was
distinguished from all the members of the family
Ophiotrichidae. Four cross-reactions occurred between families out of 150 tests attempted for all
species, and all were minimal (<1% of the homotypic assay results). A homotypic response for
the single member of the Ophionereidae was not
obtained because the appropriate assay was not
carried out.
Comparisons at the genus level within families
revealed more cross-reactivity. Within the Ophiocomidae, neither Ophiarthrum pictum nor O.
elegans cross-reacted with Ophiocoma erinaceus,
which is in the same family. However, the sperm
of O. pictum cross-reacted with the extract of
Ophiomastix caryophyllata to a significant degree
(12.5%), and Ophiocoma scolopendrina sperm
cross-reacted even more substantially with the
Ophiomastix caryophyllata extract (25%). O.
erinaceus attractant and Ophiomastix caryophyllata sperm showed a cross-reaction, as did
O. erinaceus sperm and O. caryophyllata attractant, but both were < 3.0% of the homotypic titer. Ophiocoma erinaceus sperm cross-reacted
very strongly with Ophiocoma scolopendrina at-
Ophiuroids
Figure 2 shows the results of the application of
the assay method to 22 species of ophiuroids in
five families (Ophiodermatidae, Ophiuridae,
Ophiocomidae, Ophionereidae, and Ophiotrichidae) from the Great Barrier Reef. In general,
sperm chemotaxis distinguished most of these
194
R.L. MILLER
Fig. 2. Species specificity of sperm chemotaxis in shallowwater ophiuroids of the Great Barrier Reef of Australia. The
columns and rows highlighted in bold separate groups of species in different families: species 1–3, Ophiodermatidae; 4,
Ophiuridae; 5–10, Ophiocomidae; 11, Ophionereidae; 12–22,
Ophiotrichidae. Numbers are the titers (see text for definition)
of sperm-attracting activity. Spm, sperm; -, no reaction; *, male
not available; +, female not available; titers in italics are >6.25%
of the corresponding homospecific test; blank areas indicate
the test was not done; bold titers in shaded areas indicate the
homospecific test. The isolated bold rectangle (lower right) contains all the Macrophiothrix species tested.
tractant (100%), but O. erinaceus attractant crossreacted much less with O. scolopendrina sperm
(12.5%). Despite the several cross-reactions, only
four of 37 possible cross-combinations carried out
within the Ophiocomidae showed significant
cross-reactivity (defined as < 6.25% of the homotypic test). None of these genera showed any
cross-reactivity with the genera Ophiothrix or
Macrophiothrix.
Substantial cross-reactions between genera
within the family Ophiotrichidae were also noted
between the Ophiothrix trilineata egg extract and
Macrophiothrix lorioli or M. rhabdota sperm. Both
titers were 12.6% of the homotypic reaction for
O. trilineata but 50% of the homotypic reactions
of the two species of Macrophiothrix. Of the 66
cross-combinations carried out within the family
Ophiotrichidae, only these four showed significant
cross-reactivity between species or genera.
Slight to extensive specificity at the species level
occurred between species within some genera, but
in most cases too few species were available for
the extensive testing required to confirm a pattern. Reciprocal cross-reactivity between two species in the genus Ophiarachnella was only 2%,
suggesting that substantial specificity existed between these two species. Ophiothrix trilineata
sperm cross-reacted with the egg extract from its
congener, O. ciliaris, but the cross-titer (211) was
only 6.25% of the homotypic reaction. This suggested that substantial specificity was present;
however, males of O. ciliaris were not available
for reciprocal testing. Although cross-reactivity
between two members of the genus Ophiarthrum
(O. pictum vs. O. elegans) in the Ophiocomidiae
was nearly complete, some specificity may be
present because the cross-titer between O. pictum
sperm and O. elegans extract (210) was only 6.25%
of the homotypic titer (214).
Sperm chemotaxis as a taxonomic
character in ophiuroids
In contrast to the above comparisons, testing
within a single species-rich ophiuroid genus (Macrophiothrix: Family Ophiotrichidae) revealed a
case of sperm chemotaxis specificity at the species level that may be useful as a taxonomic
character (Fig. 2). Specificity was first noticed in
the genus Macrophiothrix when two unidentified
species with strong similarity in external morphology were tested at Orpheus Island in 1985.
Complete species specificity of sperm attraction
was found. The two species were later identified
as M. sp. nov., a new species which has yet to be
formally described (A. Hoggett, personal communication), and M. lorioli.
A second test of specificity within the genus was
attempted in 1993 at Orpheus Island using 14
ECHINODERM SPERM CHEMOTAXIS
randomly collected Macrophiothrix specimens. Six
males and eight females were individually separated into labeled containers and treated as described in Materials and Methods. All possible
combinations of sperm and egg extracts were
tested for specificity of sperm chemotaxis. Following testing, each animal was preserved in
alcohol and code-labelled for subsequent independent taxonomic identification by A. Hoggett
(Table 1).
The sperm of male A (11/13B) was strongly positive only to the M. koehleri extract when tested
against ovarian extracts of M. lorioli and M. koehleri previously prepared at Lizard Island. This animal died before it could be tested against any
other extracts. Male B (11/13C) sperm was tested
against the same M. lorioli and M. koehleri egg
extracts and was very weakly positive (1.6% of
the homotypic titer) to the M. koehleri extract but
negative to the M. lorioli extract.
When the sperm of each of the remaining four
males was tested against all eight Orpheus Island female extracts, the animals sorted into
three groups:
195
All these animals were later identified as M.
koehleri.
2. Individual sperm samples from the three
males C (11/14E), E (11/14G), and F (11/15A)
cross-reacted with the Lizard Island M. lorioli extract and also responded positively only to egg
extracts from six Orpheus females, G (11/13A), I
(11/14C), K (11/14I), L (11/14J), M (11/14K), and
N (11/16A). All of these animals were later identified as M. lorioli.
3. Sperm from male B (11/13C) responded only
slightly (1.6% of the homotypic value) to the M.
koehleri extract and not at all to the M. lorioli
extract from Lizard Island. Its sperm did not respond at all to extracts from any of the M. koehleri or M. lorioli females collected at Orpheus
Island. This male was later identified as the same
species as M. sp. nov. mentioned above.
In November of the years 1992–1994, a more
complete testing of Macrophiothrix species specificity was carried out using six species collected
at Lizard Island each year. (Insufficient gonadal
material was available to permit complete testing of three additional species, since only one M.
belli (male), one M. virgata (male) and one M.
propinqua (female) were collected.) The results are
included as part of Figure 2.
Specificity of sperm attraction was nearly com-
1. Sperm from male D (11/14F) cross-reacted
with the Lizard Island M. koehleri oocyte extract
and responded positively only to Orpheus Island
extracts from females H (11/14B) and J (11/14D).
TABLE 1. Species specificity of sperm chemotaxis among fourteen randomly collected individuals of Macrophiothrix1
Females
M. lorioli2
Male
Macrophiothrix A
Macrophiothrix B
Macrophiothrix C
Macrophiothrix D
Macrophiothrix E
Macrophiothrix F
Retrial of B and D males
B males
D males
M. koehleri2
G
H
I
J
K
L
M
N
—
—
7
—
7
7
12
6
—
12
—
—
nd
—
11
—
11
11
nd
—
—
14
—
6
nd
—
13
—
12
12
nd
—
—
16
—
—
nd
—
12
—
11
12
nd
—
12
—
12
13
nd
—
12
—
11
11
—
nd
10
nd
nd
nd
—
—
6
12
—
—
—
13
—
—
—
14
—
—
—
—
—
—
nd
nd
Key to individuals
Code
Male
Species
A
B
C
D
E
F
11/13B
11/13C
11/14E
11/14F
11/14G
11/15A
M. koehleri
M. n. sp.
M. lorioli
M. koehleri
M. lorioli
M. lorioli
1
2
Code
Female
Species
G
H
I
J
K
L
M
N
11/13A
11/14B
11/14C
11/14D
11/14I
11/14J
11/14K
11/16A
M. lorioli
M. koehleri
M. lorioli
M. koehleri
M. lorioli
M. lorioli
M. lorioli
M. lorioli
Numbers are the titer (see text for definition) of sperm attracting activity. nd, test not done; —, no reaction.
M. lorioli and M. koehleri extracts prepared at Lizard Island.
196
R.L. MILLER
plete at the species level between all six species
where both sexes could be tested. The same specificity pattern and almost identical titer levels were
obtained in each of the three trials made 1 year
apart. Of the 51 combinations tested (including
the gametes obtained from the three additional
species mentioned above), only one substantial
cross-reaction was found (M. lorioli extract × M.
rhabdota sperm = 211, or 25% of the homotypic
test). This tally omits the substantial cross-reactions between the sperm of M. rhabdota or M. lorioli and the ovarian extract of Ophiothrix trilineata
described earlier. In all tests using the six Macrophiothrix species, there was either no cross-reaction or the heterotypic activity was 1.5% or less of
the homotypic test. The partial pattern using the
three additional species suggests they will most
likely follow the same specificity pattern.
Summary of results
Holothuroidea
Of the 24 species available, totaling 676 possible combinations (Table 2), 264 tests could not
be run for lack of material, and seven species could
not be tested or were homotypic negative. Of the
412 tests carried out, 252 were specific at the family or genus level, 130 showed mainly minor (<
6.25%) heterotypic reactions, 128 showed significant
cross-reactions (>6.25%) and twelve of these had
heterotypic titers significantly higher than the homotypic. Two species in the genus Bohadschia were
specific at the species level, but four heterotypic reactions between these two species and other members of the genus Holothuria were observed.
Ophiuroidea
Of the 22 species available (for a total of 484
possible combinations) (Table 2), 183 tests were
not run for lack of material, and seven species
could not be tested or were homotypic negative.
Of the 301 tests carried out, 54 of the 290 that
were specific at the family or genus levels yielded
minor (<6.25%) heterotypic cross-reactions. A further ten showed significant (>6.25%) cross-reactivity within genera or families. One test was
homotypic negative, while 237 tests were heterotypic negative. Ten of the 15 ophiuroid species
tested bidirectionally showed specificity at the species level. Six were in the genus Macrophiothrix.
Stability of sperm-attracting and
motility-activating activity
The sperm-attracting activity of most of the species used was tested for chemical stability by exposing the alcoholic extracts to normal room
temperatures for periods of up to a year as the
result of long periods between access to fresh
sperm combined with unforeseen shipping and
storage problems. Holothurian ovarian extracts
were found to be stable at room temperature for
at least 1 year, but ophiuroid extracts lost most
or all attracting activity after standing at room
temperature for over a month. Frozen samples retained up to 50% of their activity after a year.
DISCUSSION
Application of sperm diffusion models (Vogel et
al., ’82) to laboratory and field studies of fertilization success supports the conclusion that sperm
TABLE 2. Summary of sperm chemotaxis species specificity in Great Barrier Reef Holothuroids and Ophiuroids
Holothuroidea
Ophiuroidea
Total number of species
Not tested or homotypic negative1
Total number of species tested
Total all combinations
Tests not run
26
7
19
676
264 (39%)
22
7
15
484
183 (38%)
Total tests run
412 (61%)
301 (62%)
Negative
Homotypic negative1
Mass action (<6.25%)
Significant cross reactions (>6.25%)
Homotypic (within genera)
Higher than homotypic2
Family and genus level specificity
Species-level specificity
154
2
130
128
92
12
252
2/19
1
(37% of 412)
(32%)
(31%)
(61%)
(11%)
237
1
54
10
10
0
290
10/15
(79% of 301)
(18%)
(3%)
(97%)
(67%)
Homotypic negatives were caused by lack of sperm motility.
Assays yielding one dilution higher than the homotypic case are not included because the assay error is plus/minus one dilution.
3
These two species still induced three heterotypic cross-reactions.
2
ECHINODERM SPERM CHEMOTAXIS
chemotaxis is of minimal or no importance during fertilization of free-spawning organisms (Babcock et al., ’94; Benzie et al., ’94; Denny, ’88; Denny
and Shibata, ’89; Levitan, ’93, ’95; Mead and
Denny, ’95). Studies on fertilization success in the
field using corals (Oliver and Babcock, ’92) or echinoderms (Pennington, ’85; Sewell and Levitan, ’92;
Babcock et al., ’92) have not demonstrated an enhancing effect of sperm chemotaxis (but see
Babcock et al., ’94). The technical difficulties of
measuring increased fertilization success or hybridization rates at low sperm and egg concentrations are considerable in the absence of
favorable biological situations.
Many species of benthic invertebrates in the
Great Barrier Reef spawn around the time of the
mass coral spawning or during the following new
moon (Harrison et al., ’84; Babcock et al., ’86, ’92).
Shallow-water echinoderm species have been observed to spawn simultaneously at different locations in the world and may aggregate for that
purpose (Babcock and Mundy, ’92; Hendler and
Meyer, ’82; Kubota, ’80; Holland, ’80; Minchin, ’87,
’92; Pearse et al., ’88; Sewell and Levitan, ’92).
Spawning times of different species recorded during multiphylum mass spawning on the GBR and
other evidence suggested that the gametes of
many species may be mixing during these events,
though partitioning of spawning in time and space
may also occur (Babcock et al., ’92; Heyward and
Babcock, ’86; Hodgson, ’88; Oliver and Babcock, ’92).
High species specificity of fertilization indicates
that adaptation of the sperm-egg recognition system to challenges by other species’ sperm has
probably occurred. Binding of the sperm to the
egg surface membrane was previously suggested
as the location of the critical specificity-determining step in fertilization (Summers and Hylander,
’75; Strathmann, ’81). Recent work using four
sympatric, sibling species of the sea urchin Echinometra matthei has confirmed that the binding
specificity-determining interactions occur between
the sperm acrosomal protein, bindin, and the egg
membrane–bound sperm receptor (Glabe and
Lennarz, ’79; Hofmann and Glabe, ’94; Metz et
al., ’94; Palumbi and Metz, ’91). Minor alteration
in the specificity of these molecules is the most
likely explanation for formation of new species
within widely distributed populations of marine
invertebrates which have long-distance larval dispersion (Palumbi, ’92). There are other mechanisms, such as egg investments that trap nonspecific
sperm, fertilizin reactions, specially shaped micropyle openings, and possibly sperm chemotaxis,
197
that confer a partial species specificity of fertilization prior to sperm-egg membrane contact
(Lillie, ’13; Collins, ’76; Yanagimachi et al., ’92;
Miller, ’96).
Like Echinometra, proper taxonomic identification of the participating species is critical, but,
for all the species used in the present study, only
morphological criteria were available for species
identification, even though most of the genera and
species used were well-known reef inhabitants.
Specificity of sperm chemotaxis is itself a taxonomic character, though in most cases a poor one.
This was demonstrated in most of the holothurians, which can be distinguished only at the family level except in the case of Bohadschia. In
contrast, the high specificity of sperm chemotaxis
at the species level in the Macrophiothrix ophiuroids produces a close match between independently determined morphological and molecular
characters (the sperm attractants). Such a high
species specificity suggests that selection for fertilization specificity based on sperm chemotaxis
has occurred in this group.
A differential increase in fertilization success
seems likely if sperm recognized eggs of their own
species at a distance under conditions where gametes of different species mix regularly or are rare
and/or retain fertilizing capacity for some time
(Miller, ’96). This development should lead to reduction in both the frequency of hybridization and
the amount of egg competition for sperm particularly at low gamete concentrations, because species-specific sperm chemotaxis acts to increase the
effective diameter of the egg and preferentially
encourages the approach of the appropriate species’ sperm (Levitan, ’93; see also Miller, ’85a;
Miller and King, ’83).
The lack of chemotaxis specificity as demonstrated in the holothurians and other species
(Miller, ’85b, unpublished data) may be the result
of two factors, one biological and the other an artefact. First, the spawning environment may have
been spatially and/or temporally partitioned so
that encounters with other species’ sperm are less
likely or impossible. Partitioning of the spawning
environment in both time and space can have
large effects on fertilization success and reduce
hybridization (Hendler, ’91; Oliver and Babcock
et al., ’92; Sewell and Levitan, ’92) and is known
to occur in ophiuroids and other echinoderms
(Babcock, ’92; Hendler, ’91; McEuen, ’88), corals
(Shlesinger and Loya, ’85; Szmant, ’86), and other
marine invertebrates. Investigating this possibility requires more knowledge of species-specific
198
R.L. MILLER
spawning times, modes of gamete release, relative positions of simultaneously spawning adults
of different species, local hydrography, and rates
of hybridization in the field than is currently available (Babcock et al., ’92).
Second, the realease kinetics, concentrations
and variety of compounds released by intact eggs
are surely different and under more subtle control than those released by damaged or extracted
eggs, so the use of crude alcoholic extracts probably decreases the accuracy of specificity testing.
The combined effects of the large variety and
amounts of compounds released by alcohol extraction on sperm membrane–bound chemoreceptors
(particularly when fat-rich eggs are used) might
produce high heterotypic titers even between
classes (Coll et al., ’94; Miller, ’85b, Miller, unpublished data). The asymmetric behavior of
the sperm flagellum during the attraction response is controlled by attractant-induced variations in cytosolic calcium surrounding the
axoneme (Cook et al., ’94). The presence in alcoholic extracts of nonspecific substances that bind
to membrane receptors might stimulate the opening of the calcium channels in the flagellar membrane that induce chemotactic behavior.
On the other hand, concentrations of these hypothetical contaminants may not be high enough
to compromise the level of specificity in all cases.
Crude extracts prepared from the eggs of hydrozoans yielded a pattern of specificity at the generic and even species level that matched the
available taxonomy, but testing at the species level
was compromised by the relatively few species
available in each genus (Miller, ’79, ’82a). In the
present study, new sets of crude ophiuroid ovarian extracts, prepared independently in each of
3 years, produced a pattern of sperm-attractant
species specificity which closely matched the available taxonomic knowledge. It is unlikely that either of these results was generated by chance.
Sperm chemotactic behavior has also been directly observed in response to living whole eggs,
egg fragments, or egg-associated structures (Carre
and Sardet, ’81; Miller, ’66, ’78). The sperm-attracting “cupule” structure found at the animal
pole of the siphonophore egg continues to release
sperm attractant well after fertilization (Carre
and Sardet, ’81); however, there is evidence that
sperm attractants function in a biologically controlled manner in at least some other cases. In
the hydromedusan Orthopyxis, sperm attractant
is released from the egg at the time of second polar body emission. The supply of sperm attrac-
tant is rapidly shut off at fertilization (Miller, ’78).
Use of egg fragments revealed that a cytoplasmic
clock controlled the time of release of sperm attractant in the absence of a germical vesicle (Freeman and Miller, ’82). Intact eggs of the coral
Platygyra sp., placed in sperm suspensions from
the same or a different clone attracted sperm in a
manner that suggested the presence of attractant
specificity that could not be demonstrated using
crude extracts of coral eggs of the same species
(R. Miller and K. Miller, unpublished data).
Sperm attractants in the sea urchin Arbacia
punctulata and the starfish Pycnopodia helianthoides are peptides that are stable in alcoholic
solution (Ward et al., ’85; Miller, ’85b; Miller and
Vogt, ’96). The nature of the compounds acting as
ophiuroid or holothurian sperm attractants in the
alcohol extracts is at present unknown. This is
also the case for most of the sperm attractants
whose biological activities have been previously
described, including the attractants released at
specific sites on the egg surface or at specific times
(Carre and Sardet, ’81; Miller, ’78, ’85a,b). It was
not possible to determine whether sperm chemotaxis occurred directly to holothurian or ophiuroid
eggs before or after polar body emission. Reliable
spawning inducers are not known for either group,
and, although heat stress sometimes induces
spawning, the eggs may not develop normally
(Hendler, ’77, ’91; Hendler and Meyer, ’82; Miller,
unpublished observations). Attempts to use whole
eggs are unlikely to succeed without special efforts, because echinoid and asteroid sperm attractants or sperm motility activators (SAPs) (for review
see Suzuki, ’89) are associated with relatively
soluble egg jellies that are retained as egg surface
contaminants. When low concentrations of sperm
are added to single eggs, the sperm-attracting or
motility-activating jelly prevents clear observations
of the source or timing of sperm activation or attraction (Miller, unpublished observation).
The class Ophiuroidea, as exemplified by species in the tropical genus Macrophiothrix, provides
a group of sympatric organisms in which sperm
chemotaxis may play a discernable role in fertilization in nature. Studies of the behavior of low
numbers of sperm in response to maturing oocytes
and of fertilization rates during hybridization experiments should permit determination of the importance of sperm chemotaxis in fertilization
success in this group both in the laboratory and
in the field. Detailed comparisons of sperm behaviors during attraction to both homotypic and
heterotypic extracts are also needed, but the re-
ECHINODERM SPERM CHEMOTAXIS
sults may be difficult to interpret unless comparative observations of the sperm flagellum during
turning behavior are also made (Miller, ’85a,b).
ACKNOWLEDGMENTS
Numerous individuals helped in one way or another with this study. Foremost is Dr. Anne
Hoggett, whose taxonomic experience with ophiuroids was critical to the research. Ken Anthony
provided particularly valuable help at Lizard Island in 1994. Dr. Craig Mundy, Ms. Stephanie
Warrington, Ms. Debi Milham, and others helped
collect animals. Thanks also to Drs. Russell
Babcock and Peter Moran at the Australian Institute of Marine Science, Dr. Lyle Vail at the Lizard Island Research Station, Mr. Geoff Charles
at the Orpheus Island Research Station, Dr. Ian
Lawn at Heron Island Research Station, and Dr.
A.O.D. Willows, Friday Harbor Labs (University
of Washington), for their hospitality and aid during the progress of this research. This research
was supported by NSF grants INT-8714032 and
OCE-8410052.
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