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. 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