Formation and structural organization of the egg–sperm bundle of the scleractinian coral Montipora capitata
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- Padilla-Gamiño, J.L., Weatherby, T.M., Waller, R.G. et al. Coral Reefs (2011) 30: 371. doi:10.1007/s00338-010-0700-8
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The majority of scleractinian corals are hermaphrodites that broadcast spawn their gametes separately or packaged as egg–sperm bundles during spawning events that are timed to the lunar cycle. The egg–sperm bundle is an efficient way of transporting gametes to the ocean surface where fertilization takes place, while minimizing sperm dilution and maximizing the opportunity for gamete encounters during a spawning event. To date, there are few studies that focus on the formation and structure of egg–sperm bundle. This study explores formation, ultrastructure, and longevity of the egg–sperm bundle in Montipora capitata, a major reef building coral in Hawai‘i. Our results show that the egg–sperm bundle is formed by a mucus layer secreted by the oocytes. The sperm package is located at the center of each bundle, possibly reflecting the development of male and female gametes in different mesenteries. Once the egg–sperm bundle has reached the ocean surface, it breaks open within 10–35 min, depending on the environmental conditions (i.e., wind, water turbulence). Although the bundle has an ephemeral life span, the formation of an egg–sperm bundle is a fundamental part of the reproductive process that could be strongly influenced by climate change and deterioration of water quality (due to anthropogenic effects) and thus requires further investigation.
Coral reefs are productive and diverse habitats that provide shelter for an extraordinary biodiversity and services that support the economies of many island and coastal communities (Connell 1978; Moberg and Folke 1999). Coral ecosystems worldwide are severely threatened by climate change, pollution, and overexploitation (Hughes et al. 2003; Lough 2008). Both the persistence of healthy reefs and the recovery of coral populations impacted by severe environmental disturbance are dependent on gamete production, successful fertilization, development of viable offspring, and survival of new recruits (Richmond 1997). All of these processes are variable and are influenced by interactions between coral biology and spatial and temporal fluctuations in the environment (Tomascik and Sander 1987; Harrison and Wallace 1990; Richmond and Hunter 1990; Szmant and Gassman 1990; Hughes and Tanner 2000; Baird et al. 2009).
Broadcast spawning is the dominant form of sexual reproduction in scleractinian corals (Harrison and Wallace 1990; Baird et al. 2009). Broadcast spawners can be either gonochoric or hermaphroditic and can release their gametes independently or simultaneously. Approximately 65% of scleractinian coral species studied thus far are hermaphroditic broadcast spawners (Richmond and Hunter 1990; Guest et al. 2008; Baird et al. 2009), and of these, the majority package and release their gametes as positively buoyant egg–sperm bundles (Arai et al. 1993; Kinzie 1996). This is in contrast to brooding corals (which can also be gonochoric or hermaphroditic), where oocytes are fertilized inside the coral polyp and well-developed larvae are released (Harrison and Wallace 1990).
Gametogenesis and gamete structure have been examined in a number of coral species representing a range of reproductive modes (Harrison and Wallace 1990; Richmond and Hunter 1990); however, relatively few studies have focused on egg–sperm bundles. Those that have, reveal that each bundle contains anywhere from 6 to 180 oocytes depending on the species (Wallace 1985; Richmond 1997). For example, in the genus Acropora (clade Complexa), which includes mostly hermaphroditic spawners (Baird et al. 2009), bundles contain anywhere from 6 to 13 oocytes (Wallace 1985). These are arranged peripherally around a centrally located sperm mass (Wallace 1985; Vargas-Angel et al. 2006) and the gametes develop on different, but specific mesenteries (Wallace 1985). In contrast, in bundles released by Favites abdita (clade Robusta), oocytes and spermatocytes are intermingled and the gametes develop on the same mesenteries (Kojis and Quinn 1982).
Egg–sperm bundles disintegrate 10–40 min after reaching the surface of the water, releasing the gametes, and making them available for fertilization (Richmond 1997). To date, no studies have examined the ultrastructure or formation of the egg–sperm bundle. The structure and organization of the egg–sperm bundle is likely to influence the time required to break open, which has implications for fertilization success and opportunities for hybridization (Wolstenholme 2004).
Here, we use electron microscopy to address this knowledge gap in Montipora capitata (Dana 1846; family Acroporidae), a major reef building coral in Hawai‘i (Jokiel et al. 2004). This species belongs to the family Acroporidae and, like most members of this family, is a simultaneous hermaphrodite that broadcast-spawns egg–sperm bundles (Wallace and Willis 1994). This family dominates coral reef assemblages throughout the Indo-Pacific region and the Caribbean Sea and is extremely sensitive to environmental (e.g., thermal anomalies Hoegh-Guldberg 1999) and biological disturbances (e.g., crown of thorns predation, Pratchett et al. 2009). As such, the analysis of the ultrastructure and formation of egg–sperm bundles in this family contributes to our basic understanding of reproductive processes in an ecologically important group of corals and one that is increasingly threatened by climate change.
Materials and methods
Collections and preliminary analysis
Montipora capitata releases egg–sperm bundles during the new moon from late spring through summer in Hawai‘i (Hunter 1988). Egg–sperm bundles were collected from coral colonies on reefs adjacent to Moku O Lo‘e Island in Kane‘ohe Bay, Hawai‘i 1–2 days after the new moon during spawning events in June through August in 2007 and 2008. Coral fragments were collected and dissected every 5 days for 1 month prior to spawning in order to evaluate gamete maturity and symbiont acquisition by the oocytes. Pictures of coral fragments were taken using a dissecting microscope (Olympus, SZX7) equipped with an Olympus camera (MagnaFire SP S 99810). A subset of the collected gamete samples were observed under the dissecting microscope and compound microscope (Olympus, BX51), photographed and measured using Image J digital analysis software (NIH). The remaining collections were fixed for scanning and transmission electron microscopy as described below.
Transmission Electron Microscopy (TEM)
For transmission electron microscopy, specimens were fixed with 4% glutaraldehyde in 0.1 M sodium cacodylate buffer (with 0.1 M calcium chloride, 0.35 M sucrose, buffered to pH 7.4) for 48 h, washed in 0.1 M sodium cacodylate (with 0.4 M sucrose) for 3 times 30 min each, followed by postfixation with 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h. Tissue was dehydrated in a graded ethanol series (30, 50, 70, 85, 95, 100%), substituted with propylene oxide, and embedded in LX112 epoxy resin. Ultrathin (60–80 nm) sections were cut with a Reichert Ultracut E ultramicrotome, double stained with uranyl acetate and lead citrate, viewed on a LEO 912 EFTEM at 100 kV, and photographed with a Proscan frame-transfer CCD.
Field Emission Scanning Electron Microscopy (FESEM)
For scanning electron microscopy, samples were fixed, postfixed, and dehydrated in the same way as TEM samples. After ethanol dehydration, samples were critical point dried (Tousimis Samdri-795), mounted on aluminum stubs, sputter coated with gold/palladium to 5–8 nm thickness (Hummer 6.2), and viewed with Hitachi S-800 and Hitachi S-48000 field emission scanning electron microscopes.
Development of gametes
Spawning of Montipora capitata occurred between 2,145 and 2,200 h during the first quarter of the new moon in June, July, and August in 2007 and 2008. Approximately 2 h before spawning, the polyps relaxed (Fig. 1b) and expanded and were observed to produce mucus. Approximately 10–15 min before spawning, the egg–sperm bundles became visible beneath the oral disk. During spawning, the oral disk of each polyp became greatly extended, and the tentacles contracted (Fig. 1c). The egg–sperm bundles were squeezed through the polyp mouth and released into the water column. The release of bundles of the coral population in the field lasted 25–30 min.
Ultrastructure of the egg–sperm bundle
The longevity of an egg–sperm bundle is limited. It is formed a few hours before spawning (Wallace 1985) and breaks apart in less than 10–40 min after it has been released from the polyp. Although it has a short life, this bundle carries out the important function of transporting gametes to the surface and maximizing the chances of encounter between gametes with very different buoyancies (i.e., sperm is denser and sinks, whereas oocytes are generally positively buoyant). This strategy increases sperm availability and facilitates outcrossing (Harrison and Wallace 1990; Richmond 1997).
This work is the first systematic study of the ultrastructure of the egg–sperm bundles in scleractinian corals and presents important new observations to understanding bundle structure and formation. A layer of mucus is present around the oocytes and within the egg–sperm interface, and this material appears to be forming and holding the egg–sperm bundle together for ejection from the polyp coral. TEM observations revealed that there is a significant discharge of granule content from the oocytes, and this material has very similar electron density to the mucous layer, suggesting that bundle formation is achieved, at least in part, by the excretion of oocyte material.
The release of cortical material from oocytes in response to seawater has been observed in other invertebrates such as the polychaete Sabellaria vulgaris (Waterman 1936) and the crustacean Penaeus aztecus (Clark et al. 1980). In S. vulgaris, the newly shed eggs have a very irregular shape due to mechanical pressure in the coelomic cavities. After exposure to seawater, the egg membrane undergoes physical alteration, and the eggs become spherical. During this period, fertilization is limited. In P. aztecus, the contact of eggs with seawater results in a dramatic and massive release of a jelly precursor material from the cortical crypts (Clark et al. 1980). The jelly precursor material is made of 25–30% carbohydrate and 70–75% protein (Lynn and Clark 1987) and is believed to be responsible for the prevention of polyspermy and establishing a microenvironment inside the oocyte suitable for embryo development (Clark et al. 1980).
With the mucus being secreted by the oocytes, the energy investment from the coral polyp is minimized. More complex structures such as membranes (composed of phospholipids and proteins) would be both more difficult to produce in a short period of time and more energetically costly (Vance 2002; Voelker 2002). SEM observations of the mucous layer in the bundle (Figs. 5, 8) resemble SEM observations of the mucous floc and web material produced by the coral Porites astreoides (Ducklow and Mitchell 1979). Coral mucus is mostly composed of carbohydrates (Coffroth 1990) and to a lesser degree glycoproteins (Krupp 1985; Vacelet and Thomassin 1991) and lipids (Benson and Muscatine 1974; Crossland et al. 1980). Further studies of the structure and macromolecular composition in the bundle mucus layer are necessary.
TEM observations of the egg–sperm interface showed areas where mucus separated the oocytes and spermatozoa and other areas where this layer was minimal or otherwise not visualized. Regardless of the presence of mucus, no fusion of gametes was observed within the bundles. This is consistent with previous research that has found that self-fertilization is very uncommon in M. capitata (Hodgson 1988; Maté et al. 1997). It is also possible that, while inside the egg–sperm bundle, the oocytes possess biochemical blocks to self- or total fertilization or that cortical rearrangement and acquisition of spherical shape of the oocyte must occur before fertilization can take place (by which time spermatozoa have been released and dispersed through water currents to other colonies). Hodgson (1988) found evidence that oocytes of M. capitata do not self- or cross- fertilize for at least 1 min after the bundle has broken open, and self-fertilization blocks have been reported to last 3 h or more in Favia pallida, Platygyra pini, and P. ryukyensis (Heyward and Babcock 1986).
Most egg–sperm bundles broke open within 30 min after release, which is approximately the time that the spawning events lasted. Agitation of water or exposure to higher temperatures accelerated the bundle breakage. However, faster breakage does not necessarily mean more or faster fertilization. On the contrary, when the egg–sperm bundles broke quickly (i.e., by higher wave turbulence), the spermatozoa mass separated from the oocytes and sank before the spermatozoa were released. Sinking of the sperm mass thus likely reduced the likelihood of these sperm encountering oocytes because oocytes are positively buoyant and remain on the surface, while the spermatozoa sink in the water column and became diluted at depth. Thus, environmental conditions during spawning events have the potential to significantly influence egg–sperm bundle breakage and fertilization rates and may diminish or promote local reproductive success.
The release of deformed oocytes toward the end of the spawning season (August) did not coincide with unusual environmental conditions, and colonies releasing deformed oocytes were found adjacent to colonies that produced regular egg–sperm bundles. These observations raise a number of interesting questions regarding the abiotic and biotic conditions leading to the production of deformed oocytes, whether or not the deformed oocytes are viable, and the selective value of releasing deformed oocytes rather than reabsorbing them. Reabsorption of unspawned oocytes has been described for brooding (Rinkevich and Loya 1979) and spawning (Neves and Pires 2002) corals and could be an important means of conserving nutrients. If the deformed oocytes result from stress, releasing deformed oocytes could be either a response with beneficial value (i.e., release of a toxin via the gametes), or an indication of impaired reproductive capacity. Failure of bundle formation and release of prematurely aborted eggs were observed in Leptoria phrygia in response to stress (Kojis and Quinn 1982). Elucidating the factors leading to the failure of bundle formation and production of deformed eggs and their occurrence in nature could prove to be an important component of understanding the dynamics of coral populations.
The formation of an egg–sperm bundle and synchronicity of spawning are critical aspects for the reproductive success of broadcast spawners, which represent the majority of coral species (Fadlallah 1983; Harrison and Wallace 1990; Baird et al. 2009). These two aspects increase the abundance of female and male gametes in seawater and increase the chances of outcrossing. The selective advantage of the egg–sperm bundle in corals is unequivocal and is exemplified by its occurrence in many species of corals from both the Robust and Complex clades (Kerr 2005). However, it is unclear if the egg–sperm bundle has been acquired or lost several times throughout evolution. Bundles from the two coral clades show differences in structure, with bundles of Acroporids (clade complexa) having the oocytes around the sperm package, whereas bundles of Favites sp. (clade robusta) have the oocytes embedded within the sperm cluster (Kojis and Quinn 1982; Wallace 1985; Harrison and Wallace 1990; Richmond 1997). Interestingly, in both clades, the oocytes released in the bundle do not have a visible germinal vesicle (Kojis and Quinn 1982, this study), and it is unknown if this observation is associated with the process of bundle formation and/or the release of cortical material in response to seawater. Future studies on bundle structure of both clades may provide insights into different mechanisms of bundle formation and evolution of reproductive strategies in corals.
Little is published on the egg–sperm bundle due to its ephemeral lifespan. However, the failure of bundle formation could have detrimental effects on the reproduction and success of coral populations. As climate change and water quality deterioration (due to anthropogenic effects) continue, environmental cues for spawning may change and impact the different steps of the reproductive cycle. Bundle formation and breakage are critical components that can be strongly influenced by the environment and thus require further investigation.
Special thanks to F. Cox for her advice and encouragement during the earlier stages of this project, to G. Carter and M. Hagedorn for collection assistance and to K. Stender and P. Duarte-Quiroga for pictures in the field. Thanks to all the wonderful volunteers that helped to collect the samples during the spawning events, especially the 2007 Edwin W. Pauley Summer Program students at HIMB. Thanks to R. Kinzie, K. J. Eckelberger, C. Smith, R. Bidigare, and two anonymous reviewers for their helpful comments. JLPG was supported by a CONACYT Graduate Research Fellowship and the GEF-World Bank Coral Reef Targeted Research Project. This is HIMB contribution number 1397 and SOEST contribution number 7983.