Effects of temperature on fertilization and early cleavage of some tropical echinoderms,with emphasis on Echinometra mathaei

Select temperatures, above normal, are shown to reduce success of fertilization and normal early cleavage in the laboratory for the echinoderms Acanthaster planci (L.), Culcita novaeguineae Muller and Troschel, Linckia laevigata (L.), Echinometra mathaei (de Blainville), and Diadema savignyi Michelin. The data indicate that cleavage is more sensitive to increased temperature than is fertilization. Upper tolerance limits for early cleavage in most of the species examined is near 34.0°C. The early developmental stages of A. planci were the most sensitive to elevated temperature, and those of E. mathaei, the least sensitive. Further experiments with E. mathaei showed that unfertilized ova were still viable, dividing normally when fertilized after 2 h exposure at 36.0°C. The ova were significantly less viable after 3 h. Early cleavage stages of E. mathaei were resistant to 36.0°C for exposure times of up to 40 min, but were inhibited beyond this period. It is suggested that the ability of E. mathaei to develop normally at 34.0°C (6C° above ambient temperature) and to withstand limited exposure to 36.0°C may account for the wide distribution of this species in habitats which are often subjected to broad temperature fluctuations, such as reef flats.Contribution No. 46 from the University of Guam Marine Laboratory.     

Effects of copper on the coral-reef echinoid Echinometra mathaei

The effects of copper-enriched seawater on fertilization success, early cleavage, larval skeletal development, and survivorship of adults of the coral-reef echinoid Echinometra mathaei (de Blaiville) were tested. Fifty percent reduction of fertilization success and cleavage success to the 8-cell stage occurred in 0.18 and 0.42 mg added Cu⁺⁺/l, respectively. Adults had respective 48- and 96-h TL₅₀ values of 0.54 and 0.30 mg/l. Larval skeletal development was suppressed in 0.02 mg/l. The latter process appears to be very sensitive and may be the most suitable for assessing effects of stress on this species.Contribution No. 68, University of Guam Marine Laboratory.     

Reproduction of sympatric populations ofHeliocidaris erythrogramma andH. tuberculata (Echinoidea) in New South Wales

Reproduction ofHeliocidaris erythrogramma (Valenciennes) andH. tuberculata (Lamarck) was compared through examination of oogenesis, spermatogenesis and monthly measurement of the gonad index. These species occur sympatrically in the Sydney region. Their reproduction was examined at two sites near Botany Bay, New South Wales, Australia, from February 1989 through January 1990.H. erythrogramma produces buoyant, 450µm-diam eggs and the sperm have a head region 10µm in length. By contrast,H. tuberculata produces negatively-buoyant, 95µm-diam eggs and the sperm have a head region 4µm in length. Histochemical examination of the gonads revealed that periodic acid Schiff-positive (PAS +) material stored in the nutritive phagocytes appears to support vitellogenesis in both species. InH. tuberculata this material is utilized in the formation of PAS + yolk oligolecithal eggs, whereas inH. erythrogramma the PAS + material appears to be converted to lipid yolk in macrolecithal eggs.H. erythrogramma had a seasonal reproductive pattern with a 3 mo summer spawning period, whereas both populations ofH. tuberculata had a 9 mo breeding period characterized by the continual presence of nutrient reserves and vitellogenic oocytes which rapidly replaced spawned ova. Spawning ceased only for 3 mo over the summer. Due to the 9 mo spawning ofH. tuberculata it is not clear what factors serve to cue reproduction in this species.     

Dissolved histamine: a potential habitat marker promoting settlement and metamorphosis in sea urchin larvae

Many species of marine invertebrate larvae settle and metamorphose in response to chemicals produced by organisms associated with the adult habitat, and histamine is a cue for larvae of the sea urchin Holopneustes purpurascens. This study investigated the effect of histamine on larval metamorphosis of six sea urchin species. Histamine induced metamorphosis in larvae of three lecithotrophic species (H. purpurascens, Holopneustes inflatus and Heliocidaris erythrogramma) and in one planktotrophic species (Centrostephanus rodgersii). Direct comparisons of metamorphic rates of lecithotrophic and planktotrophic larvae in assays cannot be made due to different proportions of larvae being competent. Histamine (10 μM) induced metamorphosis in 95% of larvae of H. purpurascens and H. inflatus after 1 h, while the coralline alga Amphiroa anceps induced metamorphosis in 40–50% of these larvae. Histamine (10 μM) and A. anceps induced 40 and 80% metamorphosis, respectively, in the larvae of H. erythrogramma after 24 h. Histamine (10 μM) and the coralline alga Corallina sp. induced 30 and 70% metamorphosis, respectively, in the larvae of C. rodgersii after 24 h. No metamorphosis of any larval species occurred in seawater controls. Larvae of two planktotrophic species (Tripneustes gratilla and Heliocidaris tuberculata) did not metamorphose in response to histamine. Seagrasses, the host plants of H. inflatus, induced rapid metamorphosis in larvae of the two Holopneustes species, and several algae induced metamorphosis in C. rodgersii larvae. Histamine leaching from algae and seagrasses may act as a habitat marker and metamorphic cue for larvae of several ecologically important sea urchin species.     

Fertilization in a suite of coastal marine invertebrates from SE Australia is robust to near-future ocean warming and acidification

Climate change driven ocean acidification and hypercapnia may have a negative impact on fertilization in marine organisms because of the narcotic effect these stressors exert on sperm. In contrast, warmer, less viscous water may have a positive influence on sperm swimming speed and so ocean warming may enhance fertilization. To address questions on future vulnerabilities we examined the interactive effects of near-future ocean warming and ocean acidification/hypercapnia on fertilization in intertidal and shallow subtidal echinoids (Heliocidaris erythrogramma, H. tuberculata, Tripneustes gratilla, Centrostephanus rodgersii), an asteroid (Patiriella regularis) and an abalone (Haliotis coccoradiata). Batches of eggs from multiple females were fertilized by sperm from multiple males in all combinations of three temperature and three \textpH/P_\textCO2 {\text{pH}}/P_{{{\text{CO}}_{2} }} treatments. Experiments were placed in the setting of projected near-future conditions for southeast Australia, an ocean change hot spot. There was no significant effect of warming and acidification on the percentage of fertilization. These results indicate that fertilization in these species is robust to temperature and \textpH/P_\textCO2 {\text{pH}}/P_{{{\text{CO}}_{2} }} fluctuation. This may reflect adaptation to the marked fluctuation in temperature and pH that characterises their shallow water coastal habitats. Efforts to identify potential impacts of ocean change to the life histories of coastal marine invertebrates are best to focus on more vulnerable embryonic and larval stages because of their long time in the water column where seawater chemistry and temperature have a major impact on development.     

The response of two ecologically important Antarctic invertebrates (Sterechinus neumayeri and Parborlasia corrugatus) to reduced seawater pH: effects on fertilisation and embryonic development

Ocean acidification, or the lowering of seawater pH, is caused by sequestration of atmospheric CO₂ into the oceans. This study investigated the effects of present-day pH 8.0, predicted ocean surface pH for the years 2100 and 2300 (pH 7.7 and pH 7.3, respectively) and an extreme pH (pH 7.0) on fertilisation and embryogenesis in the Antarctic nemertean worm Parborlasia corrugatus and sea urchin Sterechinus neumayeri. Fertilisation success was not affected by pH in P. corrugatus across a range of sperm concentrations. Fertilisation success in S. neumayeri declined significantly in pH 7.0 and 7.3 seawater, but only at a low sperm concentration. Seawater pH had no effect on the rate of egg cleavage in S. neumayeri, or the proportion of abnormal embryos 1-day post-fertilisation. P. corrugatus embryogenesis was also relatively robust to pH changes, with a significant effect detected only when the seawater pH was decreased to 7.0. While fertilisation and early cell division were relatively robust, later development through to the gastrula was sensitive to pH. In S. neumayeri, an effect of pH on development was evident by the gastrula stage, while there were significantly more abnormal P. corrugatus embryos in pH 7.0 up to the blastula stage, and in pH 7.0 and pH 7.3 at the coeloblastula stage. Our results are similar to the observations on other marine invertebrate species where fertilisation and early embryonic development are generally robust to lowered seawater pH, while the older coeloblastula and gastrula stages are more responsive. We also found no evidence to suggest that Antarctic species are more adversely affected by lower seawater pH compared with the findings for non-Antarctic counterparts. We conclude that in the two species we examined, near-future decreases in pH (decreases of ≈0.3–0.5 pH units) may not have a significant effect on fertilisation and early embryogenesis, while predicted longer term decreases (decreases of ≈0.7–0.77 pH units) could reduce fertilisation success in S. neumayeri if sperm concentrations are low and may increase abnormalities in P. corrugatus during later embryogenesis.     

Effect of thermal stress and total residual chlorination on early life stages of the mummichog Fundulus heteroclitus

Effects of simultaneous short-term (7.5 to 60 min) thermal stress (24° to 34°C) and total residual chlorination (0.05 to 1.0 mg l^-1) on specific development stages of the mummichog Fundulus heteroclitus (Pisces: Cyprinodontidae), were investigated. For the embryonic stages, the total number of successfully hatched larvae was used as the criterion to measure effect. For the larval stages, survival 24 h after exposure was used. In the embryonic stages, temperature was the most important main variable. Only one embryonic stage (gastrula) was confounded by second-order interactions (temperature x duration of exposure x total residual chlorination). Both 0-day and 7-day-old larval stages showed significant higher-order interactions for all combinations of test parameters, suggesting the presence of synergistic effects of the three main experimental variables.Contribution No. 308 to the Gulf Breeze Environmental Research Laboratory. Contribution No. 183 to the Belle W. Baruch Institute for Marine Biology and Coastal Research.     

Temperature-time relationships for survival of embryos and larvae of Mulinia lateralis (Mollusca: Bivalvia)

The influence of temperature over 8 time intervals on survival of cleavage stages, trochophore larvae, and straighthinge veliger larvae of Mulinia lateralis (Say) was investigated using a thermal gradient apparatus. There was a direct relationship of mortality with increased period of exposure. As the clams aged, temperature tolerance increased, with cleavage stages being most sensitive to higher temperatures and straight-hinge larvae least sensitive. Multiple-regression equations were developed to allow prediction of percentage mortalities under different conditions of temperature and time exposure. Entrainment of M. lateralis embryos and larvae in the cooling-water systems of steam-electric power plants should be as short as possible to keep mortality toa minimum.Contribution No. 550 of the Natural Resources Institute, University of Maryland, and Contribution No. 531 of the Virginia Institute of Marine Science.     

Effects of perfluorooctane sulfonate on behavioural activity,regeneration and antioxidant enzymes in planarian Dugesia japonica

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and has been found to be the predominant perfluorinated chemicals (PFCs) in the environment. In the present study, planarian Dugesia japonica is employed to investigate the toxicity of this compound. The behavioural activity, survival, regeneration and antioxidant enzyme activities of the D. japonica exposed to PFOS were investigated. The results showed that the planarian D. japonica is very sensitive to low concentration pollutants including PFOS and suitable for toxicological bioassays. PFOS has severe toxicity in inducing oxidative stress, regeneration and development toxicity in planarians. The toxicity is indicated by reduced planarian locomotor velocity (pLMV), reduced appearance of auricles in regenerating animals, changed in antioxidant enzyme SOD and CAT activities. The information presented in this study will help elucidating the PFOS-induced toxicity in aquatic organisms.     

Mycosporine-like amino acids prevent UVB-induced abnormalities during early development of the green sea urchin Strongylocentrotus droebachiensis

Experiments were performed to determine how ultraviolet radiation (UVR) in the environmentally relevant range affects development of the sea urchin Strongylocentrotus droebachiensis (Müller) and whether mycosporine-like amino acids (MAAs), present in the early life stages, reduce UV-induced damage. Eggs, embryos, and larvae contained five MAAs having absorption maxima ranging from 320 to 334 nm. Eggs contained principally shinorine and porphyra-334, which absorb maximally at 334 nm and half-maximally at 312 and 348 nm, spanning much of the environmental range of biologically effective UVR. Concentrations of MAAs remained constant in unirradiated embryos through the gastrula stage, but decreased significantly in two-armed pluteus larvae. Daily exposure to combined photosynthetically active radiation (PAR, 400–700 nm) and UVR did not affect the concentration of MAAs in these embryos up to the two-armed pluteus stage. Prism larvae of sea urchins and the sand dollar Echinarachnius parma (Lamarck) did not accumulate shinorine from the surrounding seawater. Daily exposure of embryos to UVA (320–400 nm) and UVB (295–320 nm) radiation in the presence of PAR induced delays and abnormalities during development, and removing UVB eliminated this effect. Abnormalities in embryos included thickening of the blastoderm wall, filling of the blastocoel by abnormal cells, exogastrulation, and formation of abnormal spicules. The percentage of embryos that developed normally was lower in batches of embryos exposed to PAR + UVA + UVB, except in embryos from urchins maintained on MAA-rich diets. In all cases, the percentage of PAR + UVA + UVB-exposed embryos that developed normally was positively related to the concentration of MAAs in eggs from which the embryos developed. Thus, the MAAs found in S. droebachiensis embryos protect them against UVB-induced abnormalities during their development to at least the four-armed pluteus larval stage. Received: 8 May 2000 / Accepted: 29 September 2000     

Factors affecting fertilization success in two species of patellid limpet (Mollusca: Gastropoda) and development of fertilization kinetics models

Laboratory experiments were undertaken to examine fertilization success in the intertidal prosobranch limpets Patella ulyssiponensis and Patella vulgata. Alkalization of eggs (10 min in pH 9.0 sea water) prior to fertilization trials improved fertilization rates greatly. Fertilization success was found to be a function of sperm concentration, gamete age and contact time. Sperm concentration needed for optimum fertilization success in vivo ranged between 10⁵ and 10⁷ sperm ml⁻¹ for both species although at higher concentrations the number of normally developing trochophore larvae decreased. For P. vulgata, sperm longevity (at a concentration of 10⁶ sperm ml⁻¹) did not exceed 6 h, whereas eggs of both species were fertilizable for up to 12 h. Maximum fertilization success occurred after 15–30 min gamete contact time. The Vogel et al. (Math Biosci 58:189–216, 1982) fertilization kinetics model is developed to allow for non-complete fertilizations under optimal sperm concentrations, and a new parameter fitting technique is developed to improve estimates of fertilization success for short gamete contact times.     

Sexual reproduction in the compound ascidian Diplosoma listerianum (Tunicata). II. Sperm penetration through ovary wall and evidence of internal fertilization

Diplosoma listerianum differs from most ascidians in that, at ovulation, eggs are emitted at the bottom of the ovary and segregated into the tunic, so that fertilization occurs far from seawater. A fertilization canal, a hollow extension of the ovary, conducts sperm towards the egg. In the present paper, ultrastructural evidence is reported on the morphological relationship between the ovary, egg envelopes and oocyte and on the mechanism by which sperm-egg interaction is established. In the ovary, the very complex sperm, equipped with a spiral dense groove, undergo metamorphosis as the first step in a sperm reaction and then pass through the ovary epithelium insinuating themselves between the intercellular junctions which appear to be mouldable, although able to maintain the egg-ovary barrier. Sperm then reach the vitelline coat, where a further step in the sperm reaction occurs. Before the egg abandons the ovary, the sperm head is incorporated into the oocyte by a process recalling phagocytosis, with the formation of an engulfing pocket. Sperm-egg contact and incorporation in D. listerianum occur in a way, never previously reported for other ascidians, in which fusion of plasma membranes takes place immediately after sperm-egg contact. Unlike other cytoplasmic components, the dense groove persists until the sperm enters the egg. It gives a corkscrew-like configuration to the sperm head and allows close adhesion to cell membranes, facilitating sperm movement. Expulsion of numerous cortical granules and features of a cortical reaction were observed in the egg penetrated by the sperm. The mode of internal fertilization of this species in comparison with that of other tunicates and phylogenetic aspects are discussed. Ripe colonies of D. listerianum collected in the Lagoon of Venice, Italy in 1986 and 1991 and colonies reared on glass in aquaria were used for our investigations.     

Ammonia as confounding factor in toxicity tests with the sea urchin Paracentrotus lividus (Lmk)

The aim of this study was to investigate the toxicity of total ammonia towards sea urchin bioassays, in order to elucidate the role of ammonia as confounding factor in sediment quality assessment studies. New toxicity data, expressed as EC50, NOEC and LOEC are reported for Paracentrotus lividus based on experiments at differing pH values. Results confirmed that sperm cell toxicity test is not very sensitive to ammonia, while total ammonia embryotoxicity is strictly pH dependent. Accurate observations of embryotoxic effects at increasing total ammonia concentrations evidenced progressive shifts from malformed plutei to gastrula and blastula blockages. Toxicity data obtained for P. lividus are comparable with previous results with other echinoid species.     

Studies on the fusion reaction in two species of Perophora (Ascidiacea)

Histological and electron microscopical studies were made on the fusion reaction between homogeneic stolons of the colonial ascidians Perophora japonica Oka and P. sagamiensis Tokioka. When two stolons make a tip-to-side contact, the epidermal cells of the lateral portion of one stolon become thick and form a protrusion. In the fusion reaction of P. japonica, epidermal cells of each stolon at the contact area extend cell processes immediately after contact. After an occurrence of degenerated epidermal cells, blood interchange between the two stolons is initiated by a partial elimination of the epidermal cells. Even after the establishment of blood interchange, the cuticlelike structure is often observed to remain at the contact area. In the fusion reaction of P. japonica, many amoebocytes are attached to the epidermal cells until the complete elimination of epidermal cells. In the fusion reaction of P. sagamiensis, many lymphocytes as well as amoebocytes are attached to the epidermal cells at the contact area.Contribution No. 461 of the Shimoda Marine Research Center     

Uncommon diversity in developmental mode and larval form in the genus <Emphasis Type="Italic">Macrophiothrix</Emphasis> (Echinodermata: Ophiuroidea)

Development mode in the ophiuroid genus Macrophiothrix includes an unusual diversity of planktonic larval forms and feeding types. The modes of development for seven congeners that coexist in coral reef habitats at Lizard Island, Australia were compared using larvae generated from crosses over several reproductive seasons from 1999 to 2003. Three species (Macrophiothrix koehleri Clark, Macrophiothrix longipeda Lamarck, Macrophiothrix lorioli Clark) develop from small eggs (<170 μm) into typical obligately feeding planktonic (planktotrophic) pluteus larvae with four larval arm pairs. The remaining four species develop from larger eggs (≥230 μm) into either facultatively-feeding or non-feeding (lecithotrophic) larval forms. The facultative planktotroph (Macrophiothrix rhabdota Clark) retains the ability to digest and benefit from food but does not require particulate food to complete metamorphosis. Among the lecithotrophic species, Macrophiothrix caenosa Hoggett retains the pluteus morphology with four pairs of larval arms, but is incapable of feeding, depending instead on maternal provisions for larval development. The remaining two lecithotrophs have simplified larval morphologies with only a single pair of full length (Macrophiothrix nereidina Lamarck) or highly reduced (Macrophiothrix belli Doderlein) larval arms and no functional mouth or gut. This genus includes the first example of facultative planktotrophy in ophiuroids, the first example in echinoderms of a complete pluteus morphology retained by a lecithotrophic larva, and three degrees of morphological simplification among lecithotrophic larval forms. Egg volume varies 20-fold among species and is related to variation in feeding mode, larval form, and development time, as predicted for the transition from planktotrophic to lecithotrophic development.     

Biochemical changes during larval development of the xanthid crab Rhithropanopeus harrisii. III. Isozyme changes during ontogeny

Isozyme changes in the xanthid crab Rhithropanopeus harrisii (Gould) are described for 6 enzyme systems through all four zoeal stages, megalopa stage, and juveniles. There is considerable variation in the number of isozymes and their expression during the ontogeny of R. harrisii.Contribution No. 806 of the Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland, USA.     

Slow developing demersal embryos and larvae of the antarctic sea star Odontaster validus

The early development of Odontaster validus at McMurdo Sound, Antarctica, is indirect and includes equal cleavage, a convoluted blastula, a free-swimming coeloblastula, a gastrula, and a feeding bipinnaria larva. Development differs from that of other asteroids in two respects: (1) The developmental rate is extremely slow; blastulae form nearly 2 days after fertilization, gastrulation begins after 7 days, and the bipinnaria develops in about 40 to 55 days. The slow developmental rate appears to be only partly related to the low environmental temperature (-1.5°C). (2) The embryos and larvae are largely demersal. Such behavior may be an adaptation to keep the larvae out of antarctic surface waters, as does brooding in many other polar echinoderms.     

Comparative fertilization biology of gymnolaemate bryozoans

Locations and oogenic stages of eggs at the time of sperm-egg fusion were determined for nine gymnolaemate species exhibiting different reproductive strategies with respect to site of embryonic development (e.g., water column, introvert, embryo sac, or ovicell) larval type, and zooid sexuality. Ovarian, coelomic, and recently spawned oocytes obtained from freshly collected colonies between 1988 and 1995 were stained with aceto-orcein and examined for the presence of a sperm nucleus. All nine species are shown to have a similar fertilization biology in which sperm fuse with eggs in or near ovaries and egg activation is delayed until eggs are spawned. In species such as the etenostomeAlcyonidium sp. and the cheilostomeElectra pilosa (Linnaeus, 1767), which produce marierons oocytes that are spawned into the water column and have cyphonautes larvae, sperm fuse with late growth stage ovarian oocytes after germinal vesicle breakdown (GVBD) at or near ovulation. In species that produce few oocytes, brood embryos, and release coronate larvae, sperm fuse with late growth stage ovarian primary oocytes before GVBD, as in the ctenostomeBowerbankia gracilis Leidy, 1855, or with very early growth stage ovarian primary oocytes, as in the cheilostomesCribrilina corbicula (O'Donoghue, 1923),Dendrobeania lichenoides (Robertson, 1900),Hippodiplosia insculpta (Hincks, 1882),Schizoporella serialis (Heller, 1867),T ricellaria gracilis (Smitt, 1867), andWatersipora arcuata Banta, 1969. Fertilization success was high in all nine species, with 83 to 100% of oocytes at an appropriate oogenic stage containing a sperm nucleus or early male pronucleus. Gymnolaemate bryozoans may maintain high levels of fertilization success by: (1) concentrating spawned sperm from the water column using colony feeding currents; (2) localizing sperm-egg encounters in or near the ovary, (3) using sperm when available by permitting sperm to fuse with early growth stage oocytes; and (4) selfing.     

Acute toxicity of industrial endocrine-disrupting chemicals,natural and synthetic sex hormones to the freshwater planarian,Dugesia japonica

Many chemicals commonly occurring in surface water are found to be hormonally active. Fourteen compounds including four sex hormones, three synthetic hormones, one non-steroidal anti-androgen, and six industrial endocrine-disrupting chemicals were selected to examine their acute toxicities on the freshwater planarian, Dugesia japonica. Among all test chemicals, diethylstilbestrol had the highest toxicity. Relatively, the toxicities of the synthetic hormones were higher than the natural ones. Among the six industrial endocrine-disrupting chemicals tested, bisphenol A was the most toxic, with a 48-h LC₅₀ of 8.3 mg L^?1. Overall, the natural or synthetic hormones, other than estrone and estriol, were more acutely toxic to planarians than the industrial endocrine disruptors tested. The acute toxicities of the test chemicals to Dugesia japonica were in the same order of magnitude as those to Daphnia magna based on available published data. Despite this study used non-environmentally relevant levels, such kinds of chemicals do not exist singly in the aquatic environment, but typically act in synergistic and/or additive ways in the complex environmental mixtures, and the total estrogen equivalents are much higher than a single chemical. This study stands as a starting point for other acute and chronic assays, namely using mixtures.     

Ultrastructural investigations on sperm penetration and gamete fusion in the ascidians Boltenia villosa and Phallusia mammillata

Sperm-egg interactions during fertilization were studied by scanning and transmission electron microscopy in the two ascidian species, Boltenia villosa (collected in 1990 near Friday Harbor, Washington, USA) and Phallusia mammillata (collected in 1990 near Brest, France). Emphasis was put on sperm-egg vitelline coat (VC) and sperm-egg plasma membrane interaction. In both species the sperm activation as revealed by translocation of the mitochondrion was exhibited by sperm binding firmly to or penetrating the VC. Vesicles are present in the sperm apex of both species. Their persistence in late stages of VC penetration suggests for them a function rather in Sperm-egg fusion than in VC penetration. In P. mammillata sperm which are about to make contact and to start fusion with the egg plasma membrane, two types of vesicles, one located at the tip and the other slightly behind the apical cap, were detected. Function and fate of these prominent vesicles remain to be determined. Membrane fusion starts between the sperm membrane of the cap-like structure and short microvilli of the egg surface and then proceeds simultaneously at several sites of the apical region. In B. villosa conspicuous microvilli which are formed at the contact site and interdigitate with the corkscrew-like sperm represent fusion sites on the egg. In both ascidian species sperm are incorporated into the egg at an acute angle. Once fully embodied the sperm lie close and almost parallel to the egg membrane. Our findings are compared with observations in Ciona intestinalis, the only other ascidian species in which Sperm-egg interactions have been thoroughly investigated so far. We suggest that observed differences in the fusion processes are based on morphological differences in sperm structure which reflect phylogenetic modifications.