Tsuyoshi Uehara

Comparative studies of the genus echinometra from okinawa and mauritius.

Abstract Okinawan sea urchins, the genus Echinometra, are four independent species. But which species are the same species as E. mathaei and E. oblonga as described by Blainville 1825 is still open to question. To answer this question, a field survey of genus Echinometra was made in Mauritius (the type locality of E. mathaei) according to the characteristics used to classify Okinawan Echinometra: appearance, pore pairs, spicules in gonads and tubefeet, sperm shape, and distribution on a reef. The results of crossfertilization between Echinometra from Mauritius and Okinawa are also reported. Mauritian Echinometra are classified into three groups which resemble Okinawan Echinometra sp. B, D, and violet spine color Echinometra. The latter has almost the same characteristics as Okinawan Echinometra sp. B but with violet spines, a spine color not found in Okinawa. In cross-fertilization experiments, the sperm of Mauritian Echinometra sp. B-like and violet Echinometra fertilized Okinawan Echinometra sp. B with almost 100% success. However, fertilization was unsuccessful with other Okinawan Echinometra species. Therefore, it could be said that Okinawan Echinometra sp. B is the same as Mauritian Echinometra sp. B-like, and the counterparts of Okinawan Echinometra sp. A and C are not distributed in Mauritius. The descriptions of E. mathaei most match Mauritian Echinometra sp. B-like and the type locality of E. mathaei is Mauritius. Thus, it is probable that Mauritian Echinometra sp. B-like is E. mathaei. Therefore, Okinawan Echinometra sp. B, which most resembles Mauritian Echinometra sp. B-like, would be also E. mathaei. Echinometra sp. A and C would be considered to be new species. Echinometra sp. D is thought to be the same species as Mauritian black Echinometra. Whether Okinawan Echinometra sp. D is the same as E. oblonga remains as a problem for future research. Because the type locality of E. oblonga is not known and, it is suggested that the E. oblonga described in the Indo-West Pacific is a complex species.

Hybrids of Two Closely Related Tropical Sea Urchins (Genus Echinometra): Evidence Against Postzygotic Isolating Mechanisms

A series of cross-fertilization experiments were conducted with two unnamed, sympatric species of sea urchins in the Echinometra mathaei species complex, Echinometra sp. A (Ea) and Echinometra sp. C (Ec). Heterogametic fertilization success was high when eggs of Ec and sperm of Ea were involved, and low with eggs of Ea and sperm of Ec. Hybrids produced from crosses in either direction developed normally to sexually mature adults; Ea × Ea were largest in test size, followed by Ec (ova) × Ea (sperm), Ea (ova) × Ec (sperm), and Ec × Ec, respectively. Color patterns of the hybrids were closer to the maternal coloration, whereas other characters such as relative test dimensions and spine lengths, morphology of tubefoot and gonad spicules, and gamete sizes were intermediate. Fertilization rates in F1 backcrosses were high, minimizing the possibility that hybrid infertility is a postzygotic mechanism of reproductive isolation. On the other hand, intensive surveys failed to find individuals with hybrid characteristics in the field, suggesting that natural hybridization between the two species is rare. Prezygotic isolating mechanisms, such as microhabitat separation and gamete incompatibility, at least between Ea eggs and Ec sperm, most likely maintain the genetic integrity of these two closely related species.

Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO3 crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO3 nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO3 from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.

Comparative viability and growth of hybrids between two sympatric species of sea urchins (Genus Echinometra) in Okinawa

Abstract Among the four sympatric but genetically divergent forms of the sea urchins belonging to Echinometra mathaei sensu lato, Echinometra sp. A (Ea) and Echinometra sp. C (Ec) were studied through a series of cross fertilization experiments and rearing of the resulting hybrids. Detailed investigations on their larval and adult growth, development and viability were carried out. Heterosis was judged at different stages from larval to 1-year-old F 1 hybrids of Ec(ova)×Ea(sperm) and Ea(ova)×Ec(sperm) and at the same time they were compared to their parental species controls. For simplicity the maternal species is named first in case of heterogametic crosses. The hybrids mostly appeared to be intermediate between that of the parental species. The average performance traits from fertilization to 3-month-old juvenile stages (viz. fertilization rate, larval survivality, metamorphosis rate, recovery rate of juveniles) of Ec(ova)×Ea(sperm) hybrids were significantly better than their reciprocal Ea×Ec hybrid but not significantly different from their conpecifics, Ea×Ea and Ec×Ec. After 1 year of rearing, the final mean weight attained by Ea×Ea, Ec×Ea, Ea×Ec and Ec×Ec was 14.7, 13.9, 13.6 and 9.2 g, respectively. Among the crosses, the growth of both the reciprocal hybrids were much faster than the conspecific Ec×Ec and slightly less than Ea×Ea. The weight increase by Ec×Ea and Ea×Ec hybrids were 51.7% and 48.4% higher over the conspecific Ec×Ec, respectively. The fast-growing Ea species thus partially transmitted this trait to the hybrids. Improvement of quality traits in the majority of the performance traits (viz. final weight, weight gain, gonad weight) in the F 1 hybrids compared to the parental species might have important implications to the development of a sea urchin fishery.

Effects of temperature on hatching rate, embryonic development and early larval survival of the edible sea urchin, Tripneustes gratilla

The temperature tolerances of embryonic and early larval development stages of Tripneustes gratilla were investigated from 13-34°C under laboratory conditions. Zygotes showed unequal cleavage at 13°C, whereas cleavage did not occurred at 34°C. Hatching was observed between 16–31°C with maximum hatching rates observed at 22–29°C. The lower and higher temperature limits for embryonic development were approximately 22°C and 29°C, respectively. Outside of this temperature range, embryos showed abnormality at different incubation times. Early larvae of this species have the ability to survive the higher temperature limit for short periods of time. Prism and 2 arm pluteus larvae survived at temperatures between 30 and 33°C, whereas 4 arm pluteus larvae survived at temperatures between 30 and 36°C for 2 h. These results suggest that the larval temperature tolerance capability of T. gratilla is stage dependent. These findings are important for understanding the life history strategy of this sea urchin in the shallow open water environment.

Hybridization and F1 backcrosses between two closely related tropical species of sea urchins (genus Echinometra) in Okinawa

Summary Four species of sea urchins of the genus Echinometra, designated species A, B, C, and D, occur along the coast of Okinawa; they are distinguished by color pattern and other characteristics. The two most distinct species, Echinometra sp. A (Ea) with white-tipped spines and Echinometra sp. D (Ed) with nearly black spines (possibly E. oblonga), were examined for potential hybridization through a series of fertilization experiments and rearing of the resulting hybrids. Fertilization was reciprocally asymmetrical; Ed ova were readily fertilized by sperm of Ea, but less than 20% of the ova of Ea were fertilized by sperm of Ed. Nevertheless, hybrids resulting from crosses in both directions developed normally through larval and juvenile stages to produce sexually mature adults. Larvae of Ed♀×Ea♂ and Ed♂×Ed♀ were larger than those of Ea♂ × Ed♀ and Ea♂ × Ea♀, reflecting the larger eggs of Ed. However, Ea♂×Ea♀ juveniles and later stages, up to maturity, were consistently larger than those of equivalent ages...

Analysis of Proteinaceous Components of the Organic Matrix of Endoskeletal Sclerites from the Alcyonarian Lobophytum crassum

The mesoglea of alcyonarians is occupied by an abundance of minute calcitic sclerites. The sclerites of the alcyonarian Lobophytum crassum contain a water-soluble organic matrix comprising 0.48% of the sclerite weight and a water-insoluble fraction comprising 1.15% of the sclerite weight. Analysis of proteinaceous components in the soluble fraction shows a particularly high content of aspartic acid, followed by alanine, glycine, and glutamate. Aspartic acid, glycine, alanine, and glutamate are the most abundant residues in the insoluble fraction. In both cases, the fractions show the highest concentration of aspartic acid from the total proteins. In an in vitro assay, we show that the matrix proteins extracted from the calcitic sclerites induce the formation of amorphous calcium carbonate prior to its transformation into the calcitic crystalline form. We also show scanning electron micrographs of the rhombohedral calcite crystals used as template, the protein imprinted with these crystals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both matrices shows the protein fractions at 67 and 48 kDa. The soluble matrix shows two additional faint bands. Both fractions stain for a carbohydrate at 67 kDa, indicating a glycoprotein at this molecular weight. A newly derived protein sequence was subjected to bioinformatics analysis involving identification of similarities to other acidic proteins. The identification of these proteins in alcyonarian endoskeletal sclerites emphasizes the fundamental importance of such acidic proteins and sheds more light on the functions of these proteins in the processes of biocalcification.

Effects of temperature on gamete longevity and fertilization success in two sea urchin species, Echinometra mathaei and Tripneustes gratilla.

We investigated gamete longevity in two sea urchin species, Echinometra mathaei (Em) and Tripneustes gratilla (Tg), via laboratory experiments. The longevity of dry sperm at different ages (6, 12, 18, 24, and 30 h after collection) was tested at 20°, 25°, and 30°C. Sperm viability was determined by the percent cleavage of eggs after in-vitro fertilization. Dry sperm of Em remained viable longer than dry sperm of Tg at all temperatures. The viability of dilute sperm and eggs was also determined at 20°, 25°, and 30°C over varying durations (for sperm, 5, 45, and 85 min; for eggs, 1, 3, 6, 12, and 24 h). The viability of dilute Em sperm remained relatively constant over time (nearly 100%) at 20° and 25°C, but decreased rapidly at 30°C. The viability of Tg sperm declined gradually over time at 20°C and decreased sharply at 25° and 30°C. Eggs of Em and Tg remained fertilizable for 12 and 6 h, respectively. Most eggs of Em and Tg underwent normal development when exposed for less than 6 h and 3 h, respectively. These results may illuminate the different fertilization strategies of these two sea urchin species.

Experimental hybridization between two recently diverged species of tropical sea urchins, Echinometra mathaei and Echinometra oblonga

Summary Two species of tropical sea urchins, Echinometra mathaei and Echinometra oblonga, occur abundantly on Okinawan intertidal reefs; E. mathaei is found mainly on inshore reef flats while E. oblonga burrows in the offshore reef margins. The two species spawn during the same season, and hybrid embryos can be formed in the laboratory. We confirmed earlier experiments showing that fertilization rates are high between eggs of E. oblonga and sperm of E. mathaei, but significantly lower than those in homogametic crosses, whereas few eggs of E. mathaei are fertilized by sperm of E. oblonga, even at very high sperm concentrations. If they spawn together in the field, most or all ova of E. oblonga would be fertilized by conspecific sperm before encountering sperm of E. mathaei on the reef flat, so hybridization is partially minimized by habitat separation. However, many viable sperm of E. oblonga would still be available to fertilize ova of E. mathaei. Species-specific differences in sperm chemotaxis or gamete compatibility may provide prezygotic barriers that also would prevent hybridization, especially between ova of E. mathaei and sperm of E. oblonga. We found that homogametic crosses reach peak fertilization levels much sooner than heterogametic crosses, even between ova of E. oblonga and sperm of E. mathaei, indicating the presence of prezygotic barriers to hybridization in the gametes. Nevertheless, laboratory crosses between E. mathaei and E. oblonga in both directions produced viable hybrids that developed through larval and juvenile stages to sexually mature adults. Phenotypic color pattern of hybrids tended to be maternal while other characteristics were intermediate (test size, weight, spine length, growth, tubefoot and gonad spicule morphology, pedicellaria valve length, gamete size). Adult F1 hybrids from both combinations were completely fertile and exhibited high fertilization rates in backcrosses. On the other hand, intensive field surveys failed to find hybrids, showing the rarity or absence of natural hybridization. These results mirror those found with hybridization studies on other species of Echinometra on Okinawa. They further support the conclusion that prezygotic isolating mechanisms such as habitat segregation as well as species-specific sperm chemotaxis and/or gamete compatibility have a role in maintaining the genetic integrity of these species.

Comparative Studies on the Energy Metabolism in Spermatozoa of Four Closely Related Species of Sea Urchins (Genus Echinometra) in Okinawa

Abstract Sea urchins of the genus Echinometra are abundant on Okinawa reef flats in southern Japan. The Okinawan Echinometra is designated into four sympatric and closely related species: A, B, C, and D (Ea, Eb, Ec, and Ed). The sperm head size and shape gradually changes to become longer and more slender according to the following order: Ea, Eb, Ec, and Ed. To obtain information regarding speciation in Okinawan Echinometra, this study examined comparatively the energy production system of spermatozoa of Ea, Eb, Ec, and Ed. All spermatozoa contained cholesterol and several kinds of phospholipids. Glycogen, glucose, and triglyceride were present at extremely low levels. After incubation in sea-water, a decrease in the level of phosphatidylcholine (PC) was observed in all spermatozoa concomitantly with activation of motility and respiration. The hydrolysis of PC correlated with the activity of phospholipase A2. Interestingly, the amount of PC consumed, the respiratory rate, and the phospholipase A2 activity in spermatozoa of Ea and Eb were approximately two-fold higher than those of Ec and Ed. Ultrastructural studies showed that lipid bodies within mitochondria were present in the midpieces of all species of spermatozoa. They became small or disappeared after incubation in seawater. Thus, the results obtained strongly suggest that spermatozoa of Ea, Eb, Ec, and Ed all use PC located in the lipid bodies as a substrate for energy metabolism. Also, it seems likely that energy production activities in Ea and Eb spermatozoa are stronger than those in Ec and Ed. The properties of energy metabolism in different species of sea urchin may be related to their habitat.