An energy budget for Porites porites (Scleractinia)

An energy budget for Porites porites (Pallas) was determined for specimens from 10 m depth on the Fore Reef of Discovery Bay, Jamaica, between July 1984 and July 1985. Evidence for habitual zooplankton ingestion was not obtained, and P. porites appears to be largely autotrophic. Out of the daily photosynthetically fixed energy, 26% is used for animal respiration and growth, 22% for zooxanthellae respiration and growth, and <1% for colony reproduction as mature planulae; 45% remains unaccounted for. Colony respiration, net photosynthesis, colony skeleton and tissue growth, zooplankton ingestion, reproductive effort and energy content of tissues were measured. Energy loss as continuous mucus secretion was not detected, but may occur by an alternative route via mucus tunics, which occur periodically in situ and in the laboratory. The energy budget suggests that a considerable excess of photosynthetically fixed energy is produced on an ideal sunny day at 10 m depth. This surplus may be required for periodic rather than continuous energy demands, or may be essential to survive less-than-ideal days, when net photosynthetic input is reduced.Contribution No. 357 of the Discovery Bay Marine Laboratory, University of the West Indies     

Divide and broadcast: sexual reproduction in the West Indian brittle star Ophiocomella ophiactoides and its relationship to fissiparity

It is known that asexual reproduction by fission is the dominant mode of propagation in the small (disc diameter <5.0 mm) ophiocomid brittle star Ophiocomella ophiactoides (H. L. Clark). This species is, however, able to reproduce sexually as well. Sexual reproduction occurs throughout much or all of the year in a population of O. ophiactoides at Jamaica. Gonads are often present in recently split individuals, indicating that sexual and asexual reproduction could occur simultaneously. Regeneration of new gonads in the new disc half is underway by the time an individual is competent to split again. Unsexable individuals are significantly smaller than males, which in turn are significantly smaller than females. The unsexable individuals appear to be below a threshold size for gonad production. Fecundity is low, with the largest females producing a maximum of about 7 400 eggs of small (mean diameter 80 m) size. Fertilized eggs develop into planktotrophic ophioplutei which are typical of the larvae of other species of ophiocomid brittle stars in morphology, rate of development and duration of planktonic period. Egg size and larval type for O. ophiactoides are characteristic of brittle stars with the planktotrophic mode of development, but egg number is uncharacteristically low. Fissiparity in O. ophiactoides may have evolved in association with small body size, concomitant low fecundity and a high-risk planktotrophic larva as a means of ensuring reliable recruitment. Available data, although sparse, suggest that this hypothesis may be applicable to fissiparous brittle stars in general. Fission in brittle stars has a broad systematic incidence at the familial level which suggests that it has evolved recurrently. The ability both to divide and broadcast appears to be a useful combination for O. ophiactoides: it can recruit reliably at large size to occupy favourable habitats quickly, but, it can still maintain low level dispersal of larvae which could provide escape from deteriorating habitats and the potential to colonize new habitats.Contribution No. 328 of the Discovery Bay Marine Laboratory, University of the West Indies     

Resource partitioning by reef corals as determined from stable isotope composition

The pattern of resource partitioning vs depth by corals collected in February 1983 from Jamaica and the Red Sea was determined from their stable carbon isotope composition. Observations were made on isolated zooxanthellae and corresponding algae-free animal tissue from eight species at four depths over a 50 m bathymetric range. Zooxanthellae ¹³C was high in shallow water and became lower as depth increased. This trend correlated significantly with the anual integrated photosynthetic rate. The trend is interpreted according to a depletion-diffusion hypothesis; in shallow water, at high rates of photosynthesis, metabolic CO₂ is nearly depleted and the supply of CO₂ from seawater bicarbonate is limited by diffusion. Since most of the available CO₂ is fixed, isotope fractionation is minimal. In deeper water, at lower rates of photosynthesis, metabolic CO₂ is ample, and isotope fractionation is greater. Animal tissue ¹³C was slightly lower than corresponding zooxanthellae values in shallow water. As depth increased the difference between zooxanthellae and animal tissue ¹³C increased and the latter approached the ¹³C of oceanic particulate organic carbon. These data suggest that carbon is translocated at all depths and that deep-water corals draw significantly on allocthonous sources of carbon.Contribution No. 436 of the Discovery Bay Marine Laboratory of the University of the West Indies     

Genetic structure of populations of two closely related brittle stars with contrasting sexual and asexual life histories,with observations on the genetic structure of a second asexual species

Ophiocoma pumila Lütken andOphiocomella ophiactoides (H. L. Clark) are morphologically similar brittle stars with contrasting life histories, the former obligately sexual, the latter fissiparous (capable of both sexual reproduction and asexual proliferation by binary fission). Electrophoretic analysis of five polymorphic enzymes was used to assess the genetic consequences of these differing life histories and provide a genetic perspective on the taxonomic relationship between the two species. Genotypic diversity ofOphiocoma pumila collected at Discovery Bay, Jamaica, in 1985 conformed to expectations for a sexually reproducing population. In contrast, genotypic diversity ofOphiocomella ophiactoides at this site was significantly lower than expected for a sexually reproducing population, due largely to the predominance of clonal proliferation over larval recruitment. Large variation in clonal composition over a short (50 m) distance emphasized the very localized scale of clonal mixing in this species. Allozymic data are indicative of a close sibling species relationship betweenOphiocoma pumila andOphiocomella ophiactoides which suggests that the present generic separation of the two species should be re-examined. Electrophoretic analysis was also used to examine the genetic structure of sponge- and alga-dwelling populations of a second fissiparous brittle star,Ophiactis savignyi (Müller & Troschel), which was also collected at Jamaica in 1985. Striking differences in the allelic composition of sponge- and alga-dwellingO. savignyi were observed. Genotypic diversity ofO. savignyi in sponges was very low, each sponge being dominated by a single genotype. Genotypic diversity ofO. savignyi in algae was higher, although still significantly lower than expectations for a sexually reproducing population. In the light of the highly clonal composition of fissiparous brittle-star populations, the adaptive significance of clonal growth may be related to an increase in the overall fitness of dispersed clones (genets), compared to individuals of strictly sexual counterparts, through greater genotypespecific biomass and, hence, fecundity.Contribution No. 476 of the Discovery Bay Marine Laboratory of the University of the West Indies     

Effects of environmental stressors on blood-glucose levels in sea hares,Aplysia dactylomela

Effects of natural stressors such as tidepool strandings, air exposure, and low salinity on blood-glucose levels were investigated in the sea hare Aplysia dactylomela from shallow areas of Discovery Bay, Jamaica. All treatments produced large and significant elevations in blood-glucose titre, 1.5-to 2.3-fold above baseline levels of 25 to 35 g glucose ml^-1. Response times were variable, with significant elevations being manifested within 30 to 120 min from initiation of the stressor. Recovery was swift, within an hour or two of restoration to pretreatment conditions, and often involved an undershoot to levels below control values. In two experiments involving tidepool strandings and associated high body-temperatures, excessively low blood-glucose titres were followed by death of all test individuals. When sea hares were exposed to 75 and 50% seawater (100%=33S) for 1 h, maximum elevation in blood-glucose concentrations occurred 1 to 2 h from onset of exposure, coincidental with maximum dilution of the body fluids of test individuals. The responsiveness of blood-glucose titres to relatively small temperature or salinity changes, or to short-duration air exposures, suggests that monitoring this physiological parameter may be a useful and sensitive means of diagnosing a wide variety of stressors in marine gastropods.     

Effect of sunlight intensity and albinism on the covering response of the Caribbean sea urchin<Emphasis Type="Italic"> Tripneustes ventricosus</Emphasis>

At Discovery Bay, Jamaica, Tripneustes ventricosus lives in beds of the turtle grass Thalassia testudinum. Especially during daylight hours, it covers its aboral surface with fragments of this plant and other objects. Normally pigmented, wild-type sea urchins covered themselves significantly less with Thalassia when sunlight was experimentally decreased to 66% or 32% ambient intensity. Consistent with this result, naturally occurring sea urchins exhibited significantly less covering at a deep (3.5 m) site than at a shallow (1 m) site, where light intensities at the bottom were 619 and 946 mol s^–1 m^–2, respectively. The graded covering response to light intensity suggests that covering is a defense against damaging solar radiation. Albino sea urchins covered themselves significantly more with Thalassia than wild-type sea urchins in both full and 66% sunlight. In addition, at the shallow site where they accounted for about 4% of the population, they showed significantly greater covering than wild-type urchins. The greater covering response of albino sea urchins suggests a greater susceptibility to solar radiation.Communicated by P. W. Sammarco, Chauvin     

Seasonal variations in the stable isotopic composition and the skeletal density pattern of the coralPorites lobata (Gulf of Eilat,Red Sea)

The stable carbon and oxygen isotopic compositions of the massive coralPorites lobata collected from the Gulf of Eilat, Red Sea, in May 1987 were measured along the vertical growth axis. The isotopic compositions show annual periodicity. The variations along the isotopic profile, compared with the X-radiography, indicate that the high-density bands are enriched in¹⁸O and deposited during the season of lowest water temperature (winter). In contrast, the low-density bands are depleted in¹⁸O and deposited during the warmest seawater temperatures (summer). The stable carbon and the oxygen isotopic ratios are negatively correlated with a shift in phase. During the season of higher water temperatures and light intensity values (summer), the skeleton is depleted in¹⁸O and enriched in¹³C and vice versa for the winter time. The shift between the carbon and the oxygen isotopic curves reflects the shift between the seasonal light intensities and seasonal temperature variations in the shallow water. The oxygen isotope ratio was used to detect the seasonal variations in seawater temperatures. The coral aragonitic skeleton is depleted in¹⁸O compared to apparent equilibrium with ambient seawater. The disequilibria range from (ca.) -3.10 to -3.50 with an average value of 3.40. The isotope fractionation behavior during skeleton precipitation is discussed in light of the environmental variables.     

Annual structure of the copepod community and its associated pelagic environment off Discovery Bay,Jamaica

Monthly samples were collected in oceanic waters off Discovery Bay, Jamaica, in 60- and 200-m vertical hauls, using 200- and 64-m mesh plankton nets, from June 1989 to July 1991. Sixty-nine species of copepods were identified: nauplii, copepodites and adults were separately enumerated. Total copepod abundances (all stages) ranged from 695 to 4120 m^-3 in the upper 60 m, and from 483 to 3319 m^-3 in the 200-m water column, without any clear seasonal pattern. With the exception of temperature, no seasonal variations in physico-chemical (chlorophyll a, S, particulate organic carbon, particulate protein) or biological variables were evident. Nauplii, adults and copepodites of selected taxa, and two chaetognath species, showed no significant variations in body length. Significant variantions in reproductive index were detected for several species, but without seasonal trends; many species appear to be continuous or intermittent breeders. There was no evidence of seasonal pattern in overall community composition or diversity, or evidence of changes due to water mass advection. The copepod community can be divided into a recurrent group of 13 (at 60 m) to 17 (at 200 m) perennial species, present year-round, and associated ephemeral groups of 1 to 3 species, present randomly for 1 to 4 consecutive months. The most plausible explanation of these patterns is that broad areas of the Caribbean Sea are dominated by the community of perennial species, while the ephemeral species represent the superimposed influence of local mesoscale gyres.     

Short-term growth measurements of corals using an accurate buoyant weighing technique

An accurate method for determining the growth rates of the skeleton of isolated branch tips (nubbins) of corals over intervals of less than 24 h is described. The skeletal weight of the coral was estimated from its buoyant weight in seawater whose density had been accurately determined. The coral tissues accounted for between 1 and 5% of the total buoyant weight in Pocillopora verrucosa and Acropora humilis with differing relative tissue biomass. After correcting for tissue buoyant weight, predictions of skeletal weight were accurate to within 1%. The method was used to estimate the growth of sample nubbins of Porites porites of similar diameter, in 2 m of water at Discovery Bay, Jamaica. Since growth of these branch tips is apical, growth rate could be expressed without correction for the size. The mean 24 h skeletal growth rate ranged between 40 and 47 mg. Differences could be measured between day-time and night-time growth, the day: night ratio being 3.7. The method also showed that P. porites virtually ceases calcification during the 4 to 5 d periods that it becomes enclosed in a mucus tunic. Nubbins of P. porites attached to the reef at different locations showed clear differences in growth rate with depth, and between clear and turbid water sites. The growth rate of nubbins was compared with that of branch tips of whole corals by measuring the linear extension after staining with Alizarin Red S. After 3 1/2 mo, the mean linear extension was 4.1 mm in each case, indicating that the growth rate of nubbins is the same as that of branch tips of the whole colony. It is suggested that this buoyant weighing technique will find applications in laboratory experiments with calcification mechanisms and as a bioassay on reefs exposed to environmental stress.Contribution No. 464 of the Discovery Bay Marine Laboratory     

Microbial associations in sponges. III. Ultrastructure of the in situ associations in coral reef sponges

Symbiotic cyanobacteria are associated with marine sponges in three ways: the majority are free-living in the mesohyl; large aggregates occur in cyanocytes (specialized, vacuolated archeocytes); and few are present in digestive vacuoles. The cyanobacteria in Jaspis stellifera and Neofibularia irata are morphologically similar to those described in Mediterranean sponges, whereas those in Pericharax heteroraphis are different. The freeliving bacterial populations are morphologically similar, although the number of bacteria varies between the species. The fourth sponge Ircinia wistarii contains a mixed bacterial population unlike those in the other sponges. Sponge digestion of microbial associates is rare and not considered to contribute significant nutrients.     

Annual biomass and production of the oceanic copepod community off Discovery Bay,Jamaica

Monthly samples were collected in oceanic waters off Discovery Bay, Jamaica, in 60- and 200-m vertical hauls, using 200- and 64-m mesh plankton nets, from June 1989 to July 1991. Length-weight regressions were derived for twelve genera of copepods (R²=0.79 to 0.97). For eight occasions spanning the study period, biomass estimates generated from these length-weight regressions differed by only 3% from direct weight determinations. The mean ash content of copepods was 7.1%, and the energy density was 20.8 kJ g^-1 ash-free dry weight (AFDW). Mean annual biomass of the total copepod community in the upper 60 m was 1.83 mg AFDW m^-3 (range 1.14 to 2.89 mg AFDW m^-3), and for the 200-m water column was 0.96 mg AFDW m^-3 (range 0.12 to 1.99 mg AFDW m^-3). Estimates of generation times for five common taxa ranged from 16.1 to 33.4 d. None of the taxa investigated displayed isochronal development; in general, stage duration increased in later copepodite stages. Weight increments showed a significant decrease in later copepodite stages, but with strong reversal of the trend from stage 5 to adult female in most species. Daily specific growth rates also declined in later copepodite stages, and ranged from 1.49 d^-1 in stage 1–2 Paracalanus/Clausocalanus spp. to 0.04 in stage 5-female of Oithona plumifera. Progressive food limitation of somatic copepodite growth and egg production is postulated. Naupliar production was 50.4 to 59.5% of copepodite production, and egg production was 35.1 to 27.7% of copepodite production in the 60-and 200-m water columns, respectively. Total annual copepod production, including copepodites, nauplii, eggs and exuviae, was 160 kJ m^-2 yr^-1 for the upper 60 m and 304 kJ m^-2 yr^-1 for the upper 200 m. Secondary production of the copepod community in oceanic waters off Discovery Bay approaches 50% of the corresponding value in tropical neritic waters.     

Light absorption and utilization among hermatypic corals: a study in Jamaica,West Indies

The chlorophyll specific absorption coefficient ( _c) was measured for zooxanthellae from six hermatypic coral species obtained, where possible, from four depths (1, 10, 30, 50 m) on reef sites near Discovery Bay, Jamaica in February and March 1983. Measurements of photosynthetic rates versus irradiance, as well as cellular and areal chlorophyll a, were also performed on these colonies or sister colonies. Together the data were used to compare minimum quantum requirements (1/Φ m) among species and depths and to assess the importance of light utilization to the growth and depth distribution of these corals. Our data suggest that, although _c was found to decrease with depth, interspecific differences in _c do not occur for zooxanthellae from the corals investigated. Minimum quantum requirements (1/Φ m) decreased significantly with depth, thereby reflecting an increase in photosynthetic light utilization efficiency with decreasing irradiance. Interspecific differences in 1/Φ m determinations were suggested but not statistically conclusive. We conclude that interspecific differences in gross photosynthesis, and perhaps growth and depth distribution, are primarily attributable to differences in the light utilization capacity of the whole coral, as reflected by the product of _c ^′ and chlorophyll per unit surface area, and in-situ quantum efficiencies. This research was performed under the auspices of the US Department of Energy under Contract No. DE-AC02-76CH00016     

Variability in dry weight and vertical distributions of decapod larvae in the Irish Sea and North Sea during the spring

Accumulation of waterborne cadmium in Littorina littorea, Mytilus edulis and Carcinus maenas (collected in 1988 and 1989 around the island of Funen, Denmark) was investigated in a matrix of salinities (10 to 30) and calcium concentrations (2.9 to 8.9 mM Ca⁺⁺). Cadmium accumulation rates in soft parts of L. littorina, soft parts and shells of M. edulis and whole bodies and exoskeletons of C. maenas decreased with increasing salinity. Changes in the calcium concentrations accounted for 72% of the salinity effect on cadmium accumulation rates in L. littorina, whereas calcium concentrations had little or no effect on cadmium accumulation in M. edulis. Cadmium accumulation in the whole body of C. maenas was affected equally by calcium concentrations and total salinity, whereas accumulation in the exoskeleton was mainly affected by changes in total salinity. Individual variability in cadmium accumulation in the organs of C. maenas was greater than the variation attributable either to changes in ambient calcium concentrations or total salinity. An appreciable amount of the inter-individual variability in the cadmium accumulation in all three species was correlated with wet:dry weight ratios of the tissues and size of the organisms.     

Anatomy of structures associated with air-breathing in Orchestia gammarellus (Crustacea: Amphipoda: Talitridae): coxal plates and gills

The structure of the coxal gills and coxal plates of the semi-terrestrial beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda: Talitridae) is described in relation to their possible use for aerial gas exchange and ion exchange. Anatomical evidence is presented to support the hypothesis that the medial surface of the coxal plates functions as an extrabranchial aerial gas-exchange site in O. gammarellus. Thus, the effective diffusion distance across the medial (or inside-facing) surface of O. gammarellus coxal plates (mean±SD=5.4±0.3 m; n=9, cuticle thickness 4.4±0.5 m, n=21) is only a third of the equivalent distance across both the coxal gills (18.4±6.0 m, n=10; cuticle thickness 1.7±0.6 m, n=7) and the lateral (or external) surface of the coxal plates (19.4±0.7 m, n=5; cuticle thickness 8.7±0.8 m, n=7). Chloride-ion-permeable areas were located using a silver-staining technique. All ten coxal gills appeared to be equally permeable to chloride ions after examination with a light microscope. However, the coxal plates and the rest of the integument do not appear to be chloride-permeable.     

Minimal genetic variation among samples of six species of coral reef fishes collected at La Parguera,Puerto Rico,and Discovery Bay,Jamaica

Intraspecific genetic variation among samples of six species of reef fishes,Chromis cyanea, Stegastes partitus, S. planifrons, S. leucostictus, S. dorsopunicans, andThalassoma bifasciatum collected over a 2 wk period in 1990 at La Parguera, Puerto Rico, USA and Discovery Bay, Jamaica, was evaluated using starch-gel electrophoresis. On average, products of 33 protein-coding loci were resolved in each species. Levels of polymorphism (0.95 criterion) ranged from 3.1% inS. dorsopunicans to 42.4% inC. cyanea. Estimates of genetic divergence among samples and indices of genetic subdivision were small in all six study species: mean genetic distances ranged from 0.000 to 0.002 and mean fixation indices ranged from 0.004 to 0.035. Estimates of numbers of migrants per generation (mN _e) ranged from 5.1 to 11.6, indicating that substantial genetic exchange probably occurs over the relatively large geographic distance (ca. 1000 km) separating coral reef communities of La Parguera and Discovery Bay. The estimates ofmN _e may be biased by a sampling strategy involving only two localities, and should therefore be interpreted with caution. With inferences based solely on allozyme frequency data under a primary assumption of neutrality, genetic substructuring of populations of the six study species on a macrogeographic scale appears virtually nonexistent.     

A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2,4-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.     

Growth and form in the reef-building coral Montastrea annularis

The in-situ growth rate of the reef coral Montastrea annularis on a reef at Jamaica, W. Indies was determined for a 1-year period using Alizarin Red-S staining techniques. Growth rate is correlated with water depth and growth form. The flattened growth form of M. annularis allows for continued rapid increase in colony surface area at low light intensities. The geomorphology of Jamaican reefs may in part be controlled by the population ecology of M. annularis.     

Phagostimulatory properties of various chemical compounds to sea hares (Aplysia kurodai and A. dactylomela)

Phagostimulatory properties of amino acids, carbohydrates, vitamins, fatty acids, and cholesterol were assessed for sea hares (Aplysia kurodai and A. dactylomela). A. kurodai were collected at Shirahama, Japan, during March-May 1979, and A. dactylomela were collected at Barbados, West Indies, during August-November 1978. Greatest phagostimulatory responses were elicited by starch, glutamic and aspartic acids, maltose, oleic acid, and certain combinations of vitamins. Combinations of nutrients resulted in negative synergistic or masking effects of the phagostimulatory properties of the component nutrients. The widespread sensitivity and responsiveness to glutamic and aspartic acids by marine invertebrates, and especially by sea hares, are discussed from the standpoint of possible use by marine herbivores as feeding cues.     

Depth-dependent photoadaption by zooxanthellae of the reef coral Montastrea annularis

Zooxanthellae living in colonies of the Caribbean reef coral Montastrea annularis photoadapt to depth-dependent attenuation of submarine light. Studies carried out at Discovery Bay, Jamaica, show that in shallow-living coral colonies, the zooxanthellae appear photoadapted to function at high light intensities, and do poorly if transplanted to low light intensities; in contrast, zooxanthellae in deeper-living coral colonies can be damaged by high light intensities. The adaptation to decreasing light intensity and changing spectral quality appears to be accomplished by increasing the size of the photosynthetic unit (PSU), as opposed to increasing the number of PSU's per cell. Whole cell absorption increases with depth, partially offsetting the loss of light energy due to depth-dependent attenuation. Calculations of photosynthetically usable radiation, the light an alga is capable of absorbing in its own submarine habitat, suggest that the algae at different depths are optimizing rather than maximizing their ability to harvest submarine light energy.     

Distribution of glycosylglycerols and oligosaccharides in the marine environment and their ecological significance in the deep sea

Determination of low molecular weight carbohydrates in marine environments indicated that 1-O--D-galactosylglycerol, 6-O--D-galactosyl-1-O--D-galacto-sylglycerol, sucrose, laminaribiose and laminaritriose are widely distributed in seawaters, suspended and sinking particles, and sediments in coastal as well as in deep-sea waters [e.g. Mikawa Bay and Sagami Bay, Kumano Nada (offshore Japan), and northwest North Pacific Ocean, Bering Sea and Antarctic Ocean: collections during 1978–1984]. Identification of these glycosylglycerols and oligosaccharides in algal cells such as a flagellate (Olisthodisus luteus), blue-green algae (e.g.Trichodesmium sp.) and a diatom (Reptocylindrus denicus) strongly suggests that these sugars are photosynthetically produced by algae in the euphotic zone and are then rapidly transported to the deep sea as sinking particles which can be collected by sediment-trap experiments. The rapid decay rate of low molecular weight carbohydrates by microorganisms suggests that the transported sugars provide energy substrates for microorganisms living in the deep sea.Please address all requests for reprints to Dr. Handa at Nagoya University