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Stable carbon isotope ratios (δ13C) of mussel soft tissues have been widely used to characterize baseline δ13C isoscapes and identify carbon sources at the base of coastal food webs. Extending soft tissue δ13C records back in time, however, is extremely challenging due to very limited sample availability. Here, we test if the stable carbon isotopic composition of periostracum (the outermost organic layer of the shell) in the Mediterranean mussel (Mytilus galloprovincialis) can be used as an environmental archive, similar to soft tissue records. In general, spatial and seasonal variations of periostracum δ13C values are comparable to those of soft tissues, but apparently the latter are more time-averaged and smoothed. Irrespective of such offset, there is a significant linear correlation between mussel periostracum and soft tissue δ13C values (R2 = 0.608, p

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

To investigate the population dynamics of naturally recruited wild Japanese eels, fisheries data of wild individuals in Okayama Prefecture were investigated as a case study. Wild and stocked eels were discriminated using a recently developed method based on otolith stable isotopes. Of the 161 eels captured in freshwater areas where eels had been stocked, 98.1% were discriminated as stocked. In contrast, 82.8% of 128 eels captured in coastal areas where eels are not stocked were discriminated as wild. There was a significant decrease in longline and set-net catch per unit effort between 2003 and 2016 in the coastal areas where most eels were discriminated as wild, indicating ongoing depletion of wild Japanese eels in these waters.

The Yesso scallop, Patinopecten yessoensis (Jay), is one of the most important bivalve species in the Japanese and Chinese mariculture industry. In recent years, however, high incidences of scallop shell deformity and mortality have occurred with increasing frequency, but timing of onset and underlying causes are often unclear. Here, we proposed a promising δ18Oshell-based method for constraining the onset of shell deformity and mortality of P. yessoensis. Following six months of intermediate suspension culture in Funka Bay, Northern Japan, shells from healthy, deformed and dead scallops were randomly sampled. High-resolution seawater temperature time-series computed from healthy scallop shell δ18O profiles were precisely and temporally aligned to the instrumental temperature curve, thus allowing δ18Oshell-derived temperature time-series from deformed and dead scallops to be contextualized and allowing timing of scallop deformity and death to be retrieved. Irrespective of scallop shell length, onsets of deformity were anchored in February, and since then deformed scallops grew slowly in comparison to healthy individuals. Without exception, however, dead scallops had already ceased their shell building and died before February, indicating different underlying causes of scallop deformity and mortality. Perhaps most promisingly, considering that shells do not have any isotopic turn-over and once formed, temperature information is locked in. Thus, this approach holds great promise for identifying time anchor points (onsets of deformity and death) in archived scallops collected over different time scales, especially during massive mortality events.

With high-temperature stress due to climate change threatening corals in the tropics, cooler high latitudes may become a potential refuge. Already established high latitudes coral communities experience severe cold stress during winter, stress that would be fatal to most of the tropical coral species. Here we studied the physiological response of two high latitude coral species in Japan: Alveopora japonica and Porites heronensis, through field transplantation and in situ incubations. The specimens experienced 243 days at a temperature under 18 °C, which is the limit for the development of reefs and, and 110 days under 15 °C. While bleaching was observed for both species, none of the specimens died, highlighting the high resilience of temperate coral species to cold stress. The survival strategies to the cold temperature seem to differ among the species. P. heronensis exhibited decreased Fv/Fm and highly decreased metabolism (photosynthesis, calcification, respiration, ETSA) suggesting that this species could survive the winter by decreasing its basal metabolism. It also shows a mitotic index of more than 30% during the warmer period that could a prompt recovery from the stress experienced during winter. A. japonica, which showed several bleaching for around three months, also showed decreased metabolism but to a lesser extent suggesting that it was able to use an alternative source of energy. These kinds of adaptations to cold stress may not be widespread among tropical coral species, highlighting the need for better conservation of this future reservoir of biodiversity that represents marginal high latitude coral communities.

Sulfur-containing compounds are important components of all organisms, but few studies have explored sulfate utilization in corals. Our previous study found that the expression of a sulfur transporter (SLC26A11) was upregulated in the presence of Symbiodinium cells in juveniles of the reef-building coral Acropora tenuis. In this study, we performed autoradiography using 35S-labeled sulfate ions (35SO4 2−) to examine the localization and amount of incorporated radioactive sulfate in the coral tissues and symbiotic algae. Incorporated 35SO4 2− was detected in symbiotic algal cells, nematocysts, ectodermal cells and calicoblast cells. The combined results of 35S autoradiography and Alcian Blue staining showed that incorporated 35S accumulated as sulfated glycosaminoglycans (GAGs) in the ectodermal cell layer. We also compared the relative incorporation of 35SO4 2− into coral tissues and endosymbiotic algae, and their chemical fractions in dark versus light (photosynthetic) conditions. The amount of sulfur compounds, such as GAGs and lipids, generated from 35SO4 2− was higher under photosynthetic conditions. Together with the upregulation of sulfate transporters by symbiosis, our results suggest that photosynthesis of algal endosymbionts contributes to the synthesis and utilization of sulfur compounds in corals., Summary: 35S-labeled sulfate incorporated into various cells of coral demonstrates that photosynthesis of endosymbiotic algae contributes to the synthesis and utilization of sulfur compounds.

Ocean acidification can negatively impact marine bivalves, especially their shell mineralization processes. Consequently, whether marine bivalves can rapidly acclimate and eventually adapt in an acidifying ocean is now increasingly receiving considerable attention. Projecting the fate of this vulnerable taxonomic group is also pivotal for the science of sclerochronology – the study which seeks to deduce records of past environmental changes and organismal life-history traits from various geochemical properties of periodically layered hard tissues (bivalve shells, corals, fish otoliths, etc.). In this review, we provide a concise overview of the long-term and transgenerational responses of marine bivalves to elevated pCO2 manifested at different levels of biological organization, with a specific focus on responses of geochemical properties (stable carbon and oxygen isotopes, minor and trace elements and microstructures) of their shells. Without exception, positive transgenerational responses to an elevated pCO2 scenario projected for the year 2100 have been found in all five bivalve species hitherto studied, under the umbrella of two non-genetic mechanisms (increased maternal provisioning and epigenetic inheritance), suggesting that marine bivalves have remarkable transgenerational phenotypic plasticity which allows them to respond plastically and acclimate rapidly in an acidifying ocean. Rapid transgenerational acclimation, especially in terms of physiological processes, however, hinders a reliable interpretation of proxy records. Transgenerationally acclimated bivalves can actively modify the calcification physiology in response to elevated pCO2, which in turn affects the processes of almost all geochemical proxies preserved in their shells. In particular, stable carbon isotopes, metabolically regulated elements (Na, K, Cu, Zn, Fe, etc.), and shell microstructures can be highly biased. In this context, we propose a number of challenges and opportunities the field of sclerochronology may face.

The frequency and severity of coral bleaching events have increased in recent decades. Regionally, human-attributed nutrient pollution, particularly nitrate, has increased in coastal areas due to inflow from rivers and groundwater. The combined effects of increased nitrate concentrations with high temperature or high light on bleaching events and reactive oxygen species levels (ROS) were tested. Coral fragments of Montipora digitata were incubated for 3 and 6 days at different light intensities (200 and 600 μmol m −2 s −1 ), temperatures (27 and 32 °C), and nitrate concentrations (

To determine the temperature history of early life stages of chub mackerel (Scomber japonicus), otolith stable oxygen isotope ratios (δ18Ootolith) of larval and juvenile fish collected in the Kuroshio–Oyashio transition area during 2004–2015 were analyzed, and experienced temperatures were estimated accordingly. The δ18Ootolith values decreased with otolith growth during the larval stage, whereas those for juveniles were positively correlated with otolith radius and increased with increasing otolith size. Combinations of δ18Ootolith values, average otolith daily increment width, and daily age were representative of the differences between better and poorer recruitment years, the former being characterized by earlier spawning and faster growth (resulting from cooler experienced temperatures), and the latter by slower growth. Analyzed fish were sorted into larval and early juvenile growth stages by cluster analysis; the high-growth cluster exhibited higher δ18Ootolith values than the low-growth cluster. The conversion of δ18Ootolith values to water temperature indicated that the higher growth cluster experienced a water temperature ca. 2.0 °C cooler during the later juvenile stage. Therefore, our results suggest the presence of a growth positive spiral, wherein individuals with high initial growth proactively enter cooler water temperature areas, accessing a highly nutritious diet resulting in further rapid growth.

A new polyacetylene carboxylic acid named montiporic acid D (1) was isolated along with a known polyacetylene alcohol, (Z)-13,15-hexadecadien-2,4-diyn-1-ol (2) from scleractinian coral Montipora digitata. The structures of compounds were determined by analyses of NMR and MS spectra.

All corals have a common structure: two tissue layers enclose a lumen, which forms the gastric cavity. Few studies have described the processes occurring inside the gastric cavity and its chemical and biological characteristics. Here, we show that the coral gastric cavity has distinct chemical characteristics with respect to dissolved O2, pH, alkalinity, and nutrients (vitamin B12, nitrate, nitrite, ammonium, and phosphate) and also harbors a distinct bacterial community. From these results, the gastric cavity can be described as a semi-closed sub-environment within the coral. Dissolved O2 shows very low constant concentrations in the deepest parts of the cavity, creating a compartmentalized, anoxic environment. The pH is lower in the cavity than in the surrounding water and, like alkalinity, shows day/night variations different from those of the surrounding water. Nutrient concentrations in the cavity are greater than the concentrations found in reef waters, especially for phosphate and vitamin B12. The source of these nutrients may be internal production by symbiotic bacteria and/or the remineralization of organic matter ingested or produced by the corals. The importance of the bacteria inhabiting the gastric cavity is supported by the finding of a high bacterial abundance and a specific bacterial community with affiliation to bacteria found in other corals and in the guts of other organisms. The findings presented here open a new area of research that may help us to understand the processes that maintain coral health.

In order to investigate the incorporation of Sr, Mg, and U into coral skeletons and its temperature dependency, we performed a culture experiment in which specimens of the branching coral (Porites cylindrica) were grown for 1 month at three seawater temperatures (22, 26, and 30 C). The results of this study showed that the linear extension rate of P. cylindrica has little effect on the skeletal Sr/Ca, Mg/Ca, and U/Ca ratios. The following temperature equations were derived: Sr/Ca (mmol/ mol) = 10.214(±0.229) � 0.0642(±0.00897) � T (C) (r 2 = 0.59, p < 0.05); Mg/Ca (mmol/mol) = 1.973(±0.302) + 0.1002 (±0.0118) � T (C) (r 2 = 0.67, p < 0.05); and U/Ca (lmol/mol) = 1.488(±0.0484) � 0.0212(±0.00189) � T (C) (r 2 = 0.78, p < 0.05). We calculated the distribution coefficient (D) of Sr, Mg, and U relative to seawater temperature and compared the results with previous data from massive Porites corals. The seawater temperature proxies based on D calibrations of P. cylindrica established in this study are generally similar to those for massive Porites corals, despite a difference in the slope of DU calibration. The calibration sensitivity of DSr, DMg, and DU to seawater temperature change during the experiment was 0.64%/C, 1.93%/C, and 1.97%/C, respectively. These results suggest that the skeletal Sr/Ca ratio (and possibly the Mg/Ca and/or U/Ca ratio) of the branching coral P. cylindrica can be used as a potential paleothermometer. 2011 Elsevier Ltd. All rights reserved.

Various stresses induce the formation of reactive oxygen species (ROS) in biological cells. In addition to stress-induced ROS, we studied the photochemical formation of hydroxyl radicals (˙OH), the most potent ROS, in coral tissues using phosphate buffer-extracted solutions and a simulated sunlight irradiation system. ˙OH formation was seen in extracts of both coral host and endosymbiont zooxanthellae. This study is the first to report quantitative measurements of ˙OH photoformation in coral tissue extracts. Our results indicated that whether or not coral bleaching occurred, coral tissues and symbiotic zooxanthellae have the potential to photochemically produce ˙OH under sunlight. However, no significant difference was found in the protein content-normalized formation rates of ˙OH between corals incubated under different temperatures and irradiance conditions. ˙OH formation rates were reduced by 40% by reducing the UV radiation in the illumination. It was indicated that UV radiation strongly affected ˙OH formation in coral tissue and zooxanthellae, in addition to its formation through photoinhibition processes.

Coral reefs are deteriorating worldwide due to various stresses, including pollution of hazardous chemicals such as antifouling chemicals. Occurrence and impacts of a new antifouling biocide Irgarol-1051 (2-methylthio- 4-tert- butylamino -6-cyclopropylamino -s-triazine) have been studied in coral reef waters around Okinawa Islands, Japan. The average concentration of Irgarol detected at commercial and fisheries Ports was 24.70 ± 9.88 ng/L. In Naha Bay, the average concentration of Irgarol was 10.00±12.98 ng/L. The average level detected around the Ports has already approaches the environmental risk limit for the marine organisms. Irgarol was detected in waters at the frequency of 92.3 % (24/26) of the total samples collected during two sampling campaigns (September and December, 2007) at the Ports. In Naha Bay, Irgarol was detected by 40.5 % (17/42) of the total water samples collected monthly from Sep., 2007 to Feb., 2008, indicating that Irgarol is widely detected along the coastal shorelines of Okinawa Island. The eco- toxicological study revealed that the rate of photosynthesis in the coral Galaxea fascicularis was significantly reduced by 18 % and 121 % relative to control when the corals were exposed to 1000 and 10,000 ng/L of Irgarol, respectively. The calcification rate dropped by 98.3 % relative to control when the corals were exposed to 10,000 ng/L of Irgarol. The results of the present study report the wide occurrence of new antifouling biocide Irgarol around coastal areas of Okinawa Island. However, the contamination does not pose serious threat on the photosynthesis and calcification of corals under short term exposure (96 h).

Coral metabolism reflects the physiological condition of a coral colony. We studied coral metabolism using a continuous-flow, complete mixing (CFCM) experimental system. Small-size Goniastrea aspera coral colonies were incubated in the CFCM system with and without hydrogen peroxide (H2O2) added to the supplied seawater (0 µM H2O2 for 12 days; 0, 0.3, 3.0, and 30 µM H2O2 for 3 days, for each treatment) Without addition of H2O2, coral metabolism, including photosynthesis (gross primary productivity) and calcification, was relatively stable and there were no significant metabolic changes, suggesting that, without H2O2 added to the CFCM system, the corals did not suffer significant stress from the experimental system over a 12-day incubation period. When H2O2 was added, large decreases in photosynthesis and calcification were observed. The non-parametric Mann–Whitney U-test showed that there were statistically significant differences in photosynthesis after addition of 3.0 µM and 30 µM H2O2, compared with the control. We also found statistically significant differences in net calcification after addition of 30 µM H2O2. Thus, the incubation experiments suggest that higher H2O2 concentrations in seawater clearly influence coral metabolism. However, the results also suggest that the current seawater H2O2 level in Okinawa is not likely to pose significant acute effects on the metabolic activities of corals.

We examined quantitative changes in the metabolism of the coral Galaxea fascicularis caused by increases in both hydrogen peroxide (H2O2) concentration and seawater temperature. Seawater temperatures were maintained at 27 or 31°C in a well-controlled incubation chamber, and three levels of H2O2 concentration (0, 0.3, 3.0 μM) were used in experimental treatments. Gross primary production, calcification rates and respiration rates were all affected by increased H2O2 concentrations and high seawater temperatures. Individual treatments of high H2O2 or elevated seawater temperature alone caused significant declines in coral photosynthesis and calcification rates within the 3-day incubation period. The synergistic effect of high H2O2 combined with high seawater temperature resulted in a 134% increase in respiration rates, which surpassed the effect of either H2O2 or high seawater temperature alone. Our results suggest that both high H2O2 concentrations and elevated temperatures in seawater can strongly affect coral metabolism; however, these effects cannot be estimated by simply summing the effects of individual stress parameters.

The distribution of corals in Japan covers a wide range of latitudes, encompassing tropical to temperate zones. However, coral communities in temperate zones contain only a small subset of species. Among the parameters that determine the distribution of corals, temperature plays an important role. We tested the resilience to cold stress of three coral species belonging to the genus Acropora in incubation experiments. Acropora pruinosa, which is the northernmost of the three species, bleached at 13 °C, but recovered once temperatures were increased. The two other species, A. hyacinthus and A. solitaryensis, which has a more southerly range than A. pruinosa, died rapidly after bleaching at 13 °C. The physiological effects of cold bleaching on the corals included decreased rates of photosynthesis, respiration and calcification, similar to the physiological effects observed with bleaching due to high temperature stress. Contrasting hot bleaching, no increases in antioxidant enzyme activities were observed, suggesting that reactive oxygen species play a less important role in bleaching under cold stress. These results confirmed the importance of resilience to cold stress in determining the distribution and northern limits of coral species, as cold events causing coral bleaching and high mortality occur regularly in temperate zones.

Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades - including A and D - have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages.

The coral species, Montipora digitata and seagrass, Thalassia hemprichii , co-inhabit the southern portion of the reef moat in Bise, Okinawa, Japan. To elucidate the biogeochemical relationship between coral and seagrass in mixed communities of the coral reef ecosystem, the carbon metabolisms and the inorganic nitrogen flux rates were estimated in various reef habitats. We used benthic chambers to investigate sandy, seagrass, coral-seagrass mixed communities, coral, and acorn worm habitats. Relatively high concentrations of nitrate and nitrite ions (NO x ) were observed in all habitats due to coastal groundwater inflow. The uptake rate constant of NO x was the highest in the coral-seagrass habitat and was significantly different from the rate constant in the seagrass habitat, indicating that seagrass benefits from co-inhabitation with coral. Dissolution of CaCO 3 was observed in the seagrass and coral-seagrass communities. This decline in basal coral carbonate substrate may contribute to increased fragmentation and dispersal of the coral habitat. On a biogeochemical scale, the coral-seagrass relationship benefits the seagrass in terms of NO x availability and benefits the coral in terms of carbonate dissolution, increasing fragmentation, and furthering habitat development.

The effects of elevated temperature and high pCO2 on the metabolism of Galaxea fascicularis were studied with oxygen and pH microsensors. Photosynthesis and respiration rates were evaluated from the oxygen fluxes from and to the coral polyps. High-temperature alone lowered both photosynthetic and respiration rates. High pCO2 alone did not significantly affect either photosynthesis or respiration rates. Under a combination of high-temperature and high-CO2, the photosynthetic rate increased to values close to those of the controls. The same pH in the diffusion boundary layer was observed under light in both (400 and 750 ppm) CO2 treatments, but decreased significantly in the dark as a result of increased CO2. The ATP contents decreased with increasing temperature. The effects of temperature on the metabolism of corals were stronger than the effects of increased CO2. The effects of acidification were minimal without combined temperature stress. However, acidification combined with higher temperature may affect coral metabolism due to the amplification of diel variations in the microenvironment surrounding the coral and the decrease in ATP contents.

From Cladonia rangiferina were isolated two novel abietane diterpenoids, hanagokenols A (1) and B (2). Also in this investigation, four known abitetane diterpenoids (3-6), four known labdane diterpenoids (7-10), one known isopimarane diterpenoid (11), and six known aromatic compounds were isolated. These structures were elucidated primarily through extensive NMR experiments. Hanagokenol A (1) was a unique abietane diterpene having an ether linkage between C-6 and C-18 of sugiol. Hanagokenol B (2) is also a unique secoabietane diterpene, having gamma-lactone which occurred by cleavage and subsequently oxidation between C-6/C-7 of 12-hydroxydehydroabietinol. Furthermore, all the isolated compounds (1-17) were tested for the antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).

From Cladonia rangiferina were isolated two novel abietane diterpenoids, hanagokenols A (1) and B (2). Also in this investigation, four known abitetane diterpenoids (3-6), four known labdane diterpenoids (7-10), one known isopimarane diterpenoid (11), and six known aromatic compounds were isolated. These structures were elucidated primarily through extensive NMR experiments. Hanagokenol A (1) was a unique abietane diterpene having an ether linkage between C-6 and C-18 of sugiol. Hanagokenol B (2) is also a unique secoabietane diterpene, having gamma-lactone which occurred by cleavage and subsequently oxidation between C-6/C-7 of 12-hydroxydehydroabietinol. Furthermore, all the isolated compounds (1-17) were tested for the antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).

Chromone glucosides, takanechromones A-C (1, 2 and 5) and chromanone glucosides, named takanechromanones A and B (3 and 4), were isolated from the methanolic extracts of Hypericumsikokumontanum together with 27 known compounds. Their structures were established based on spectroscopic evidence. The isolated compounds and some chromone derivatives were assayed for antimicrobial activity against Helicobacter pylori and cytotoxicity against human cancer cell lines.

A small-scale chamber experimental system was designed to study the effects of temperature on colony-level coral metabolism. The system continuously supplies fresh seawater to the chamber, where it is mixed immediately and completely with the seawater already present. This continuous-flow complete-mixing system (CFCM system), in conjunction with theoretical equations, allows quantitative determination of chemical uptake and release rates by coral under controlled environmental conditions. We used the massive hermatypic coral Goniastrea aspera to examine variations in pH, total alkalinity, and total inorganic carbon for 16 days at 27 degrees C under controlled light intensities (300 and 0 micromol m(-2) s(-1)). We confirmed the stability of the CFCM system with respect to coral photosynthetic and calcification fluxes. In addition, we obtained daily photosynthetic and calcification rates at different temperatures (27 degrees C, 29 degrees C, 31 degrees C, and 33 degrees C). When seawater temperature was raised from 31 degrees C to 33 degrees C, the gross primary production rate (Pgross) decreased 29.5%, and the calcification rate (G) decreased 85.7% within 2 days. The CFCM system allows quantitative evaluation of coral colony chemical release and uptake rates, and metabolism.

Modern scleractinian coral skeletons are commonly composed of aragonite, the orthorhombic form of CaCO3. Under certain conditions, modern corals produce calcite as a secondary precipitate to fill pore space. However, coral construction of primary skeletons from calcite has yet to be demonstrated. We report a calcitic primary skeleton produced by the modern scleractinian coral Acropora tenuis. When uncalcified juveniles were incubated from the larval stage in seawater with low mMg/Ca levels, the juveniles constructed calcitic crystals in parts of the primary skeleton such as the septa; the deposits were observable under Raman microscopy. Using scanning electron microscopy, we observed different crystal morphologies of aragonite and calcite in a single juvenile skeleton. Quantitative analysis using X-ray diffraction showed that the majority of the skeleton was composed of aragonite even though we had exposed the juveniles to manipulated seawater before their initial crystal nucleation and growth processes. Our results indicate that the modern scleractinian coral Acropora mainly produces aragonite skeletons in both aragonite and calcite seas, but also has the ability to use calcite for part of its skeletal growth when incubated in calcite seas.

The enhanced toxicity of 6-mercaptopurine with endotoxin has been confirmed with BALB/c male mice. Striking increases in thioinosinic acid were seen in mice pretreated with endotoxin before injection of 6-mercaptopurine, and were in agreement with the time course of the inhibiting effect of endotoxin on sleeping time. However, there were no significant differences in hypoxanthineguanine phosphoribosyltransferase between control mice and endotoxin-treated mice. Furthermore, there were significant increases in xanthine oxidase after treatment with endotoxin. It is clear that metabolism of 6-mercaptopurine might be modified by activity of the drug-metabolizing enzyme in mouse liver. Based on the effect of endoxotin on 6-mercaptopurine metabolism and the enzymes responsible for 6-mercaptopurine, it is suggested that death caused by 6-mercaptopurine-endotoxin combinations is due to an enhancement of the lethal effect of 6-mercaptopurine. Simultaneous administration of 6-mercaptopurine and endotoxin resulted in a parallel potentiation of either 6-mercaptopurine toxicity or endotoxin toxicity. This interaction was abolished in mice made resistant to the effects of endotoxin. Based on time of death, it is suggested that death caused by 6-mercaptopurineendotoxin combinations is due to an enhancement of the lethal effect of endotoxin. These results indicate that the underlying mechanism of the interaction may involve a dualistic enhancement of each agent.

Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes.

The metabolism of antipyrine was studied in five patients with acute leukemia, before and after treatment and in relapse, to ascertain the effects of treatment on hepatic microsomal enzyme activity. The mean antipyrine half-life was significantly longer in patients after treatment (15.0 +/- 3.7 h) than in patients before treatment (8.8 +/- 0.7 h) (P less than 0.02). Furthermore, the mean antipyrine half-life in patients after treatment was also significantly longer than in patients in relapse (9.2 +/- 2.7 h) (P less than 0.05). Similarly, the mean metabolic clearance rate of antipyrine was significantly lower in patients after treatment (26.9 +/- 2.2 ml/h/kg) than in patients before treatment (46.7 +/- 13.1 ml/h/kg) (P less than 0.02). The mean apparent volume of distribution was not significantly different during the course of acute leukemia. The mean transaminase activity in patients after treatment was significantly higher than that in patients before treatment and in relapse. Thus, treatment with antileukemic agents and blood transfusions might alter hepatic microsomal enzyme activity.