Production of Deuterated Zeaxanthin by <Emphasis Type="Italic">Flavobacterium Multivorum</Emphasis> and its Detection by Resonance Raman and Mass Spectrometric Methods

Flavobacterium multivorum, a zeaxanthin-producing organism, was grown aerobically in a medium prepared with deuterated water. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) and resonance Raman spectroscopy (RRS) analysis revealed 75% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 600. Deuterated zeaxanthin upon excitation with a 488 nm laser exhibited characteristic resonance Raman vibrational modes at 1161 and 1504 cm⁻¹ as compared to 1007, 1159 and 1525 cm^–1 for undeuterated zeaxanthin. HPLC/APCI-MS and HPLC/RRS were specific and sensitive with limits of detection of 2.5 pg and 50 ng, respectively.     

Comparative Tolerances of Various <Emphasis Type="Italic">Beauveria bassiana</Emphasis> Isolates to UV-B Irradiation with a Description of a Modeling Method to Assess Lethal Dose

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm⁻² (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm⁻². Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I _s). For all isolates, the I _s − D observations fit well with the survival model I _s = 1/[1 + exp(a + bD)] (0.94 ≤ r ² ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm⁻² for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm⁻² measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

A resonance Raman band assignable to the O–O stretching mode in the resting oxidized state of bovine heart cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

In the resting oxidized state (the fully oxidized “as-isolated” state) of cytochrome c oxidase (CcO) preparation, a resonance Raman band is observed at 755 cm^-1 upon 647.1 nm excitation in resonance with an absorption band at 655 nm. Addition of cyanide eliminates the Raman band concomitant with loss of the absorption band at 655 nm. These results strongly suggest that the Raman band at 755 cm^-1 originates from the O−O stretching mode of the bridging peroxide (Fe−O^-−O^-−Cu) in the O₂ reduction site of the fully oxidized “as-isolated” CcO. Although the peroxide bridged structure has been proposed on the basis of X-ray crystallography and reductive titration experiments, the present vibrational spectroscopic analyses reveal conclusively the chemical nature of the bridging ligand at the O₂ reduction site of the fully oxidized “as-isolated” bovine heart CcO.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P⁺B_A⁻ in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P⁺/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P⁺B_A⁻ stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm⁻¹ reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B_A⁻ at 1020 nm is registered in RCs of both mutants. The formation of the B_A⁻ band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm⁻¹. Only a partial stabilization of the P⁺B_A⁻ state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P⁺ and B_A⁻ is proposed to explain rapid stabilization of the P⁺B_A⁻ state in native RCs. Small oscillatory components at ∼330–380 cm⁻¹ in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P⁺B_A⁻ free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P⁺ and B_A⁻ is assumed to be another potential contributor to the mechanism of P⁺B_A⁻ stabilization.     

Zooplankton boom and ice amphipod bust below melting sea ice in the Amundsen Gulf, Arctic Canada

Early summer in the Arctic with extensive ice melt and break-up represents a dramatic change for sympagic–pelagic fauna below seasonal sea ice. As part of the International Polar Year-Circumpolar Flaw Lead system study (IPY-CFL), this investigation quantified zooplankton in the meltwater layer below landfast ice and remaining ice fauna below melting ice during June (2008) in Franklin Bay and Darnley Bay, Amundsen Gulf, Canada. The ice was in a state of advanced melt, with fully developed melt ponds. Intense melting resulted in a 0.3- to 0.5-m-thick meltwater layer below the ice, with a strong halocline to the Arctic water below. Zooplankton under the ice, in and below the meltwater layer, was sampled by SCUBA divers. Dense concentrations (max. 1,400 ind. m⁻³) of Calanus glacialis were associated with the meltwater layer, with dominant copepodid stages CIV and CV and high abundance of nauplii. Less abundant species included Pseudocalanus spp., Oithona similis and C. hyperboreus. The copepods were likely feeding on phytoplankton (0.5–2.3 mg Chl-a m⁻³) in the meltwater layer. Ice amphipods were present at low abundance (<10 ind. m⁻²) and wet biomass (<0.2 g m⁻²). Onisimus glacialis and Apherusa glacialis made up 64 and 51% of the total ice faunal abundance in Darnley Bay and Franklin Bay, respectively. During early summer, the autochthonous ice fauna becomes gradually replaced by allochthonous zooplankton, with an abundance boom near the meltwater layer. The ice amphipod bust occurs during late stages of melting and break-up, when their sympagic habitat is diminished then lost.     

Effect of UV-B radiation on the growth and antioxidant enzymes of Antarctic sea ice microalgae <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. ICE-L

The effect of ultraviolet-B (UV-B) radiation on Antarctic phytoplankton has become an attractive ecological issue as a result of annual springtime ozone depletion. The effects of UV-B radiation on the growth and antioxidant enzymes were investigated using Antarctic sea ice microalgae Chlamydomonas sp. ICE-L as the material in this study. The results demonstrated that UV-B radiation could notably inhibit the growth, especially at high UV-B radiation intensity (70 μW cm⁻²). Malondialdehyde and O₂ ^·− content in ICE-L increased rapidly in early days (1–3 days) exposed to UV-B radiation enhancement, then decreased rapidly. In the stress of UV-B radiation enhancement, the superoxide dismutase, peroxidase and Catalase activities of 1–4 days in ICE-L were obviously higher than those in the control, and their activities became higher at high UV-B radiation intensity (70 μW cm⁻²). These enzymes activity of 7 days would kept stable at low UV-B radiation intensity (35 μW cm⁻²), but kept high level at high UV-B radiation intensity (70 μW cm⁻²). However, the ascorbate peroxidase activity in ICE-L kept stable under the stress of UV-B radiation enhancement. The above experimental results indicated that the antioxidant enzyme system played an important role in the adaptation of Antarctic ice microalgae under the UV-B radiation change of Antarctic ecosystems.     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

Properties of native and hydrophobic laccases immobilized in the liquid-crystalline cubic phase on electrodes

Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS²⁻), K _M^app, and the maximal current, I _max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal current density and the efficiency of the catalysis, I _max/K _M^app, were found to be 73 μA cm⁻² and 208.2 μA cm⁻² mM⁻¹, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS²⁻ in the cubic-phase environment. Rate constants were calculated to be 7.5 × 10⁴ and 3.6 × 10⁴ M⁻¹ s⁻¹ for native and hydrophobic laccase, respectively.     

Leaf traits variation during leaf expansion in <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The morphological, anatomical and physiological variations of leaf traits were analysed during Quercus ilex L. leaf expansion. The leaf water content (LWC), leaf area relative growth rate (RGR_l) and leaf dry mass relative growth rate (RGR_m) were the highest (76±2 %, 0.413 cm² cm⁻² d⁻¹, 0.709 mg mg⁻¹ d⁻¹, respectively) at the beginning of the leaf expansion process (7 days after bud break). Leaf expansion lasted 84±2 days when air temperature ranged from 13.3±0.8 to 27.6±0.9 °C. The net photosynthetic rate (P _N), stomatal conductance (g _s), and chlorophyll content per fresh mass (Chl) increased during leaf expansion, having the highest values [12.62±1.64 μmol (CO₂) m⁻² s⁻¹, 0.090 mol (H₂O) m⁻² s⁻¹, and 1.03±0.08 mg g⁻¹, respectively] 56 days after bud break. Chl was directly correlated with leaf dry mass (DM) and P _N. The thickness of palisade parenchyma contributed to the total leaf thickness (263.1±1.5 μm) by 47 %, spongy layer thickness 38 %, adaxial epidermis and cuticle thickness 9 %, and abaxial epidermis and cuticle thickness 6 %. Variation in leaf size during leaf expansion might be attributed to a combination of cells density and length, and it is confirmed by the significant (p<0.001) correlations among these traits. Q. ilex leaves reached 90 % of their definitive structure before the most severe drought period (beginning of June — end of August). The high leaf mass area (LMA, 15.1±0.6 mg cm⁻²) at full leaf expansion was indicative of compact leaves (2028±100 cells mm⁻²). Air temperature increasing might shorten the favourable period for leaf expansion, thus changing the final amount of biomass per unit leaf area of Q. ilex.     

Passive and iontophoretic transport enhancement of insulin through porcine epidermis by depilatories: Permeability and fourier transform infrared spectroscopy studies

The effect of thioglycolate-based depilatory lotions was studied on the in vitro passive and iontophoretic permeability of insulin through porcine epidermis and biophysical changes in the stratum corneum (SC) lipids and proteins. The porcine epidermis and Franz diffusion cells modified for iontophoresis were used for the in vitro transport studies. Cathodal iontophoresis was performed at 0.2 mA/cm² current density. Resistance of the control- and depilatory-lotion-treated epidermis was determined according to Ohmslaw. Biophysical changes were studied on porcine SC before (control) and after treatment with the depilatory lotions using Fourier transform infrared (FT-IR) spectroscopy. Asymmetric (∼2915 cm⁻¹) and symmetric (∼2848 cm⁻¹) Carbon-Hydrogen (C-H) stretching absorbances were studied to estimate the extent of lipid extraction. Fourier self-deconvolution and second derivative procedures were applied to amide I band (1700–1600 cm⁻¹) in order to estimate quantitatively the changes in the secondary structure of the SC protein. The passive permeability of insulin was significantly (P<.05) increased through depilatory-lotion-treated (ie, Better Off, Marzena, and Sally Hansen) epidermis in comparison to control. Iontophoresis significantly enhanced (P<.05) the permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis. Further, we were able to achieve the desired flux of insulin (5.25 U/cm²/d) through Better Off-treated epidermis using 0.2 mA/cm² current density and 100 U/mL donor concentration of insulin. The SC treated with depilatory lotions showed a decrease in peak areas of C-H stretching absorbances in comparison with untreated SC. Depilatory lotion treatment also decreased (P<.05) the epidermal resistance in comparison with the control epidermis. The decrease in the α-helix conformation and the increase in the random and turn structures were observed in the SC proteins due to depilatory lotion treatment. The changes in the secondary structure of proteins and lipid extraction from the SC are suggested as the cause of the decrease in the epidermal resistance and the increase in the passive and iontophoretic permeability of insulin through depilatory-pretreated epidermis in comparison with the control epidermis.     

Drought tolerance in eggs and juveniles of the Iberian slug, <Emphasis Type="Italic">Arion lusitanicus</Emphasis>

The spread of invasive species is an increasing problem world wide. The invasive slug Arion lusitanicus has spread to most parts of Europe, where it often is considered as a serious pest. There is a need for better knowledge of its ecophysiology to be able to predict the effect of climatic factors, such as temperature and humidity on the population dynamics and abundance. The aim of the present study was to assemble data on the water balance and drought tolerance of eggs and juveniles of A. lusitanicus. Both eggs and juveniles had little capacity to prevent evaporative water loss and lost water when the ambient humidity fell below 99.8 and 99.5%, respectively. The water conductance of the cuticle of juveniles was 242 μg cm⁻² h⁻¹ mmHg⁻¹ and resembles that of other slug species. Both eggs and juveniles of A. lusitanicus tolerate a substantial water loss. There was no difference in water loss resistance between eggs and juveniles, but the eggs were slightly more tolerant to water loss than the juveniles. The percent water loss causing 50% mortality was 72% for the juveniles and 81% for the eggs. Despite A. lusitanicus’ tolerance of substantial water loss, its survival depends on humid habitats.     

Assessing Primary and Bacterial Production Rates in Biofilms on Pebbles in Ishite Stream, Japan

Various measurements of microbial productivity in streambed pebble biofilms were analyzed almost monthly for 1 year to quantify the importance of primary production as an autochthonous source of organic matter utilized to support heterotrophic bacterial production in the dynamic food web within this natural microbial habitat. Bacterial density varied from 0.3 × 10⁸ to 1.4 × 10⁸ cells cm⁻², and chlorophyll a concentration ranged from 0.7 to 25.9 μg cm⁻², with no coupled oscillation between seasonal changes in these two parameters. In bottle incubation experiments, the instantaneous bacterial growth rate of bacteria was significantly correlated with their production rate [measured by frequency of dividing cells (FDC)] as follows: ln μ = 0.138FDC − 3.003 (n = 15, r ² = 0.445, p < 0.001). FDC values in the pebble biofilms increased with fluctuations during the study period, ranging from 3.6% to 9.2%. Bacterial production rates largely fluctuated between 0.15 to 0.92 μg C cm⁻² h⁻¹, and its seasonal pattern was similar to that of bacterial density. Net primary production measured between May 2002 to November 2002 attained minimum level (0.5 μg C cm⁻² h⁻¹) in June and maximum level (1.9 μg C cm⁻² h⁻¹) in August. Percentages of bacterial production to net primary production ranged between 21% and 120%. Because this ratio extends both below and above 100% for these parameters, it is likely that both autochthonous and allochthonous supplies of organic matter are important for production of bacteria in the pebble biofilms that develop in rapidly flowing fresh water streams.     

Characterization of Novel Orange Fluorescent Protein Cloned from Cnidarian Tube Anemone <Emphasis Type="Italic">Cerianthus</Emphasis> sp.

A novel orange fluorescent protein (OFP) was cloned from the tentacles of Cnidarian tube anemone Cerianthus sp. It consists of 222 amino acid residues with a calculated molecular mass of 25.1 kDa. A BLAST protein sequence homology search revealed that native OFP has 81% sequence identity to Cerianthus membranaceus green fluorescent protein (cmFP512), 38% identity to Entacmaea quadricolor red fluorescent protein (eqFP611), 37% identity to Discosoma red fluorescent protein (DsRed), 36% identity to Fungia concinna Kusabira-orange fluorescent protein (KO), and a mere 21% identity to green fluorescent protein (GFP). It is most likely that OFP also adopts the 11-strand β-barrel structure of fluorescent proteins. Spectroscopic analysis indicated that it has a wide absorption spectrum peak at 548 nm with two shoulders at 487 and 513 nm. A bright orange fluorescence maximum at 573 nm was observed when OFP was excited at 515 nm or above. When OFP was excited well below 515 nm, a considerable amount of green emission maximum at 513 nm was also observed. It has a fluorescence quantum yield (Φ) of 0.64 at 25°C. The molar absorption coefficients (ɛ) of folded OFP at 278 and 548 nm are 47,000 and 60,000 M-1⁻¹ • cm-1⁻¹, respectively. Its fluorescent brightness (ɛ Φ) at 25°C is 38,400 M⁻¹-1 • cm⁻¹-1. Like other orange-red fluorescent proteins, OFP is also tetrameric. It was readily expressed as soluble protein in Escherichia coli at 37°C, and no aggregate was observed in transfected HeLa cells under our experimental conditions. Fluorescent intensity of OFP is detectable over a pH range of 3 to 12.     

Phenotypic plasticity for skeletal growth,density and calcification of <Emphasis Type="Italic">Porites lobata</Emphasis> in response to habitat type

A reciprocal transplant experiment (RTE) of the reef-building coral Porites lobata between shallow (1.5 m at low tide) back reef and forereef habitats on Ofu and Olosega Islands, American Samoa, resulted in phenotypic plasticity for skeletal characteristics. Transplants from each source population (back reef and forereef) had higher skeletal growth rates, lower bulk densities, and higher calcification rates on the back reef than on the forereef. Mean annual skeletal extension rates, mean bulk densities, and mean annual calcification rates of RTE groups were 2.6–9.8 mm year⁻¹, 1.41–1.44 g cm⁻³, and 0.37–1.39 g cm⁻² year⁻¹ on the back reef, and 1.2–4.2 mm year⁻¹, 1.49–1.53 g cm⁻³, and 0.19–0.63 g cm⁻² year⁻¹ on the forereef, respectively. Bulk densities were especially responsive to habitat type, with densities of transplants increasing on the high energy forereef, and decreasing on the low energy back reef. Skeletal growth and calcification rates were also influenced by source population, even though zooxanthella genotype of source colonies did not vary between sites, and there was a transplant site x source population interaction for upward linear extension. Genetic differentiation may explain the source population effects, or the experiment may have been too brief for phenotypic plasticity of all skeletal characteristics to be fully expressed. Phenotypic plasticity for skeletal characteristics likely enables P. lobata colonies to assume the most suitable shape and density for a wide range of coral reef habitats.     

Phytoplankton grazing by epi- and infauna inhabiting exposed rocks in coral reefs

Exposed rocks with no visible macro-fauna are abundant in all coral reefs. Depletion of phytoplankton cells and pigments by the minute crypto fauna inhabiting the outer few centimeters of such rocks was experimentally studied over an annual cycle in the Gulf of Aqaba, Red Sea. Different substrata were introduced into small (3.6 L), well mixed, tanks that were fed by running seawater pumped directly from the reef at a rate of 11±1 L h⁻¹. A steady-state reduction in phytoplankton abundance and chlorophyll a concentration of 38±26% (mean ± 1 SD) was found for untreated rocks but not for sand, gravel, or killed controls. Average areal clearance rate by untreated rocks was 17.3±8.0 ml cm⁻² h⁻¹. Conservative extrapolation of this rate to the whole reef community suggests that the fauna inhabiting exposed rocks clears 2.1±0.9 m³ m⁻² d⁻¹ at Eilat. Phytoplankton removal by untreated rocks varied from 1.5 ng chlorophyll a cm⁻² h⁻¹ during the oligotrophic summer conditions to 6 ng chlorophyll a cm⁻² h⁻¹ during the spring bloom. These values correspond to a potential nitrogen gain of 1.3 and 5.2 mmol N m⁻² day⁻¹, respectively. Cryptic reef-rock fauna can have a key role in the biogeochemical functioning of coral reef communities.     

Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil

To examine the influence of vesicular-arbuscular (VA) mycorrhizal fungi on phosphorus (P) depletion in the rhizosphere, mycorrhizal and non-mycorrhizal white clover (Trifolium repens L.) were grown for seven weeks in a sterilized calcareous soil in pots with three compartments, a central one for root growth and two outer ones for hyphae growth. Compartmentation was accomplished by a 30-μm nylon net. The root compartment received a uniform level of P (50 mg kg⁻¹ soil) in combination with low or high levels of P (50 or 150 mg kg⁻¹ soil) in the hyphal compartments. Plants were inoculated withGlomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or remained uninfected. Mycorrhizal inoculation doubled P concentration in shoot and root, and increased dry weight, especially of the shoot, irrespective of P levels. Mycorrhizal contribution accounted for 76% of total P uptake at the low P level and 79% at the high P level, and almost all of this P was delivered by the hyphae from the outer compartment. In the non-mycorrhizal plants, the depletion of NaHCO₃-extractable P (Olsen-P) extended about 1 cm into the outer compartment, but in the mycorrhizal plants a uniform P depletion zone extended up to 11.7 cm (the length of the hyphal compartment) from the root surface. In the outer compartment, the mycorrhizal hyphae length density was high (2.5–7 m cm⁻³ soil) at the various distances (0–11.7 cm) from the root surface. Uptake rate of P by mycorrhizal hyphae was in the range of 3.3–4.3×10⁻¹⁵ mol s⁻¹ cm⁻¹.