ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

Controversies over the scientific name of the principal mosquito vector of yellow fever virus – expediency versus validity

The history of the scientific name of the yellow fever mosquito, the vector of yellow fever virus, ranges from 1757 to the early twenty‐first century. In his 1757 work Iter Palæstinum, Frederic Hasselquist gave the name Culex aegypti to a mosquito species responsible for fierce attacks on humans in Egypt. That name was never later ascribed to Hasselquist as author, but to Linnaeus, although the name never appeared in any of Linnaeus’ publications. In Cuba, at the end of the nineteenth century, the vector of the unknown infectious agent of yellow fever was first identified as Culex mosquito and later more validly named Stegomyia fasciata. Mosquito taxonomists differed strongly about the name of the mosquito through much of the twentieth century. Interventions by the International Commission on Zoological Nomenclature imposed a biologically invalid specific name, and in the early twenty‐first century a phylogenetic analysis of the culicid tribe Aedini restored the genus Stegomyia from a century earlier. That action was short‐lived. A phylogenetic reassessment resulted in the return of Stegomyia to subgeneric rank in Aedes; thus, the name of the yellow fever mosquito survives in the traditional classification of convenience as the trinomial Aedes (Stegomyia) aegypti (Linnaeus).     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

UV‐B INDUCTION OF UV‐B PROTECTION IN ULVA PERTUSA (CHLOROPHYTA)1

The green macroalga Ulva pertusa Kjellman produced UV‐B absorbing compounds with a prominent absorption maximum at 294 nm in response only to UV‐B, and the amounts induced were proportional to the UV‐B doses. Under a 12:12‐h light:dark regime, the production of UV‐absorbing compounds occurred only during the exposure periods with little turnover in the dark. There was significant reduction in growth in parallel with the production of UV‐B absorbing compounds. The polychromatic action spectrum for the induction of UV‐B absorbing compounds in U. pertusa exhibits a major peak at 292 nm with a smaller peak at 311.5 nm. No significant induction was detected above 354.5 nm, and radiation below 285 nm caused significant reduction in the levels of UV‐B absorbing compounds. After UV‐B irradiation at 1.0 W·m^?2 for 9 h, the optimal photosynthetic quantum yield of the samples with UV‐B absorbing compounds slightly increased relative to the initial value, whereas that of thalli lacking the compounds declined to 30%–34% of the initial followed by subsequent recovery in dim light of up to 84%–85% of the initial value. There was a positive and significant relationship between the amount of UV‐B absorbing compounds with antioxidant activity as determined by the α,α‐diphenyl‐β‐picrylhydrazyl scavenging assay. In addition to mat‐forming characteristics and light‐driven photorepair, the existence and antioxidant capacity of UV‐B absorbing compounds may confer U. pertusa a greater selective advantage over other macroalgae, thereby enabling them to thrive in the presence of intense UV‐B radiation.     

EFFECT OF NITRATE CONCENTRATION ON THE RELATIONSHIP BETWEEN PHOTOSYNTHETIC OXYGEN EVOLUTION AND ELECTRON TRANSPORT RATE IN ULVA RIGIDA (CHLOROPHYTA)1

The electron transport rate (ETR) versus gross photosynthesis (GPS) relationship varies as a function of species, temperature, irradiance, and inorganic carbon levels, but less is known about the effect of nitrogen supply on this relationship. The objective of this study was to evaluate the effect of nitrate concentration on the ETR versus GPS relationship in Ulva rigida C. Agardh from the Mediterranean Sea. Chlorophyll content and tissue absorptance increased 2‐fold as nitrate in the media increased from 0 to 50 μM. Whereas internal N content increases 3‐fold at 50 μM, internal C increased slightly. Oxygen evolution and ETR, evaluated as in vivo chl fluorescence using pulse amplitude modulated fluorometry, in general saturated at irradiances above 100 μmol photons·m^?2·s^?1. Both maximum ETR and GPS values increased as nitrate concentration increased. In general, the ETR versus GPS relationship showed a linear response to increasing nitrate with little variance of the data. This relationship, however, became more variable at high irradiances and high nitrate concentrations. The ETR/GPS ratio was close to the theoretical value of 4 at low nitrate concentrations, and the ratio decreased exponentially when nitrate concentration in the media increased. The variations of ETR/GPS under different inorganic nitrogen supply are discussed in terms of the effect of nitrate on the photosynthesis and respiration relationship.     

Affinity, capacity and oxygen sensitivity of two different mechanisms for bicarbonate utilization in Ulva lactuca L. (Chlorophyta)

Ulva lactuca, collected on the west coast of Sweden at the end of May, was able to utilize the HCO₃ ^? pool of seawater only through extracellular dehydration via carbonic anhydrase, followed by uptake of the CO₂ formed. A decrease in the CO₂ supply via this mechanism resulted in the gradual development of an additional method of HCO₃ ^? utilization, namely a direct uptake of HCO₃ ^? . Photosynthesis could then be supported by either a ‘HCO₃ ^? dehydration mechanism’ or a ‘HCO₃ ^? uptake mechanism’. Through selective inhibition of either of these mechanisms, the physiological properties of the other could be assessed. These properties suggest that the HCO₃ ^? uptake mechanism of U. lactuca is important under conditions when low concentrations of inorganic C, high pH and high external O₂ concentrations would limit photosynthesis supported by the HCO₃ ^? dehydration mechanism. Such conditions may occur during intense irradiation of the alga in rockpools or in shallow bays with low rates of water exchange. The results are discussed in relation to a possible coupling between mechanisms for inorganic C acquisition and cell structure (or even morphology) of green macroalgae. They also illustrate some necessary precautions when using Michaelis–Menten kinetics for estimations of V_max and K_1/2 values.     

Determination of the nutritional value of proteins obtained from Ulva armoricana

The in vitro digestibility of Ulva armoricana proteins by trypsin, chymotrypsin and human intestinal juice was determined to evaluate their nutritional value. The amino acid composition of the protein fraction and its changes during a sampling period from October to February were also studied. Some differences in the protein pattern shown by SDS PAGE were found in different months, such as the presence of a 54 kDa protein in February. The protein fraction is composed mainly of aspartic and glutamic acids (24–35% of protein fraction, according to season) and the essential amino acids constitute 27–36% of the total fraction. The efficiencies of trypsin and chymotrypsin in Ulva protein digestion are comparable. Only four proteins with apparent molecular weights of 86, 68, 40, and 29 KDa are digested by these proteolytic systems. The proteins from the October sample were more sensitive to chymotrypsin than those from the February sample. For instance, two proteins with apparent molecular weights of 100 and 67 kDa were weakly digested by chymotrypsin in the February extract, were fully digested in the October sample. The February sample differed from two others in the presence of glycosylated proteins, most of which have apparent molecular weights higher than 43 KDa. With the October sample, the activity of human intestinal juice was more effective than two other proteolytic systems. This is especially evident with a 27 kDa protein, which was only partially digested by the intestinal liquid and not digested by chymotrypsin or trypsin. However, human intestinal juice in the February apparently did not attack the 27 kDa protein. These data suggest a change in protein structure making it less sensitive to human intestinal juice. The glycosylation of protein extract, which was especially marked in February, could explain the differences in behaviour of U. armoricana proteins in response to the digestive action of human enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Surface sensing and stress-signalling in Ulva and fouling diatoms – potential targets for antifouling: a review

Understanding the underlying signalling pathways that enable fouling algae to sense and respond to surfaces is essential in the design of environmentally friendly coatings. Both the green alga Ulva and diverse diatoms are important ecologically and economically as they are persistent biofoulers. Ulva spores exhibit rapid secretion, allowing them to adhere quickly and permanently to a ship, whilst diatoms secrete an abundance of extracellular polymeric substances (EPS), which are highly adaptable to different environmental conditions. There is evidence, now supported by molecular data, for complex calcium and nitric oxide (NO) signalling pathways in both Ulva and diatoms being involved in surface sensing and/or adhesion. Moreover, adaptation to stress has profound effects on the biofouling capability of both types of organism. Targets for future antifouling coatings based on surface sensing are discussed, with an emphasis on pursuing NO-releasing coatings as a potentially universal antifouling strategy.     

Seasonal changes of fatty acid composition and thermotropic behavior of polar lipids from marine macrophytes

Major glyco- and phospholipids as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from five species of marine macrophytes harvested in the Sea of Japan in summer and winter at seawater temperatures of 20-23 and 3 degrees C, respectively. GC and DSC analysis of lipids revealed a common increase of ratio between n-3 and n-6 polyunsaturated fatty acids (PUFAs) of polar lipids from summer to winter despite their chemotaxonomically different fatty acid (FA) composition. Especially, high level of different n-3 PUFAs was observed in galactolipids in winter. However, the rise in FA unsaturation did not result in the lowering of peak maximum temperature of phase transition of photosynthetic lipids (glycolipids and phosphatidylglycerol (PG)) in contrast to non-photosynthetic ones [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)]. Different thermotropic behavior of these lipid groups was accompanied by higher content of n-6 PUFAs from the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG in both seasons. Seasonal changes of DSC transitions and FA composition of DGTS studied for the first time were similar to PC and PE. Thermograms of all polar lipids were characterized by complex profiles and located in a wide temperature range between -130 and 80 degrees C, while the most evident phase separation occurred in PGs in both seasons. Polarizing microscopy combined with DSC has shown that the liquid crystal - isotropic melt transitions of polar lipids from marine macrophytes began from 10 to 30 degrees C mostly, which can cause the thermal sensitivity of plants to superoptimal temperatures in their environment.