INFLUENCE OF ULTRAVIOLET RADIATION ON THE EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN AND DNA POLYMERASE α IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

To evaluate the effects of UV radiation on the expression of DNA replication‐related genes in phytoplankton, the mRNA levels of DNA polymerase α and proliferating cell nuclear antigen (PCNA) in a marine diatom, Skeletonema costatum (Greville) Cleve, were studied using the methods of real‐time quantitative PCR. Treating the algal cultures with UVC radiation for 15 min caused severe mortality during the 24‐h period after treatment. A significant amount of thymine dimers was detected in the treated cultures, and the mRNA levels of DNA polymerase α and PCNA increased by as much as 140 and 23 pmol·(g total RNA)^?1, respectively, compared with the control experiments. In contrast, massive cell deaths did not occur in cultures receiving UVA/B radiation, and the formation of thymine dimers was inconspicuous. Also, UVA/B did not enhance the expression levels of DNA polymerase α or PCNA. Based on the calculation of biologically effective UV doses, daily exposure to sunlight may increase the expression of DNA polymerase α or PCNA genes in S. costatum by 12% at sea surface. This level of increase does not seriously affect the value of using these genes as growth indicators, but caution is needed in the extrapolation of this conclusion to all phytoplankton species.     

AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.     

SINGLE‐CELL PULSE AMPLITUDE MODULATION FLUORESCENCE MEASUREMENTS OF THE GIANT DIATOM ETHMODISCUS (BACILLARIOPHYCEAE)1

Diurnal changes in effective yield (ΔF:F_m′), rapid light curves (RLCs), and induction/dark recovery time series were measured on individual cells of the giant diatom Ethmodiscus Castracane using active fluorescence (pulse amplitude modulation fluorometry). Unlike the co‐occurring diatom Hemiaulus and bulk phytoplankton, there was no observable diurnal down‐regulation of yield or relative electron transport rates in Ethmodiscus. Yields were constant at or near maximum values (0.7–0.8). Increases in ΔF:F_m′ during the initial actinic levels are consistent with dark nonphotochemical quenching mechanisms. Sustained actinic illumination (660 μmol photon·m^?2·s^?1) resulted in a ΔF:F_m′ of 0.2–0.3, but rapid recovery to near‐maximum values occurred in subsequent dark periods. Such recovery occurred even after exposure to full sunlight for 28 min, but not at 60 min. Thus, the lack of diurnal down‐regulation in Ethmodiscus is apparent, not real, and is an artifact of the time scale of sample extraction from net tows. These positively buoyant cells showed no evidence of routine photodamage, probably due to mixing and reduction in the average light exposure. The general patterns seen in RLCs from light‐and dark‐adapted higher plants are significantly different from those observed in Ethmodiscus. These results suggest that active fluorescence characteristics require careful examination to differentiate habitat‐ and taxon‐specific characteristics from light‐history effects. It is unclear whether the rapid recovery seen in Ethmodiscus is unique. The differences seen between Hemiaulus and Ethmodiscus from the same samples suggest that changes in community yield values measured in countertop systems could be the result of species replacement in addition to experimental or environmental perturbations.     

VERTICAL MIGRATION BY RHIZOSOLENIA SPP. (BACILLARIOPHYCEAE): IMPLICATIONS FOR FE ACQUISITION

Available data support a mechanism of buoyancy-mediated vertical migration by large-sized diatoms of Rhizosolenia spp. as a means to access "new" nitrogen from deep waters. To assess whether phytoplankton simultaneously satisfy their Fe requirements by this mechanism, field samples collected during summer 1996 at stations located along a transect through the central North Pacific gyre were assayed for the presence of flavodoxin and ferredoxin via Western blot analysis. All samples, regardless of their buoyancy status and the station from which they were collected, had accumulated flavodoxin but not ferredoxin. To understand better the significance of the field results, cultures of Rhizosolenia formosa H. Peragallo were grown in the laboratory with varying levels of total Fe (200 nM–10,000 nM). Fe had little effect on the physiological and photochemical parameters measured for each treatment. Growth rates did not exceed 0.17 d ^{− 1} and values of F_v/F_m ranged from 0.48 to 0.62. In addition, R. formosa accumulated only flavodoxin at each level of Fe addition. From these results, it appears that for some rhizosolenids, flavodoxin is constitutively expressed. The underlying basis for the constitutive nature of this flavodoxin is unclear at present, although it is likely that it is ultimately related to chronic Fe deficit incurred in natural waters.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

THE COMPLEX POLYSACCHARIDES OF THE RAPHID DIATOM PINNULARIA VIRIDIS (BACILLARIOPHYCEAE)1

A combination of carbohydrate analysis and atomic force microscopy (AFM) was used to characterize the polysaccharides of the pennate diatom, Pinnularia viridis (Nitzsch) Ehrenberg. Polymeric substances were fractionated into those in the spent culture medium (SCM) and those sequentially extracted from the cells with water at 45° C (WW), NaHCO₃ containing EDTA at 95° C (HB), and 1 M NaOH containing NaBH₄ at 95° C. Carbohydrate, protein, and sulfate were detected in all the fractions, but their relative proportions differed significantly. Nineteen sugars were identified, including pentoses, hexoses, 6‐deoxyhexoses, O‐methylated sugars, aminohexoses, and traces of uronic acids. To some extent, the same constituent monosaccharides and a proportion of the linkage patterns occurred in all four fractions, indicating the fractions contained a spectrum of highly heterogeneous but structurally related polysaccharides. Several carbohydrates were enriched in specific fractions. A soluble, partially substituted, 3‐linked galactan was slightly enriched in the SCM. The WW fraction was highly enriched in 3‐linked glucan, presumably derived from chrysolaminaran. Chemical and AFM data for the WW and HB fractions indicated that compositional differences were associated with substantial changes in the morphology and properties of the cell surface mucilage. Soluble polymers relatively enriched in fucose conferred a degree of softness and compressibility to the mucilage, whereas most of the mucilage comprised firmer more gelatinous polymers comparatively enriched in rhamnose. The frustule residue dissolved during extraction with NaOH, and a partially substituted 3‐linked mannan, together with relatively large amounts of protein, was obtained.     

UNCOUPLING OF SILICON COMPARED WITH CARBON AND NITROGEN METABOLISMS AND THE ROLE OF THE CELL CYCLE IN CONTINUOUS CULTURES OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) UNDER LIGHT,NITROGEN, AND PHOSPHORUS CONTROL1

The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light‐ (E), nitrogen‐ (N), and phosphorus‐ (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2‐ to 6‐fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions.     

REJUVENATION OF MELOSIRA GRANULATA (BACILLARIOPHYCEAE) RESTING CELLS FROM THE ANOXIC SEDIMENTS OF DOUGLAS LAKE,MICHIGAN, II. ELECTRON MICROSCOPY 1

Detailed cytological changes that accompany the rejuvenation of resting cells of Melosira granulata were studied with the electron microscope. Dormant and viable cells that we previously classified as the condensed state generally contain definable chloroplasts, mitochondria, a nucleus and other cytoplasmic remnants. However, there appears to be a continuous cytoplasmic degradation spectrum and some cells which appear intensely colored with the light microscope have discontinuous chloroplast membranes and few other cytoplasmic remnants. Rejuvenation of viable dormant cells is initially accompanied by the accumulation of both lipids and polyphosphates. In the earliest stages of expansion, these storage products are dispersed throughout the cell. In later stages of expansion, the lipids appear to be coalesced into larger droplets which are easily identified at the light microscope level. The fully expanded stage is characterized by the normal complement of organelles and their arrangement at the periphery of the cells and central cytoplasmic bridge. These cells appear both anabolically and catabolically active as evidenced by the abundance of endoplasmic reticulum, ribosomes and secretory and lytic vesicles. Prior to cell division, both lipids and polyphosphates a re reduced or absent in the cells. The ultrastructural features of the dormant, condensed state in resting cells of M, granulata are similar to those described for hypnospores. A rejuvenation sequence that produces cytological features common to resting state formation could provide a population of cells which could easily revert should environmental conditions become adverse.     

STRUCTURE,LIFE HISTORY AND SYSTEMATICS OF RHOICOSPHENIA (BACILLARIOPHYTA). V. INITIAL CELL AND SIZE REDUCTION IN RH. CURVATA AND A DESCRIPTION OF THE RHOICOSPHENIACEAE FAM. NOV.1

The initial epivalve of Rhoicosphenia curvata (Kütz.) Grun. differs from vegetative valves in having a strongly arched section, a wide hyaline marginal strip, no pseudosepta, an unthickened margin, and a terminal raphe fissure at the head pole. The initial epivalve is of the D type, with short raphe fissures. The epicingulum consists of three bands as usual, but they are narrower and more delicate than those of vegetative cells. The initial hypovalve and hypocingulum are similar in every way to those of vegetative cells, except for the rounded section of the hypovalve. During size reduction the almost isopolar outline of the initial valves and their immediate descendants gives way to an increasingly strong heteropolarity, and this is accompanied by changes in the relative lengths of the raphe slits and the shape of the central area. Different populations have different gametangium and initial cell sizes, suggesting the presence of races within the species. The structure of the initial cell indicates that Rhoicosphenia is less closely related to the monoraphid genera than to the gomphocymbelloid genera, confirming conclusions reached from studies of the vegetative cell and auxospore formation. Rhoicosphenia should therefore be separated into a new family, the Rhoicospheniaceae, which is described.     

Do benthic and planktonic diatoms produce equivalent effects in crustaceans?

Hippolyte inermis Leach 1814 is a benthic shrimp characterized by a peculiar mechanism of sex reversal influenced by diatom foods. In fact, the appearance of primary females in spring is due to an apoptotic early disruption of the androgenic gland and of the male gonad, triggered by still unknown compounds present in diatoms of the genus Cocconeis. The influence of diatoms on the reproductive ecology and life cycle of planktonic crustaceans has been demonstrated previously: some planktonic diatoms produce aldehydes inducing apoptosis in the embryos and in the larvae of marine copepods, reducing their viability. Both benthic and planktonic diatoms therefore produce compounds having an apoptotic effect on some tissues of target crustaceans, although the ecological significance of the two processes is different: deleterious for copepod populations, regulative for shrimps associated with Posidonia oceanica. In the present article we experimentally administered specific planktonic diatoms, their fractions and compounds known to induce apoptosis in planktonic copepods, to H. inermis postlarvae, to check whether the apoptotic effect is due to an identical family of diatom compounds, and to establish whether the processes observed in the plankton and in the benthos, respectively, are analogous or homologous, from an ecological point of view. Our results indicated that diatom compounds acting in the two systems are different, since both planktonic diatoms and their aldehydes had negligible effects on the sex ratios of cultured shrimps.