Microphytobenthos are the major intertidal primary producers in many estuarine systems and assessment of their biomass is essential in ecological studies. Algal biomass can be assessed by the use of proxy measurements, e.g. chlorophyll a (Chl a), using a lengthy and destructive extraction procedure. Fluorescence is a non-destructive and in situ alternative, recently applied to microphytobenthos, using the minimum fluorescence signal (Fo) as a measurement of Chl a. In this study, Fo after 15 min of dark adaptation (Fo15) showed a significant positive correlation with Chl a (r = 0.84, p<0.001, n = 24). The relationship between Fo15 and Chl a extracted from sediment samples of increasing sample depth (0.2–2 mm) became less significant as sediment depth increased. The strongest relationship between Fo15 and Chl a, expressed as concentration (mg m−2) and content (mg kg−1), was found when all data were combined, but nearly comparable levels of association were found for in situ measurements using sediment cores up to 2.0 mm deep. Results suggested that full dark adaptation was not always achieved because a significant negative correlation was observed between Fv/Fm and ambient irradiance for seasonal data. In addition, Fo15 showed a more significant correlation with Chl a than with Fm15, suggesting some residual level of non-photochemical quenching after 15 min in the dark. Despite the incomplete dark adaptation, the highly significant relationships between Fo15 and both Chl a concentration and content suggest that the use of Fo15 is a suitable in situ proxy method of estimating algal biomass.

The crude phlorotannins from the brown alga Eisenia bicyclis showed inhibitory activity against 10 of 13 kinds of glycosidases present in the viscera of the turban shell Turbo cornutus. Phloroglucinol and its oligomers – eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), and an unidentified tetramer – were isolated from the crude phlorotannins by column and thin-layer chromatography. Phlorofucofuroeckol A, dieckol and 8,8′-bieckol inhibited α-fucosidase, β-galactosidase and β-mannosidase partially purified from T. cornutus, while phloroglucinol, eckol and the unidentified tetramer were weakly active. Dieckol was a competitive inhibitor of α-fucosidase with an inhibition constant (Ki) of 0.12 mM. The amounts of phlorotannins released after the immersion of freshly collected E. bicyclis in seawater or deionized water were estimated by high-performance liquid chromatography. Nearly all the phlorotannins were exuded into the medium following the death of the algae, whereas no phlorotannins were detected in the medium of living algae. These findings indicate that the phlorotannins deter the feeding of marine herbivorous gastropods by inhibiting the glycosidases.

Nutrient manipulation experiments were conducted on a natural planktonic community in outdoor mesocosms. Inorganic nitrogen (N) and silicon (Si) were added to achieve N[ratio ]Si ratios of 1[ratio ]1 and 4[ratio ]1. Total particulate carbon (PC) biomass of the microbial assemblage was determined by elemental analysis. Cell volume measurements by microscope on individual components of the community (bacteria, diatoms, photosynthetic nanoflagellates, heterotrophic nanoflagellates, dinoflagellates and ciliates) were also made. We applied published C[ratio ]volume relationships to determine the volume estimated C content (CBV) of these microbial groups and hence of the total assemblage. The total CBV and total PC were compared to test the applicability of C[ratio ]volume relationships under different nutrient regimes both before and after nutrient exhaustion. For initial N[ratio ]Si ratios of 1[ratio ]1, prior to nutrient exhaustion, the relationship between CBV and PC was linear with a gradient of approximately 1, (0.99±0.06), indicating that the published C[ratio ]volume relationships accurately predicted the C content of the microbial assemblage. For N[ratio ]Si ratios of 4[ratio ]1, a linear relationship was again evident between CBV and PC (slope: 1.36±0.08). However, statistical comparison using a general linear model indicated that the gradient of this relationship differed significantly from that when the N[ratio ]Si ratio was 1[ratio ]1, and hence CBV overestimated elemental C. For both N[ratio ]Si ratios, subsequent to nutrient exhaustion (N or Si), and hence when the diatom fraction of the microbial assemblage was in yield-limited post-exponential phase, the two measures of biomass were not well correlated. This indicated that measured cytoplasmic cell volume was a poor indicator of C biomass within the microbial assemblage in nutrient-deplete conditions.

Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

New holococcolith–heterococcolith life-cycle associations are documented based on observations of combination coccospheres. Daktylethra pirus is shown to be a life-cycle phase of Syracosphaera pulchra and Syracolithus quadriperforatus a life-cycle phase of Calcidiscus leptoporus. In addition, new observations from cultures confirm the life-cycle associations of Crystallolithus braarudii with Coccolithus pelagicus and of Zygosphaera hellenica with Coronosphaera mediterranea. In all four cases previous work has shown that the heterococcolithophorid species is associated with another holococcolithophorid. Two other examples of a heterococcolithophorid being associated with two holococcolithophorids have previously been identified, so this seems to be a common phenomenon. The six examples are reviewed to determine whether a single underlying mechanism is likely to be responsible for all cases. It is concluded that there is no single mechanism but rather that the six examples fall into three categories: (a) in two cases the holococcolith types are probably simply ecophenotypic morphotypes; (b) in two other cases the holococcolith types are discrete and are paralleled by morphometric differences in the heterococcolith types; (c) in the final two cases the holococcolith types are discrete but are not paralleled by any obvious morphological variation in the heterococcolith morphology. We infer that cryptic speciation may be widespread in heterococcolithophorid phases and that study of holococcolithophorid phases can provide key data to elucidate this phenomenon.

The aim of the current investigation was to test the general convention that the Dasyaceae, Delesseriaceae and Rhodomelaceae are all monophyletic families of the red algal order Ceramiales. Phylogenetic relationships among 45 ceramialean taxa were determined, including eight ceramiacean, 18 dasyacean, nine delesseriacean and eight rhodomelacean species, plus two of uncertain ceramialean affinities, based on 34 anatomical characters and nuclear small subunit (SSU) rDNA sequences. Results from our ‘total-evidence’ approach were consistent with the notion that the Dasyaceae, Delesseriaceae and Rhodomelaceae have evolved from a common ancestor within a paraphyletic Ceramiaceae. Our data indicate, however, that the Rhodomelaceae alone is monophyletic at the family level, with both the Dasyaceae and Delesseriaceae polyphyletic. In particular we resolved two independent and well-supported lineages for the included Dasyaceae, viz., a Dasya group and a Heterosiphonia group, which were as distinct from one another as they were from the Rhodomelaceae and the various lineages of Delesseriaceae. The molecular data alone were equivocal on the issue of monophyly of the Dasyaceae. We therefore advocate conservative taxonomic revisions as an interim step towards eventual resolution of familial-level taxonomy in the Ceramiales. Based on our results, the Heterosiphonioideae H.-G. Choi, Kraft, I.K. Lee et G.W. Saunders subfam. nov. is proposed for Heterosiphonia and five closely related genera, and the Dasyoideae Schmitz et Falkenberg is emended for the remaining taxa. Although the Dasyoideae is a natural group, it is in need of a thorough systematic reinvestigation at the generic level. Our analyses indicate that the genus Dasya is polyphyletic or paraphyletic in excluding Dasysiphonia, Eupogodon and Rhodoptilum and that Heterosiphonia japonica also has affinities to this group, taxonomic issues that will be addressed in detail elsewhere.

Examination of material obtained from the Urdaibai estuary (North Spain) during a 2 year phytoplankton sampling programme revealed a rare species of the genus Chaetoceros, C. salsugineus Takano. The morphology of this small (4.0×5.5 μm) member of the subgenus Hyalochaetae has been described using both light and electron microscopy. Regarding the original material, two new features have been observed: longer chains up to 24 cells and a new type of aperture, bisected by the fusion of central protuberances of adjacent valves. This bloom-forming diatom reached high densities in the poly-euhaline zone of the estuary closely related with high temperatures, denoting its neritic and summer preferences. Maximal cell densities (106–107 cells l−1) were reached in August and September when the water column was completely mixed and the salinity and temperature were about 31 psu and 20 °C, respectively. This is the first report of C. salsugineus in Europe and contributes to the knowledge of the morphological and ecological features of this species. Relationships with other small species of the subgenus Hyalochaetae are also discussed.

Quantification of the iron nutritional status of phytoplankton is of great interest not only for the study of the oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a molecular marker for iron stress, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin from Scenedesmus vacuolatus have been immunoquantified in cells grown under different iron nutritional conditions. Flavodoxin and ferredoxin levels correlate with the iron availability, and the calculated flavodoxin index can be used as an iron-stress marker. Other physiological parameters such as copper deficiency, heterotrophic or mixotrophic growth, nitrogen source and salt stress were also tested as potential factors influencing flavodoxin expression. Salt stress and heterotrophic growth conditions alter flavodoxin and ferredoxin expression. Once flavodoxin expression is repressed by iron (and severe deficiency alleviated), S.vacuolatus still increases its ferredoxin from 0.5 to 1.6 mol of ferredoxin per mole of ferredoxin-NADP+ reductase, and this ratio can be used for the evaluation of mild deficiency.

The effects on photosynthesis of CO2 and desiccation in Porphyra haitanensis were investigated to establish the effects of increased atmospheric CO2 on this alga during emersion at low tides. With enhanced desiccation, net photosynthesis, dark respiration, photosynthetic efficiency, apparent carboxylating efficiency and light saturation point decreased, while the light compensation point and CO2 compensation point increased. Emersed net photosynthesis was not saturated by the present atmospheric CO2 level (about 350 ml m−3), and doubling the CO2 concentration (700 ml m−3) increased photosynthesis by between 31% and 89% at moderate levels of desiccation. The relative enhancement of emersed net photosynthesis at 700 ml m−3 CO2 was greater at higher temperatures and higher levels of desiccation. The photosynthetic production of Porphyra haitanensis may benefit from increasing atmospheric CO2 concentration during emersion.

The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcóllar mine in April 1998, threatening the Doñana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance was 2.12×10−5 mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHM-resistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of microalgal populations in contaminated environments when the population size is large enough.