An immunofluorescence study on lectin binding sites in gametes of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae)

The distribution of binding sites for the lectins WGA, DSA. RCA I, PNA, AAA, MAA. SNA, GNA. and Con A in gametes of both sexes of the brown alga, Ectocarpus siliculosus (Dillw.) Lyngbye, was investigated by fluorescence microscopy. Digoxigenin-conjugated lectins and an FITC-anti-digoxigenin antibody were used as a high sensitivity detection system. Organelles and other distinct cellular domains could be distinguished by their binding specificities. Glycoconjugates associated with one flagellum were found to be associated with the axoneme by lectin binding to isolated flagellar apparatuses. In addition, changes in the distribution of carbohydrate epitopes during the attachment of gametes to the substratum were revealed by differential lectin binding.     

Salt Tolerance of Ectocarpus siliculosus (Dillw.) Lyngb.: Comparison of Gametophytes,Sporophytes and Isolates of Different Geographic Origin

Forty Ectocarpus siliculosus isolates from a wide geographical range, including gametophyte and sporophyte plants, have all been acclimated to the same salinity for several years. Their salinity tolerances in respect of cell viability, photosynthesis and dark respiration were evaluated over the salinity range: 8 to 96 ‰. Significant differences in the physiological tolerances to salt stress compared with viability measurements were evident. Genotypic differences in salt tolerances between groupings of the isolates, and also differences in responses of gametophyte and sporophyte generations were found. However, diploid and haploid sporophyte material had similar tolerances. Triploid and tetraploid sporophytes did not have improved tolerances over those of diploid plants. Culture plants originating from low salinities in the Baltic Sea had broader tolerances than field material collected from Baltic waters of similar salinity.     

Taxonomic value of a betaine lipid in Ectocarpus (Phaeophyceae) and the first record of a sexual population of E. fasciculatus for the Pacific Ocean

The betaine lipid DGTA differentiates between two species of Ectocarpus: it is present in E. fasciculatus Harvey, and lacking in E. siliculosus (Dillwyn) Lyngbye, Two ectocarpoid isolates from the coast of Chile, which could not be identified to species level, were found to belong to opposite DGTA types. Culture experiments showed that these plants were sporophytes. Their meiospores produced gametophytes of the species predicted by the lipid analysis. Promoted by this new approach, a sexual population of Ectocarpus fasciculatus has been detected for the first time in the Pacific Ocean.     

Effect of Extracellular Ca2+ and Ca2+-Antagonists on the Movement and Chemoorientation of Male Gametes of Ectocarpus siliculosus (Phaeophyceae)

Chemoorientation in male gametes of Ectocarpus siliculosus in response to sexual pheromones is effected by two distinct mechanisms, chemokinesis and chemoklinotaxis. These are characterized by a strongly asymmetric bending pattern of the anteriorly-directed flagellum and transient unilateral bending of the hind flagellum, respectively. Removal of extracellular Ca²⁺ showed that normal flagellar movement and chemokinesis require millimolar concentrations of Ca²⁺ in the medium. The response to pheromones is strongly inhibited by La³⁺, whereas the Ca²⁺-channel drugs, verapamil and nifedipine, have only little effect. Nifedipine nethertheless effectively inhibited accumulation at pheromone sources. These results are interpreted as an indication for the involvement of two pharmacologically distinct Ca²⁺-channels in chemokinesis and chemoklinotaxis. The calmodulin-antagonist, trifluoperazine, induces, at low concentrations, the same flagellar response in chemokinesis as the pheromone, the mechanism of action remaining unknown.     

REFLECTIVE PROPERTIES OF THE STIGMA IN MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) STUDIED BY CONFOCAL LASER SCANNING MICROSCOPY1

The reflection properties of the stigma in male gametes of Ectocarpus siliculosus (Dillw.) Lyngbye were investigated using confocal laser scanning microscopy in the epireflection contrast mode. The complex reflection pattern obtained after optical xy (horizontal) and xz (vertical) sectioning was consistent with stigma ultrastructure as revealed by serial thin sections. The intensity and pattern of the reflection signal varied with the orientation of the cell/stigma to the incident laser light. Maximal reflection occurred only in approximately normal orientation of the stigma to the light source. Focusing of reflected light from an elongated concave depression of the stigma on the region of the flagellar swelling was observed in xy and xz sections of living and fixed gametes. The results indicate the importance of mechanisms (focusing) other than quarter-wave interference reflection in signal amplification by the eyespot of flagellate algae.     

Reinstatement of Ectocarpus crouaniorum Thuret in Le Jolis as a third common species of Ectocarpus (Ectocarpales,Phaeophyceae) in Western Europe,and its phenology at Roscoff,Brittany

Based on morphological characters, cross‐fertility and molecular systematics, two species are currently recognized in the ubiquitous temperate brown algal genus Ectocarpus: the type species E. siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey. We studied diversity, cross‐fertility and ecology of Ectocarpus in megatidal areas in northwest France (Western Europe) and propose to reinstate a third species, E. crouaniorum Thuret in Le Jolis. Genotyping of 67 individuals from five localities, including the type locality of E. crouaniorum, using internal transcribed spacer 1 (ITS1) length as a marker, showed that the three species co‐occurred whenever the habitat was suitable. Our survey also revealed a single putative field hybrid between E. crouaniorum and E. siliculosus, and a single individual of a further Ectocarpus genotype. In laboratory experiments, E. crouaniorum was crossed with E. siliculosus and E. fasciculatus. In 12 of 13 crosses, the zygotes did not develop (postzygotic sterility); in one experiment a viable hybrid was produced after crossing a female E. crouaniorum with a male E. siliculosus, but this hybrid was unable to form meiospores. Phylogenetic analysis of five molecular markers from the nuclear, mitochondrial and plastid genomes (in total 1818 bp) confirmed genetic separation of the three species. Ecologically, E. crouaniorum was confined to high intertidal pools and run‐offs, where the gametophyte was common from spring to summer. Another characteristic was that it usually occurred as an epiphyte of up to 12 cm in length on erect thalli of Scytosiphon lomentaria. Sporophytes of E. crouaniorum were found all year long; they were <3 cm in size or microscopic and were epilithic in the same habitat. The presence of a third species of Ectocarpus in Western Europe suggests that species diversity in this genus is larger than recognized during the last 40 years.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales,Phaeophyceae)

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).     

Entry of the DNA virus, Ectocarpus fasciculatus virus type 1 (Phycodnaviridae), into host cell cytosol and nucleus

The process by which Ectocarpus fasciculatus virus type 1 (EfasV‐1) infects zoospores of its brown algal host was studied by electron microscopy. Upon virus attachment to the target cell, the integral membrane component of the viral capsid fuses with the host plasma membrane, and the 140‐nm viral DNA‐protein core enters the cytosol. Within 5 min after infection, particles resembling viral cores appeared in the nucleus. The entry mechanism of EfasV‐1 into the host nucleus remains enigmatic.     

Laminarionema elsbetiae gen. et sp. nov. (Ectocarpales, Phaeophyceae), a new endophyte in Laminaria sporophytes

Laminarionema elsbetiae gen. et sp, nov, (Ectocarpaceae, Ectocarpales. Phaeophyceae), a new endophyte of Laminaria japonica Areschoug (Laminariales, Phaeophyceae), is described from Muroran. Hokkaido, Japan. Laminarionema elsbetiae grows in the host tissues forming networks in the epidermal and subcortical layers as well as penetrating into the cortical and medullary layers. Only phaeophycean hairs emerge from the surface of the host tissue. No reproductive cells were found in field material. However, under host-free culture the species formed three morphologically different reproductive structures. Macrosporangia containing a single large motile spore were formed under long and short day conditions below 20°C, transformed from vegetative cells, conical to elongated in shape, 50–75 μm in length and ca. 10 μm in diameter. Microsporangia were linear to lanceolate, sometimes branched, formed under long and short day conditions below 15°C. Unilocular sporangia were more or less irregular in shape, formed under short day conditions of 5–15°C, 60–75 μm in length and 40–45 μm in diameter. Sexual fusion between macro- and microspores was not seen. In mixed cultures of L. elsbetiae with young sporophytes of L Japonica Areschoug as well as Saccorhiza dermatodea (de la Pylaie) J. Agardh, L elsbetiae infected both hosts, grew in the same manner as in natural hosts, and formed macrosporangia between host epidermal cells.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

Body musculature of Beauchampiella eudactylota (Gosse, 1886) (Rotifera: Euchlanidae) with additional new data on its trophi and overall morphology

This study presents the results of confocal laser scanning microscopy and fluorescence‐labelled phalloidin used to visualize the system of body musculature in Beauchampiella eudactylota. Moreover, the poorly known trophi of B. eudactylota are described based on scanning electron microscopy. In total, four paired longitudinal muscles (musculi longitudinales I–IV) and three circular muscles (musculi circulares I–III) were identified. Among these are the musculus longitudinalis ventralis, the musculus longitudinalis dorsalis and the musculus circumpedalis as documented in previous studies for other rotifer species. Compared to other species, B. eudactylota is characterized by the low number of lateral longitudinal muscles and the absence of some longitudinal muscles (musculi longitudinales capitum) and circular muscles (corona sphincter, musculus pars coronalis). Moreover, scanning electron microscopic data on the trophi of B. eudactylota reveal a number of striking similarities to the trophi in some species of Epiphanidae. This suggests that either (1) these similarities represent plesiomorphic characters present both in Epiphanidae and B. eudactylota or (2) they are synapomorphic features of B. eudactylota and some species of Epiphanidae, which would question the monophyly of Euchlanidae.     

The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.     

Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast

The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.     

UV‐radiation and elevated temperatures induce formation of reactive oxygen species in gametophytes of cold‐temperate/Arctic kelps (Laminariales,Phaeophyceae)

Enhanced UV‐radiation (UVR) through stratospheric ozone depletion and global warming are crucial stressors to marine macroalgae. Damages may arise through formation of reactive oxygen species (ROS) in gametophytes of ecologically important kelps, brown algae of the order Laminariales, Such stress‐induced damages may have a negative impact on their fitness and further impact their following life stages. In our study, gametophytes of three kelp species Alaria esculenta (L.) Grev., Laminaria digitata (Huds.) Lamour., Saccharina latissima (L.) Lane, Mayes, Druehl, Saunders from the Arctic, and of L. hyperborea (Gunnerus) Foslie from the North Sea were exposed to photosynthetically active radiation, UV‐A, and UV‐B radiation and four temperatures (2–18°C). ROS are formed predominantly in the peripheral cytoplasm and in chloroplasts especially after exposure to UVR. Superoxide (O₂*^‐) is additionally formed in small, globular cytoplasmic structures, possibly mitochondria. In the surrounding medium O₂*^‐‐concentration increased markedly at elevated temperatures and under UV stress in some cases. Ultrastructural damage was negligible pointing to a high stress tolerance of this developmental stage. Our data indicate that stress tolerant gametophytes of three Arctic kelp species should sustain their crucial function as seed bank for kelp populations even under prospective rising environmental perturbations.     

Characteristics of the Infection of Tilletia laevis Kühn (syn. Tilletia foetida (Wallr.) Liro.) in Compatible Wheat

Tilletia laevis Kühn (syn. Tilletia foetida (Wallr.) Liro.) causes wheat common bunt, which is one of the most devastating plant diseases in the world. Common bunt can result in a reduction of 80% or even a total loss of wheat production. In this study, the characteristics of T. laevis infection in compatible wheat plants were defined based on the combination of scanning electron microscopy, transmission electron microscopy and laser scanning confocal microscopy. We found T. laevis could lead to the abnormal growth of wheat tissues and cells, such as leakage of chloroplasts, deformities, disordered arrangements of mesophyll cells and also thickening of the cell wall of mesophyll cells in leaf tissue. What’s more, T. laevis teliospores were found in the roots, stems, flag leaves, and glumes of infected wheat plants instead of just in the ovaries, as previously reported. The abnormal characteristics caused by T. laevis may be used for early detection of this pathogen instead of molecular markers in addition to providing theoretical insights into T. laevis and wheat interactions for breeding of common bunt resistance.